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evidence suggests that overall the actions of the transcription factor stat3 in the heart are beneficial .
some key studies involving genetic mouse models supporting a role for stat3 in ischemic protection and preventing heart failure are listed in table 1 . for a comprehensive overview of this rather complicated topic ,
the reader is referred to several recent articles that chronicle the significant contribution that stat3 plays in cardiac development , protection and remodeling .
of particular significance , this transcription factor has been implicated in the protection of cardiac myocytes that is provided by ischemic and pharmacological pre- and postconditioning , delayed ischemic preconditioning and post - infarct remodeling .
not surprisingly , many of the beneficial actions of stat3 in the heart are ascribed to its transcriptional activity .
stat3 activation in the heart has been implicated ( often based on circumstantial evidence ) in the upregulation of anti - apoptotic ( bcl - xl ) , anti - oxidant ( mnsod and metallothioneins ) and pro - angiogenic ( vegf and ve - cadherin ) genes , as well as production of protective paracrine factors by endothelial cells .
some studies have also attributed anti - fibrotic and anti - inflammatory actions to stat3 signaling in the heart through suppression of gene expression .
stat3 might suppress gene expression by well characterized means such as by competing with other transcription factors or cofactors .
alternatively , stat3 might be linked to inhibition or induction of mirnas that in turn determine the mrna expression profile of cardiac cells .
involvement of stat3 in both early preconditioning and postconditioning would not by design involve gene expression . in this
regard , recent evidence suggests that stat3 has direct non - transcriptional actions at the level of the mitochondrion that are protective of cardiac function by limiting excessive reactive oxygen species ( ros ) generation .
these mitochondrial actions of stat3 are poorly understood , but may have significance not only for both preconditioning and postconditioning , but heart failure as well .
the nontranscriptional role of stat3 may be related to the separate observations that stat3 can serve as a scaffold protein and is redox - sensitive .
the latter attribute of stat3 may manifest itself by formation of higher order complexes that conceivably could affect its association with other proteins and subcellular distribution . on the other hand
, there is evidence that stat3 activation in cardiac myocytes may be harmful in certain cases .
stat3 has been linked to pathological cardiac hypertrophy through both canonical ( phosphorylation on y705 ) and noncanonical ( unphosphorylated stat3 accumulation in the nucleus ) means ; although , as noted by others , convincing evidence linking stat3 activation under normal levels of expression to actual physical hypertrophy of cardiac myocytes is scant . however , a recent study reported that uncontrolled stat3 activation downstream of a mutant intractable gp130 , the common receptor of interleukin-6 ( il-6 ) cytokines , is harmful to the heart in myocardial infarction by causing excessive inflammation ( upregulation of il-6 and complement - activating mannose - binding lectin c ) , ventricular rupture , and heart failure .
a genetic reduction in cardiac myocyte stat3 levels was able to rescue the mutant gp130 phenotype .
in addition , hearts of mice with cardiac myocyte - targeted deletion of socs3 , the stat3-induced inhibitor of gp130 , were characterized by development at about 25 weeks of age of cardiac contractile dysfunction , various ventricular arrhythmias and signs of heart failure that were preceded by abnormalities in ca handling and troponin i hypophosphorylation .
given previous reports linking stat3 signaling to cardiac remodeling , it is notable that only minimal histological abnormalities were seen in socs3 cardiac knockout ( ko ) failing hearts , although cardiac myocyte hypertrophy was present .
unfortunately studies often approach the question of the role of stat3 in the heart from the perspective that all stat3 signaling is equivalent , particularly when it comes to signaling by the il-6 type cytokines , which share the gp130 signaling protein .
however , we propose that the context of how stat3 is activated and also what other signaling events are occurring at the same time will impact on the outcome ultimately linked to stat3 activation .
as an extension of this proposition , a better understanding of the events that regulate stat3 activation and its transcriptional and nontranscriptional ( mitochondrial ) behaviors is imperative .
to complicate matters further , stat3 activation for the purposes of transcription , and likely for its mitochondrial actions , may involve either tyrosine ( y705 ) or serine ( s727 ) phosphorylation alone , both ( more typically / canonically ) or neither , and is now known to be impacted as well by acetylation and cellular redox status . in canonical signaling , s727 phosphorylation occurs in either the cytoplasm or nucleus on stat3 that is likely already phosphorylated on y705 , as prior s727 phosphorylation seems to block y705 phosphorylation .
thus , stat3 s727 phosphorylation in the cytoplasm may function as a switch favoring the mitochondrial actions of stat3 over its canonical nuclear actions , although this is conjecture and will need to be investigated .
a number of factors have been shown to induce stat3 activation in the heart , including prolactin , granulocyte colony - stimulating factor ( g - csf ) , tumor necrosis factor- ( tnf ) , erythropoietin ( epo ) , opioids , leptin , angiotensin ii and insulin .
pre- and postconditioning - induced stat3 activation is attributable to the release of paracrine factors , such as tnf and il-6 .
the increase in ros that accompanies ischemia - reperfusion ( ir ) in the heart is also associated with marked stat3 activation ; whether ros activates stat3 via an effect on upstream jak kinases , phosphatases or largely through the upregulation or release of paracrine factors is not known .
notably , cardiac ir is associated with the production of il-6 cytokines , which are prominent activators of stat3 signaling .
they can be further classified according to those that signal through gp130 homodimers ( il-6 and il-11 ) , gp130 heterodimers with the lif receptor [ lif , cardiotrophin-1 ( ct-1 ) , ciliary neurotrophic factor ( cntf ) , cardiotrophin - like cytokine ( clc ) and oncostatin m ( osm ) in humans ] , or gp130 heterodimers with the osm receptor ( osm ) .
additional ligand - binding proteins are needed for il-6 , il-11 , cntf , clc and probably ct-1 .
lif likely binds first to the lif receptor ( lifr / cd118 ) , which then dimerizes with gp130 .
lifr is ubiquitously expressed in the normal heart and unlike gp130 does not appear to be downregulated ( at least appreciably ) in human heart failure , although cardiac levels of lif are increased in heart failure .
the il-6 family cytokines are frequently described as characterized by functional redundancy , particularly with regard to signaling in the heart .
although different actions among them have been noted , the differences are commonly explained away as due to differences in expression levels of receptors or duration of stat3 activation .
however , marked qualitative differences have been noted among the gp130-related cytokines as far as gene induction and functional impact , in particular between il-6 and lif , which are not amenable to a straightforward explanation based on the strength of their respective signaling responses . at an early time , it was noted that il-6 and lif differ as far as the induction of acute - phase genes .
more recently , lif and il-6 have been shown to counter - regulate development of the t lymphocyte lineages with il-6 coupling moreover to the activation of a gene response that downregulates lif signaling .
how il-6 and lif might exert dissimilar actions is not known . based on conjecture , lifr likely modifies the character of the gp130 signal , but how this occurs is not known .
both contain three box regions with the two membrane proximal ones being important for association with a jak family kinase and the distal one playing a role in stat3 and src - family kinase hck activation ( and possibly reinforcing erk activation at least for gp130 ) .
gp130 has four yxxq motifs that upon tyrosine phosphorylation by a jak family kinase serve as docking sites for stat3 ( and potentially stat1 ) ; the cytoplasmic domain of lifr is shorter and has three yxxq motifs .
schematic of the cytoplasmic regions of human gp130 and lifr showing the relative locations of the three box motifs and the stat3 yxxq binding motifs .
the yxxv domain is important for linking the receptors to pi3k / akt and shp2/mapk signaling pathways and for termination of signaling by recruiting either shp2 or socs3 .
socs3 terminates signaling by inhibiting jak activity directly through the n - terminal kinase inhibitory region ( kir ) or by facilitating the ubiquitination and proteasomal degradation of signaling components .
both gp130 and lifr contain a more membrane proximal yxxv motif that upon phosphorylation is important in signal termination by recruiting shp2 or socs3 .
shp2 terminates signaling through its tyrosine phosphatase activity , but also serves as a scaffold protein linking gp130 and lifr to additional signaling pathways ( fig
socs3 is induced by stat3 and terminates lif / il-6 signaling by inhibiting jak activity directly through the n - terminal kinase inhibitory region ( kir ) or by facilitating ubiquitination and proteasomal degradation of the jaks , other signaling components , or receptors .
a significant difference between the yxxv site of lifr and gp130 is that the former shows little affinity for socs3 , while the latter binds both socs3 and shp2 .
this might explain why lif - induced stat3 signaling is in general sustained ( although at a reduced level from the initial increase ) , while il-6 activation of stat3 is brief ( personal observation ) .
basic signaling similarities and differences of gp130 ( right , blue ) and lifr ( left , orange ) .
the tyrosine phosphatase activity of shp2 is thought to contribute to termination of receptor signaling .
the shp2 site on gp130 also binds socs3 , which terminates signaling by inhibiting jak activity .
the tyrosine phosphatase shp1 associates with jak1 and contributes as well to termination of gp1320 and lifr signaling .
gp130 contains an acidic domain ( light blue ) comprising amino acids 771 to 811 that binds the src - family kinase hck and couples to erk1/2 and pyk2 ( not shown ) activation .
for gp130 , s782 phosphorylation may regulate cell surface expression . at least for gp130 ,
pkc that is associated with stat3 may phosphorylate gp130 on t890 , helping to stabilize stat3-gp130 association .
in addition to jak - stat3 signaling , lifr and gp130 couple to the activation of 2 major signaling pathways : shp2/mapk and phosphatidylinositol 3-kinase ( pi3k)/akt . whether lifr and gp130 couple equally well to both of these pathways is not known .
tyrosine phosphorylation of shp2 leads to its interaction with grb2 ( growth factor receptor bound protein ) and sos ( son of sevenless ) , which triggers the ras / raf / mek / erk cascade ( fig . 2 ) .
tyrosine phosphorylated shp2 was linked to erk5 activation via grb2-associated binder-1 ( gab1 ) , a scaffolding / docking protein which likely contributes to protective signaling as well ( fig . 3 ) .
erk5 activation is thought to be responsible for the unique hypertrophic phenotype of longitudinal elongation produced by the il-6 type cytokines on cardiac myocytes .
details on how lifr and gp130 couple to pi3k / akt signaling is not known , although the pi3k regulatory subunit p85 likely associates with shp2 via gab1 as well ( fig . 3 ) .
pi3k in turn leads to activation of akt and a diverse series of signaling pathways .
stat3 appears to serve as a scaffold protein that helps assemble an activation module with jaks activating stat3 and indirectly , pi3k and akt , and in turn pi3k activating
similar events pertain to lifr ( orange , left ) , but for simplicity are not shown .
the scaffold protein gab1 forms a central point of a signaling complex linking jak1-p85 ( the regulatory subunit of pi3k ) , shp2-p85 , and stat3-p85 .
the pi3k catalytic subunit p110 is activated resulting in creation of phosphorylated phosphatidylinositol binding sites for akt .
once at the membrane , akt is activated by phosphorylation by phosphoinositide dependent protein kinase 1 ( pdpk1 ) and mammalian target of rapamycin complex 2 ( mtorc2 ) .
akt plays a role in both the trigger and mediator phases of pre- and postconditiong .
both kinases phosphorylate and inhibit glycogen synthase kinase 3 ( gsk3 ) leading to inhibition of mitochondrial permeability transition pore ( mptp ) opening , which can cause cell death . erk1/2 and
akt can inhibit gsk3 as well via nitric oxide synthase 3 ( nos3 ) activation .
activation of stat3 constitutes the survivor activating factor enhancement ( safe ) pathway for cardiac ischemic protection .
a mitochondrial role for stat3 has been proposed to explain short - term actions and may preferentially involve stat3 phosphorylated on s727 ( green circle ) .
gab1 and shp2 are also linked to cardiac hypertrophy caused by the il-6 type cytokines via erk5 activation .
notably , pi3k / akt signaling is essential for conferring cardioprotection in response to ischemic pre- and postconditioning stimuli , being involved in both the trigger and mediator phases .
its involvement in the latter forms part of the reperfusion injury salvage kinase ( risk ) pathway that confers protection by attenuating opening of the mitochondrial permeability transition pore ( mptp ) through as yet undefined means ( fig . 3 ) .
a second set of intracellular signaling events that operates independently of the risk pathway as a trigger for protection and also confers protection during reperfusion by targeting mptp opening was recently identified and involves stat3 activation .
details on how the safe pathway works at the level of the mitochondrion are not known .
thus , the il-6 family of cytokines have in theory the potential of conferring protection to cardiac myocytes from ir injury by activating both the safe and risk pathways ( fig . 3 ) .
indeed , the lifr ligand ct-1 was shown to protect isolated cardiac myocytes and the adult rat heart from injury when added either just prior to ischemia or at reoxygenation / reperfusion .
this was not the case with human heart muscle preparations which required longer exposure to ct-1 to confer protection indicating that gene expression was involved .
a carboxyl - terminal acidic domain of gp130 was shown to couple to cellular proliferation and inhibition of stem cell differentiation through the binding and activation of src family kinases , in particular hck ( fig . 2 ) .
neither is it known if hck ( or another src family member ) modulates gp130 signaling ; however , such a scenario might explain the increased tyrosine phosphorylation of gp130 in the face of reduced tyrosine phosphorylation of jak2 in hearts of patients with end - stage dilated cardiomyopathy .
the focus of our research has been lif , which has been shown to be produced by cardiac myocytes and to have protective effects on heart cells .
pretreatment of adult or neonatal cardiac myocytes with lif protected against hypoxia - reoxygenation or doxorubicin - induced injury at a later ( 612 h ) time point .
lif treatment was also shown to protect the heart from ir injury or myocardial infarction .
these beneficial actions of lif are attributed in part to the stimulation of angiogenesis and upregulation of mnsod , bcl - xl and vegf .
lif was also shown to have effects on the growth , metabolism , contractility and ca handling of cardiac myocytes , which might overall be considered disadvantageous ; however , because these studies mainly relied on cultured cells or isolated muscle the physiological significance of these effects is uncertain .
several recent observations support the conclusion that production of lif by cardiac myocytes may have physiological importance under stress conditions in myocardial repair and regeneration beyond a protective action on the cells themselves . in the mouse
, lif was found to contribute to the homing of bone marrow - derived cardiac progenitors to the infarcted myocardium and the differentiation of resident cardiac stem cells into endothelial cells .
second , in a rat genetic model of heart failure , myocardium - produced lif was shown to cause cholinergic transdifferentiation of cardiac sympathetic nerves , which might represent a means of protecting the heart from excessive sympathetic drive .
two recent studies seem to provide evidence supporting the old adage that too much of a good thing is harmful in the case of stat3 activation in the injured or aging myocardium . or do they ? in one study , sustained activation of stat3 in cardiac myocytes following a stress stimulus ( mi ) was achieved by expressing a mutant gp130 protein ( y757f ) that does not bind socs3 in mice that also have a heart targeted deletion of gp130 .
of note , the socs3 recruitment site is important for shp2 binding and thus coupling of gp130 to mapk , and possibly to pi3k / akt , signaling would be expected to be impaired as well .
indeed , hearts harboring the mutant gp130 protein showed no increase over wild type mice in either erk or akt activation following mi , although stat3 py levels were markedly enhanced .
the other study employed cardiac myocyte - targeted socs3 ko mice , which not only showed increased stat3 py levels , but increased akt , erk , and p38 mapk activation .
these signaling pathways were enhanced under basal conditions . the fact that stat3 activation was enhanced under basal conditions in the socs3 ko model , but
not the gp130 y757f model , indicates that something was driving gp130 signaling ( which could be characterized as complete ) in the socs3 ko model in a feed - forward manner .
consequently , the socs3 ko mice developed cardiac hypertrophy and heart failure with age without additional stress . in these mice ,
imposition of stress by transverse aortic constriction further enhanced gp130 signaling and caused cardiac dysfunction .
together , both studies would seem to refute the oft - cited supposition that sustained stat3 activation is beneficial and anti - inflammatory ( as seen with il-10 ) , while brief stat3 activation is harmful and pro - inflammatory ( as seen with il-6 ) .
this is somewhat surprising as the genetic reprogramming of il-6 signaling in socs3 ko macrophages was attributed to enhanced stat1 activation , owing perhaps to enhanced recruitment of the two more membrane distal yxxq stat binding sites of gp130 that are capable of activating both stat1 and stat3 .
however , increased stat1 activation was not found in the heart in either the socs3 ko ( preliminary observation ; personal communication , dr yajima ) or the gp130 y757f model .
this would have neatly explained deleterious consequences of enhanced gp130 signaling as several lines of evidence have shown that stat1 has pro - apoptotic actions in cardiac myocytes , both as a transcription factor and as a signaling molecule .
although both models were associated with increased stat3 activation ( tyrosine phosphorylation ) there are marked differences in the phenotypes of the two models that are illuminating and indicate that prolonged stat3 activation was not functionally equivalent in both ( table 2 ) .
of note , the gp130 y757f model was associated with enhanced cardiac inflammation , while the socs3 ko model was not ( although inflammation in the gp130 y757f model was assessed in hearts subjected to mi , whereas in the socs3 ko model inflammation was assessed under basal conditions ) .
since inflammation was observed in the instance where there was no concomitantly enhanced pi3k / akt signaling , enhanced stat3 signaling may not be inflammatory perhaps as long as it is balanced ( both in magnitude and temporally ) by enhanced pi3k signaling .
consistent with this possibility is the finding that cardiac myocyte - targeted stat3 overexpression was associated with cardiac hypertrophy , but was protective against doxorubicin - induced heart failure .
symbols / abbreviations : , no difference compared with wild type ; cm , cardiac myocyte ; nr , not reported ; wt , wild type ; , increase ; , decrease . while stat3 signaling is important for the anti - inflammatory actions of il-10 , the basis for why il-10-induced stat3 signaling is anti - inflammatory is still unresolved
; however , evidence was recently presented that a subset of stat3-induced anti - inflammatory genes in macrophages in response to il-10 treatment is dependent upon pi3k - mediated signaling . in this scenario ,
cytokine - induced activation of inflammatory genes by pi3k / akt - mediated activation of nfb ( or by stat3 ) may be offset by the actions of pi3k / akt on stat3 signaling ( as long as the latter is sustained ) .
moreover , nfb p65 and p50 are known to physically interact with functional consequences regardless of whether stat3 is tyrosine and/or serine phosphorylated .
conceivably , different phosphorylation ( or acetylation ) profiles of stat3 might be associated with different gene expression profiles linked to stat - nfb association .
pi3k signaling and jak - stat signaling could converge at multiple sites in both the cytoplasm and nucleus to affect expression of a particular gene , but of note the pi3k / akt pathway also induces acetylation of lysine residues on stat3 that affect dimerization and transcriptional behavior . in this regard ,
the gp130 y757f model was associated with increased levels of cardiac stat3 , which may have been due to stat3-induced stat3 expression .
accumulating evidence shows that accumulation of unphosphorylated stat3 in the nucleus can drive expression of a set of pro - inflammatory genes , including il-6 , which was in fact increased in the gp130 y757f model .
thus , under certain conditions stat3 activation sets into play a series of events that lead to a sustained inflammatory response .
coupling of lif and the other il-6 family cytokines to a balanced pi3k - stat3 response requires jak activation .
the increased stat3 activation ( i.e. , increased tyrosine phosphorylation ) that is observed with ir need not reflect a jak - mediated event as protein tyrosine phosphatases are known to be inactivated by oxidative stress .
in fact , we found that oxidative stress inhibits lif - induced jak1 and jak2 activity in cardiac myocytes . since then site - directed mutagenesis experiments were performed to demonstrate that two nearby cysteine residues in the amino - teminus region of jak2 s catalytic domain act together as a redox - sensitive switch .
the presence of this switch would thus permit the catalytic activity of jak2 to be directly regulated by the redox state of the cell . of note , these cysteine residues are highly conserved in both jak1 and jak2 among mammalian and most lower - order species .
stat3 transcriptional response may also be directly affected by oxidative stress due to the presence of redox - sensitive cysteines .
we recently presented evidence that oxidative stress may attenuate lif - induced gene expression in human microvascular endothelial cells by causing serine phosphorylation and degradation of stat3 's binding partner and transcriptional cofactor p300/cbp .
stat3 deletion reduced respiration of cardiac myocyte mitochondria due to 50% decrease in activities of complexes i and ii .
these findings are consistent with reports of increased ros formation by complex i with decreased activity , as seen in the heart with ischemia or age . besides regulating the electron transport chain , stat3 associates with matrix - localized cyclophilin d , the target of mitochondrial permeability transition pore ( mptp ) inhibitor cyclosporine .
in fact , mitochondria from stat3 hearts undergo mptp opening at lower calcium levels , which has significance for ischemia - reperfusion injury . recently
, cardiac myocyte - specific overexpression of mitochondria - targeted stat3 was found to partially block complexes i and ii with no increased basal ros production ; however , there was no ischemia - induced ros release from complex i and less reduction in complex i activity with ischemia .
how stat3 regulates complexes i / ii is unknown , but some intermediary process is likely , since the ratio of complexes i and ii to mitochondrial stat3 is ~10 .
impaired activity of complexes i and ii of mitochondria from stat3 pro - b cells could be restored by a mimetic of constitutively serine phosphorylated stat3 ( stat3 y705f / s727d ) , while stat3 y705f / s727a was ineffective .
also , a number of studies report that stat3 ps727 is enriched in mitochondria compared with the cytoplasm ; however , this may not be the case for all species .
the question of whether ischemic pre- or post - conditioning dynamically regulate mitochondrial stat3 levels or phosphorylation profile has not been determined .
conceivably , translocation of stat3 to mitochondria could function as part of a feedback loop to control mitochondrial generation of ros .
the transcription factor stat3 has been implicated in a protective and reparative response in the heart .
thus , increasing duration or intensity of stat3 activation ought to minimize damage and improve heart function under conditions of stress .
however , given the many ways that stat3 is post - translationally modified and its interaction with other signaling networks , the assumption that enhanced stat3 activity is solely a reflection of increased tyrosine phosphorylation must be construed as a gross over - simplification .
understanding the beneficial importance of stat3 signaling to heart function will require a greater appreciation of the context in which it is activated . in this regard ,
defining the differences in the impact on the heart between gp130 homodimer and heterodimer stat3 activation will be illuminating . | multiple lines of evidence suggest that the transcription factor stat3 is linked to a protective and reparative response in the heart .
thus , increasing duration or intensity of stat3 activation ought to minimize damage and improve heart function under conditions of stress .
two recent studies using genetic mouse models , however , report findings that appear to refute this proposition .
unfortunately , studies often approach the question of the role of stat3 in the heart from the perspective that all stat3 signaling is equivalent , particularly when it comes to signaling by il-6 type cytokines , which share the gp130 signaling protein .
moreover , stat3 activation is typically equated with phosphorylation of a critical tyrosine residue . yet , stat3 transcriptional behavior is subject to modulation by serine phosphorylation , acetylation , and redox status of the cell .
unphosphorylated stat3 is implicated in gene induction as well .
thus , how stat3 is activated and also what other signaling events are occurring at the same time is likely to impact on the outcome ultimately linked to stat3 .
notably stat3 may serve as a scaffold protein allowing it to interact with other singling pathways . in this context , canonical gp130 cytokine signaling may function to integrate stat3 signaling with a protective pi3k / akt signaling network via mutual involvement of jak tyrosine kinases .
differences in the extent of integration may occur between those cytokines that signal through gp130 homodimers and those through heterodimers of gp130 with a receptor chain .
signal integration may have importance not only for deciding the particular gene profile linked to stat3 , but for the newly described mitochondrial stabilization role of stat3 as well .
in addition , disruption of integrated gp130-related stat3 signaling may occur under conditions of oxidative stress , which negatively impacts on jak catalytic activity . for these reasons ,
understanding the importance of stat3 signaling to heart function requires a greater appreciation of the plasticity of this transcription factor in the context in which it is investigated . | Introduction
Leukemia Inhibitory Factor (LIF) and LIF Receptor
STAT3Too Much of a Good Thing?
Redox Sensitivity
Mitochondrial STAT3
Conclusions and Future Directions | evidence suggests that overall the actions of the transcription factor stat3 in the heart are beneficial . some key studies involving genetic mouse models supporting a role for stat3 in ischemic protection and preventing heart failure are listed in table 1 . for a comprehensive overview of this rather complicated topic ,
the reader is referred to several recent articles that chronicle the significant contribution that stat3 plays in cardiac development , protection and remodeling . of particular significance , this transcription factor has been implicated in the protection of cardiac myocytes that is provided by ischemic and pharmacological pre- and postconditioning , delayed ischemic preconditioning and post - infarct remodeling . not surprisingly , many of the beneficial actions of stat3 in the heart are ascribed to its transcriptional activity . stat3 activation in the heart has been implicated ( often based on circumstantial evidence ) in the upregulation of anti - apoptotic ( bcl - xl ) , anti - oxidant ( mnsod and metallothioneins ) and pro - angiogenic ( vegf and ve - cadherin ) genes , as well as production of protective paracrine factors by endothelial cells . some studies have also attributed anti - fibrotic and anti - inflammatory actions to stat3 signaling in the heart through suppression of gene expression . alternatively , stat3 might be linked to inhibition or induction of mirnas that in turn determine the mrna expression profile of cardiac cells . involvement of stat3 in both early preconditioning and postconditioning would not by design involve gene expression . in this
regard , recent evidence suggests that stat3 has direct non - transcriptional actions at the level of the mitochondrion that are protective of cardiac function by limiting excessive reactive oxygen species ( ros ) generation . these mitochondrial actions of stat3 are poorly understood , but may have significance not only for both preconditioning and postconditioning , but heart failure as well . the nontranscriptional role of stat3 may be related to the separate observations that stat3 can serve as a scaffold protein and is redox - sensitive . the latter attribute of stat3 may manifest itself by formation of higher order complexes that conceivably could affect its association with other proteins and subcellular distribution . on the other hand
, there is evidence that stat3 activation in cardiac myocytes may be harmful in certain cases . stat3 has been linked to pathological cardiac hypertrophy through both canonical ( phosphorylation on y705 ) and noncanonical ( unphosphorylated stat3 accumulation in the nucleus ) means ; although , as noted by others , convincing evidence linking stat3 activation under normal levels of expression to actual physical hypertrophy of cardiac myocytes is scant . however , a recent study reported that uncontrolled stat3 activation downstream of a mutant intractable gp130 , the common receptor of interleukin-6 ( il-6 ) cytokines , is harmful to the heart in myocardial infarction by causing excessive inflammation ( upregulation of il-6 and complement - activating mannose - binding lectin c ) , ventricular rupture , and heart failure . in addition , hearts of mice with cardiac myocyte - targeted deletion of socs3 , the stat3-induced inhibitor of gp130 , were characterized by development at about 25 weeks of age of cardiac contractile dysfunction , various ventricular arrhythmias and signs of heart failure that were preceded by abnormalities in ca handling and troponin i hypophosphorylation . given previous reports linking stat3 signaling to cardiac remodeling , it is notable that only minimal histological abnormalities were seen in socs3 cardiac knockout ( ko ) failing hearts , although cardiac myocyte hypertrophy was present . unfortunately studies often approach the question of the role of stat3 in the heart from the perspective that all stat3 signaling is equivalent , particularly when it comes to signaling by the il-6 type cytokines , which share the gp130 signaling protein . however , we propose that the context of how stat3 is activated and also what other signaling events are occurring at the same time will impact on the outcome ultimately linked to stat3 activation . as an extension of this proposition , a better understanding of the events that regulate stat3 activation and its transcriptional and nontranscriptional ( mitochondrial ) behaviors is imperative . to complicate matters further , stat3 activation for the purposes of transcription , and likely for its mitochondrial actions , may involve either tyrosine ( y705 ) or serine ( s727 ) phosphorylation alone , both ( more typically / canonically ) or neither , and is now known to be impacted as well by acetylation and cellular redox status . thus , stat3 s727 phosphorylation in the cytoplasm may function as a switch favoring the mitochondrial actions of stat3 over its canonical nuclear actions , although this is conjecture and will need to be investigated . a number of factors have been shown to induce stat3 activation in the heart , including prolactin , granulocyte colony - stimulating factor ( g - csf ) , tumor necrosis factor- ( tnf ) , erythropoietin ( epo ) , opioids , leptin , angiotensin ii and insulin . pre- and postconditioning - induced stat3 activation is attributable to the release of paracrine factors , such as tnf and il-6 . the increase in ros that accompanies ischemia - reperfusion ( ir ) in the heart is also associated with marked stat3 activation ; whether ros activates stat3 via an effect on upstream jak kinases , phosphatases or largely through the upregulation or release of paracrine factors is not known . notably , cardiac ir is associated with the production of il-6 cytokines , which are prominent activators of stat3 signaling . they can be further classified according to those that signal through gp130 homodimers ( il-6 and il-11 ) , gp130 heterodimers with the lif receptor [ lif , cardiotrophin-1 ( ct-1 ) , ciliary neurotrophic factor ( cntf ) , cardiotrophin - like cytokine ( clc ) and oncostatin m ( osm ) in humans ] , or gp130 heterodimers with the osm receptor ( osm ) . lifr is ubiquitously expressed in the normal heart and unlike gp130 does not appear to be downregulated ( at least appreciably ) in human heart failure , although cardiac levels of lif are increased in heart failure . the il-6 family cytokines are frequently described as characterized by functional redundancy , particularly with regard to signaling in the heart . although different actions among them have been noted , the differences are commonly explained away as due to differences in expression levels of receptors or duration of stat3 activation . however , marked qualitative differences have been noted among the gp130-related cytokines as far as gene induction and functional impact , in particular between il-6 and lif , which are not amenable to a straightforward explanation based on the strength of their respective signaling responses . more recently , lif and il-6 have been shown to counter - regulate development of the t lymphocyte lineages with il-6 coupling moreover to the activation of a gene response that downregulates lif signaling . based on conjecture , lifr likely modifies the character of the gp130 signal , but how this occurs is not known . the yxxv domain is important for linking the receptors to pi3k / akt and shp2/mapk signaling pathways and for termination of signaling by recruiting either shp2 or socs3 . shp2 terminates signaling through its tyrosine phosphatase activity , but also serves as a scaffold protein linking gp130 and lifr to additional signaling pathways ( fig
socs3 is induced by stat3 and terminates lif / il-6 signaling by inhibiting jak activity directly through the n - terminal kinase inhibitory region ( kir ) or by facilitating ubiquitination and proteasomal degradation of the jaks , other signaling components , or receptors . this might explain why lif - induced stat3 signaling is in general sustained ( although at a reduced level from the initial increase ) , while il-6 activation of stat3 is brief ( personal observation ) . basic signaling similarities and differences of gp130 ( right , blue ) and lifr ( left , orange ) . the shp2 site on gp130 also binds socs3 , which terminates signaling by inhibiting jak activity . in addition to jak - stat3 signaling , lifr and gp130 couple to the activation of 2 major signaling pathways : shp2/mapk and phosphatidylinositol 3-kinase ( pi3k)/akt . tyrosine phosphorylation of shp2 leads to its interaction with grb2 ( growth factor receptor bound protein ) and sos ( son of sevenless ) , which triggers the ras / raf / mek / erk cascade ( fig . tyrosine phosphorylated shp2 was linked to erk5 activation via grb2-associated binder-1 ( gab1 ) , a scaffolding / docking protein which likely contributes to protective signaling as well ( fig . erk5 activation is thought to be responsible for the unique hypertrophic phenotype of longitudinal elongation produced by the il-6 type cytokines on cardiac myocytes . details on how lifr and gp130 couple to pi3k / akt signaling is not known , although the pi3k regulatory subunit p85 likely associates with shp2 via gab1 as well ( fig . stat3 appears to serve as a scaffold protein that helps assemble an activation module with jaks activating stat3 and indirectly , pi3k and akt , and in turn pi3k activating
similar events pertain to lifr ( orange , left ) , but for simplicity are not shown . the scaffold protein gab1 forms a central point of a signaling complex linking jak1-p85 ( the regulatory subunit of pi3k ) , shp2-p85 , and stat3-p85 . once at the membrane , akt is activated by phosphorylation by phosphoinositide dependent protein kinase 1 ( pdpk1 ) and mammalian target of rapamycin complex 2 ( mtorc2 ) . gab1 and shp2 are also linked to cardiac hypertrophy caused by the il-6 type cytokines via erk5 activation . notably , pi3k / akt signaling is essential for conferring cardioprotection in response to ischemic pre- and postconditioning stimuli , being involved in both the trigger and mediator phases . its involvement in the latter forms part of the reperfusion injury salvage kinase ( risk ) pathway that confers protection by attenuating opening of the mitochondrial permeability transition pore ( mptp ) through as yet undefined means ( fig . a second set of intracellular signaling events that operates independently of the risk pathway as a trigger for protection and also confers protection during reperfusion by targeting mptp opening was recently identified and involves stat3 activation . details on how the safe pathway works at the level of the mitochondrion are not known . a carboxyl - terminal acidic domain of gp130 was shown to couple to cellular proliferation and inhibition of stem cell differentiation through the binding and activation of src family kinases , in particular hck ( fig . neither is it known if hck ( or another src family member ) modulates gp130 signaling ; however , such a scenario might explain the increased tyrosine phosphorylation of gp130 in the face of reduced tyrosine phosphorylation of jak2 in hearts of patients with end - stage dilated cardiomyopathy . the focus of our research has been lif , which has been shown to be produced by cardiac myocytes and to have protective effects on heart cells . lif treatment was also shown to protect the heart from ir injury or myocardial infarction . lif was also shown to have effects on the growth , metabolism , contractility and ca handling of cardiac myocytes , which might overall be considered disadvantageous ; however , because these studies mainly relied on cultured cells or isolated muscle the physiological significance of these effects is uncertain . several recent observations support the conclusion that production of lif by cardiac myocytes may have physiological importance under stress conditions in myocardial repair and regeneration beyond a protective action on the cells themselves . second , in a rat genetic model of heart failure , myocardium - produced lif was shown to cause cholinergic transdifferentiation of cardiac sympathetic nerves , which might represent a means of protecting the heart from excessive sympathetic drive . two recent studies seem to provide evidence supporting the old adage that too much of a good thing is harmful in the case of stat3 activation in the injured or aging myocardium . in one study , sustained activation of stat3 in cardiac myocytes following a stress stimulus ( mi ) was achieved by expressing a mutant gp130 protein ( y757f ) that does not bind socs3 in mice that also have a heart targeted deletion of gp130 . of note , the socs3 recruitment site is important for shp2 binding and thus coupling of gp130 to mapk , and possibly to pi3k / akt , signaling would be expected to be impaired as well . the other study employed cardiac myocyte - targeted socs3 ko mice , which not only showed increased stat3 py levels , but increased akt , erk , and p38 mapk activation . the fact that stat3 activation was enhanced under basal conditions in the socs3 ko model , but
not the gp130 y757f model , indicates that something was driving gp130 signaling ( which could be characterized as complete ) in the socs3 ko model in a feed - forward manner . in these mice ,
imposition of stress by transverse aortic constriction further enhanced gp130 signaling and caused cardiac dysfunction . together , both studies would seem to refute the oft - cited supposition that sustained stat3 activation is beneficial and anti - inflammatory ( as seen with il-10 ) , while brief stat3 activation is harmful and pro - inflammatory ( as seen with il-6 ) . this is somewhat surprising as the genetic reprogramming of il-6 signaling in socs3 ko macrophages was attributed to enhanced stat1 activation , owing perhaps to enhanced recruitment of the two more membrane distal yxxq stat binding sites of gp130 that are capable of activating both stat1 and stat3 . however , increased stat1 activation was not found in the heart in either the socs3 ko ( preliminary observation ; personal communication , dr yajima ) or the gp130 y757f model . this would have neatly explained deleterious consequences of enhanced gp130 signaling as several lines of evidence have shown that stat1 has pro - apoptotic actions in cardiac myocytes , both as a transcription factor and as a signaling molecule . although both models were associated with increased stat3 activation ( tyrosine phosphorylation ) there are marked differences in the phenotypes of the two models that are illuminating and indicate that prolonged stat3 activation was not functionally equivalent in both ( table 2 ) . of note , the gp130 y757f model was associated with enhanced cardiac inflammation , while the socs3 ko model was not ( although inflammation in the gp130 y757f model was assessed in hearts subjected to mi , whereas in the socs3 ko model inflammation was assessed under basal conditions ) . since inflammation was observed in the instance where there was no concomitantly enhanced pi3k / akt signaling , enhanced stat3 signaling may not be inflammatory perhaps as long as it is balanced ( both in magnitude and temporally ) by enhanced pi3k signaling . while stat3 signaling is important for the anti - inflammatory actions of il-10 , the basis for why il-10-induced stat3 signaling is anti - inflammatory is still unresolved
; however , evidence was recently presented that a subset of stat3-induced anti - inflammatory genes in macrophages in response to il-10 treatment is dependent upon pi3k - mediated signaling . in this scenario ,
cytokine - induced activation of inflammatory genes by pi3k / akt - mediated activation of nfb ( or by stat3 ) may be offset by the actions of pi3k / akt on stat3 signaling ( as long as the latter is sustained ) . moreover , nfb p65 and p50 are known to physically interact with functional consequences regardless of whether stat3 is tyrosine and/or serine phosphorylated . conceivably , different phosphorylation ( or acetylation ) profiles of stat3 might be associated with different gene expression profiles linked to stat - nfb association . pi3k signaling and jak - stat signaling could converge at multiple sites in both the cytoplasm and nucleus to affect expression of a particular gene , but of note the pi3k / akt pathway also induces acetylation of lysine residues on stat3 that affect dimerization and transcriptional behavior . in this regard ,
the gp130 y757f model was associated with increased levels of cardiac stat3 , which may have been due to stat3-induced stat3 expression . accumulating evidence shows that accumulation of unphosphorylated stat3 in the nucleus can drive expression of a set of pro - inflammatory genes , including il-6 , which was in fact increased in the gp130 y757f model . thus , under certain conditions stat3 activation sets into play a series of events that lead to a sustained inflammatory response . the increased stat3 activation ( i.e. , increased tyrosine phosphorylation ) that is observed with ir need not reflect a jak - mediated event as protein tyrosine phosphatases are known to be inactivated by oxidative stress . since then site - directed mutagenesis experiments were performed to demonstrate that two nearby cysteine residues in the amino - teminus region of jak2 s catalytic domain act together as a redox - sensitive switch . the presence of this switch would thus permit the catalytic activity of jak2 to be directly regulated by the redox state of the cell . stat3 transcriptional response may also be directly affected by oxidative stress due to the presence of redox - sensitive cysteines . we recently presented evidence that oxidative stress may attenuate lif - induced gene expression in human microvascular endothelial cells by causing serine phosphorylation and degradation of stat3 's binding partner and transcriptional cofactor p300/cbp . these findings are consistent with reports of increased ros formation by complex i with decreased activity , as seen in the heart with ischemia or age . how stat3 regulates complexes i / ii is unknown , but some intermediary process is likely , since the ratio of complexes i and ii to mitochondrial stat3 is ~10 . the question of whether ischemic pre- or post - conditioning dynamically regulate mitochondrial stat3 levels or phosphorylation profile has not been determined . conceivably , translocation of stat3 to mitochondria could function as part of a feedback loop to control mitochondrial generation of ros . the transcription factor stat3 has been implicated in a protective and reparative response in the heart . thus , increasing duration or intensity of stat3 activation ought to minimize damage and improve heart function under conditions of stress . however , given the many ways that stat3 is post - translationally modified and its interaction with other signaling networks , the assumption that enhanced stat3 activity is solely a reflection of increased tyrosine phosphorylation must be construed as a gross over - simplification . understanding the beneficial importance of stat3 signaling to heart function will require a greater appreciation of the context in which it is activated . in this regard ,
defining the differences in the impact on the heart between gp130 homodimer and heterodimer stat3 activation will be illuminating . | [
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percutaneous coronary intervention ( pci ) is a minimally invasive procedure for treating patients with advanced coronary artery disease . with this technique ,
a catheter system is introduced into patients circulation system through their femoral or radial artery under local anesthesia .
a preshaped guiding catheter is positioned into the ostium of the coronary artery . through this catheter
, a guide wire serving to deploy devices , such as balloon catheters and stents , is advanced into the branches of the artery .
once a stenosis is targeted , the balloon is deployed at the lesion site to fix the vessel blockage .
pci is normally performed in a catheterization laboratory under the guidance of x - ray angiography ( xa ) that coronary arteries are opacified with contrast agent . however , due to contrast agent toxicity , its injection times are limited , such that guide wire and device advancement into the target lesion is performed under
interventional cardiologists have to mentally reconstruct the position of coronary arteries and stenosis based on previous images , which increases the risk of failure for the procedures , especially in difficult cases . to address this problem , shechter et al .
proposed to model the coronary motion with surrogate signals from contrast - enhanced images , and hence , the guidance in vessel - free images becomes feasible by prospective motion correction with such a model .
as the motion of coronary arteries is mainly affected by patient s breathing motion and cardiac heart beat , it is reasonable to model the coronary motion with surrogates which are correlated with patients respiratory and cardiac motion .
with such an aim , we have been focusing on respiratory - induced coronary motion modeling .
therefore , the purpose of our study was to develop and evaluate a method for fast and robust extraction of respiratory motion surrogates from x - ray angiograms for pci .
signals from external apparatus , such as navigators or bellows , have been used in many studies for respiratory motion modeling .
one commonly used surrogate is diaphragm superio - inferior ( si ) motion [ 35 ] .
this is extracted by drawing a rectangular roi on diaphragm border followed by manual tracking the diaphragm or automatic calculating the 1d translation .
these methods use morphological operation to preprocess xa images followed by a second - order curve fitting to the diaphragm border .
studies on other respiratory - related objects , e.g. , coronary sinus catheter and tracheal bifurcation , can be seen in [ 4 , 8 ] .
these methods require specific objects being present in images , which is not always the case in xa images for pci .
dimension reduction techniques have been used for studying respiratory motion as well . in , an automatic method based on principal component analysis ( pca )
this method first creates a mask using hessian - based vesselness filtering and analyzes pixels inside the mask with pca technique . in another study ,
hierarchical manifolding learning was used to find correlation between image regions and respiratory motion . in this work
, we developed a real - time , pca - based method for extracting a respiratory surrogate from coronary xa sequences .
our contributions are threefold : first , we develop a method that is simple to implement and runs in real time on common pc hardware ; second , we evaluate the method on several clinical datasets , comparing the results with manual ground truth and existing methods ; third , we assess the usability of the method on monoplane and biplane image data in both retrospective and prospective scenarios .
coronary motion analysis in frames of xa sequence is complicated by the existence of both respiratory and cardiac motion in images .
therefore , respiratory motion surrogate extraction could possibly benefit from elimination of the objects representing cardiac motion from xa images .
in this situation , respiratory motion becomes the major source of intensity change in xa sequence and could be analyzed with methods having source decomposition capability , such as principle component analysis . to give an overview , our proposed method consists of two major steps .
first , images are downsampled and processed with morphological closing to remove coronary arteries , guiding catheters , etc .
next , pixel intensity changes in the vessel - removed images are analyzed with principal component analysis to extract respiratory motion information .
depending on the original image size , the downsampling factor is chosen to be 4 if original size is \documentclass[12pt]{minimal }
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\begin{document}$$150\times 150$$\end{document}150150 , which already allows fast processing in later steps and still preserves enough original information that we need for subsequent analysis .
next , as we are interested in respiratory motion only , we remove structures that show cardiac motion . to this end ,
similar to , a morphological closing is applied to the downsampled image with a circular structuring element in order to remove any tubular and curvilinear structures , such as coronary arteries , guiding catheters , guide wires and stitches .
the size of the structuring element is chosen based on the maximal diameter of coronary arteries and guiding catheters .
dodge reported that the lumen diameter of the left main artery measures \documentclass[12pt]{minimal }
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\begin{document}$$4.5\pm 0.5$$\end{document}4.50.5 mm . in another study using transthoracic echocardiography , the average wall thickness of left anterior descending artery was \documentclass[12pt]{minimal }
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\begin{document}$$4.5\pm 0.9$$\end{document}4.50.9 mm . having a maximum coronary diameter of 57 mm and a maximum magnification of 1.5 , we use a structuring element of around 11 mm in diameter ( roughly 78 pixels in radius in the downsampled images ) to remove the curvilinear structures .
this size is shown to be adequate and guarantee a complete removal of vessels and guiding catheters from our images . with the mentioned operations ,
objects representing cardiac motion are effectively removed from downsampled xa images , while the diaphragm border and lung tissues still remain .
morphological closing might cause circular artifacts which , however , have lower contrast than the arteries , which is sufficient to prevent the subsequent analysis from being contaminated by cardiac motion . an example morphological - closed image is showed in fig .
b image processed with morphological closing : guiding catheter and coronary arteries are removed morphological closing operation on an xa image . a original xa .
b image processed with morphological closing : guiding catheter and coronary arteries are removed principal component analysis ( pca ) is typically used for dimension reduction . it transforms a multivariate dataset to a new orthogonal coordinate system such that most variance of this dataset could be represented by a few coordinates .
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\begin{document}$$\textit{n}\times 1$$\end{document}n1 projection vector .
so each frame in such sequence is represented by an element in vector p. the assumption underlying our approach is that respiratory motion is the major source of variation in these sequences where cardiac motion is eliminated .
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\begin{document}$$150\times 150$$\end{document}150150 , which already allows fast processing in later steps and still preserves enough original information that we need for subsequent analysis .
next , as we are interested in respiratory motion only , we remove structures that show cardiac motion . to this end ,
similar to , a morphological closing is applied to the downsampled image with a circular structuring element in order to remove any tubular and curvilinear structures , such as coronary arteries , guiding catheters , guide wires and stitches .
the size of the structuring element is chosen based on the maximal diameter of coronary arteries and guiding catheters .
dodge reported that the lumen diameter of the left main artery measures \documentclass[12pt]{minimal }
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\begin{document}$$4.5\pm 0.5$$\end{document}4.50.5 mm . in another study using transthoracic echocardiography , the average wall thickness of left anterior descending artery was \documentclass[12pt]{minimal }
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\begin{document}$$4.5\pm 0.9$$\end{document}4.50.9 mm . having a maximum coronary diameter of 57 mm and a maximum magnification of 1.5
, we use a structuring element of around 11 mm in diameter ( roughly 78 pixels in radius in the downsampled images ) to remove the curvilinear structures .
this size is shown to be adequate and guarantee a complete removal of vessels and guiding catheters from our images . with the mentioned operations ,
objects representing cardiac motion are effectively removed from downsampled xa images , while the diaphragm border and lung tissues still remain .
morphological closing might cause circular artifacts which , however , have lower contrast than the arteries , which is sufficient to prevent the subsequent analysis from being contaminated by cardiac motion . an example morphological - closed image is showed in fig .
b image processed with morphological closing : guiding catheter and coronary arteries are removed morphological closing operation on an xa image . a original xa .
it transforms a multivariate dataset to a new orthogonal coordinate system such that most variance of this dataset could be represented by a few coordinates .
hence , reducing its dimension is normally achieved by preserving only a few coordinates in the new coordinate system without losing much information .
similar to , we first use the pca technique on morphological - closed images to obtain principal components for each sequence . representing a frame of an xa sequence with an n\documentclass[12pt]{minimal }
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thus , an xa sequence consisting of n frames is represented as a \documentclass[12pt]{minimal }
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next , we project the xa sequence on the first principal component \documentclass[12pt]{minimal }
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\begin{document}$$\textit{n}\times 1$$\end{document}n1 projection vector .
so each frame in such sequence is represented by an element in vector p. the assumption underlying our approach is that respiratory motion is the major source of variation in these sequences where cardiac motion is eliminated .
for our experiments , we used anonymized imaging data that were acquired from department of cardiology at erasmus mc ( university medical center rotterdam ) in rotterdam , the netherlands . xa images of eight patients who underwent a pci procedure that were acquired with siemens axiom - artis biplane system were analyzed .
the frame rate of all sequences is 15 frames per second . the number of frames per series ranges from 55 to 244 , corresponding to 3.716.3 s of imaging time .
all eight patients have in total 1898 frames . from our image data , five are \documentclass[12pt]{minimal }
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the diaphragm can clearly be seen in seven patients in both images of the biplane data , whereas the diaphragm border is not visible in the other one . in that case , some lung tissues motion can still be observed and served as the main indicator of respiratory motion . contrast agent injection and fading during imaging can be seen in all sequences .
we first manually selected a rectangular roi in original xa sequence on diaphragm border or where there is lung tissue motion , see fig .
2a . stacking all frames into a image volume and inspecting the sagittal view of the roi , we observed a profile representing the change of diaphragm position , see fig .
manual labeling diaphragm or lung tissue s motion track was subsequently done on the sagittal view of roi image ( fig .
this labeling step resulted in a vector of real numbers representing the diaphragm position in the roi over time and it served as the ground truth data in our study.fig .
2ground truth data . a drawing a rectangular roi on diaphragm border . b sagittal view of roi .
c manual labeling of diaphragm border ground truth data . a drawing a rectangular roi on diaphragm border . b sagittal view of roi .
c manual labeling of diaphragm border for retrospective evaluation , we used all frames of an xa sequence to compute the first principal component .
the resultant projection vector p represents respiratory motion of such sequence and was compared with the previously mentioned ground truth diaphragm labeling .
this comparison was quantified by calculating the correlation coefficient between projection vector p and ground truth vector . in order to gain insight of the usability of our method on different system
, the retrospective analysis was tested with both monoplane and biplane data . for biplane data , we combined information from both planes by simply using the concatenated matrix \documentclass[12pt]{minimal }
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\begin{document}$$\textit{x}=\bigl ( { \begin{matrix}x_a \\ x_b\end{matrix } } \bigr ) $ $ \end{document}x=(xaxb ) in the same approach we have described in section principal component analysis . by doing so
in addition , we compared the performance of our method , called vessel removed in later sections , with a recently published method that uses a masked - pca approach .
masked - pca technique was designed for retrospective cardiac and respiratory motion gating on interventional cardiac x - ray images . in order to extract respiratory motion surrogate with this method
, we slightly changed its implementation by directly using the projection vector on the first principal component without filtering it .
we also investigated other possible variations of pca - based methods , e.g. , running pca on the downsampled images without morphological closing ( called downsampled image ) , and running pca with an inverted mask of the mask created in ( called inverted mask ) . in all cases , the correlation coefficient of the resulting respiratory motion surrogate and the ground truth was calculated to quantify the performance on respiratory motion extraction .
we also evaluated whether the motion surrogate derived from our method could be used for prospective respiratory - induced coronary motion modeling in pci . in this experiment
, we only utilized a part of the sequence to build the pca - derived model and used it to make predictions on subsequent frames . considering a scenario in coronary motion modeling ,
we would build a model based on frames with contrast agent and use it to improve alignment of preoperative data onto xa on frames where contrast agent has been flushed away .
this makes it reasonable to choose frames before contrast agent starts fading for the pca learning phase and use frames without contrast for the prediction phase .
similar to retrospective experiment , we used the correlation coefficient to quantitatively evaluate the results with monoplane and biplane data and also compared with performances of other previously mentioned methods .
all experiments were implemented in matlab 2013b on an intel core2 2.66 ghz computer with 4 gb ram running windows .
for our experiments , we used anonymized imaging data that were acquired from department of cardiology at erasmus mc ( university medical center rotterdam ) in rotterdam , the netherlands . xa images of eight patients who underwent a pci procedure that were acquired with siemens axiom - artis biplane system were analyzed .
the frame rate of all sequences is 15 frames per second . the number of frames per series ranges from 55 to 244 , corresponding to 3.716.3 s of imaging time .
all eight patients have in total 1898 frames . from our image data , five are \documentclass[12pt]{minimal }
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the diaphragm can clearly be seen in seven patients in both images of the biplane data , whereas the diaphragm border is not visible in the other one . in that case , some lung tissues motion can still be observed and served as the main indicator of respiratory motion . contrast agent injection and fading during imaging can be seen in all sequences .
we first manually selected a rectangular roi in original xa sequence on diaphragm border or where there is lung tissue motion , see fig .
2a . stacking all frames into a image volume and inspecting the sagittal view of the roi , we observed a profile representing the change of diaphragm position , see fig .
manual labeling diaphragm or lung tissue s motion track was subsequently done on the sagittal view of roi image ( fig .
this labeling step resulted in a vector of real numbers representing the diaphragm position in the roi over time and it served as the ground truth data in our study.fig .
c manual labeling of diaphragm border ground truth data . a drawing a rectangular roi on diaphragm border .
for retrospective evaluation , we used all frames of an xa sequence to compute the first principal component .
the resultant projection vector p represents respiratory motion of such sequence and was compared with the previously mentioned ground truth diaphragm labeling .
this comparison was quantified by calculating the correlation coefficient between projection vector p and ground truth vector . in order to gain insight of the usability of our method on different system
, the retrospective analysis was tested with both monoplane and biplane data . for biplane data , we combined information from both planes by simply using the concatenated matrix \documentclass[12pt]{minimal }
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\setlength{\oddsidemargin}{-69pt }
\begin{document}$$\textit{x}=\bigl ( { \begin{matrix}x_a \\ x_b\end{matrix } } \bigr ) $ $ \end{document}x=(xaxb ) in the same approach we have described in section principal component analysis . by doing so
in addition , we compared the performance of our method , called vessel removed in later sections , with a recently published method that uses a masked - pca approach .
masked - pca technique was designed for retrospective cardiac and respiratory motion gating on interventional cardiac x - ray images . in order to extract respiratory motion surrogate with this method
, we slightly changed its implementation by directly using the projection vector on the first principal component without filtering it .
we also investigated other possible variations of pca - based methods , e.g. , running pca on the downsampled images without morphological closing ( called downsampled image ) , and running pca with an inverted mask of the mask created in ( called inverted mask ) . in all cases ,
the correlation coefficient of the resulting respiratory motion surrogate and the ground truth was calculated to quantify the performance on respiratory motion extraction .
we also evaluated whether the motion surrogate derived from our method could be used for prospective respiratory - induced coronary motion modeling in pci . in this experiment
, we only utilized a part of the sequence to build the pca - derived model and used it to make predictions on subsequent frames . considering a scenario in coronary motion modeling ,
we would build a model based on frames with contrast agent and use it to improve alignment of preoperative data onto xa on frames where contrast agent has been flushed away .
this makes it reasonable to choose frames before contrast agent starts fading for the pca learning phase and use frames without contrast for the prediction phase .
similar to retrospective experiment , we used the correlation coefficient to quantitatively evaluate the results with monoplane and biplane data and also compared with performances of other previously mentioned methods .
all experiments were implemented in matlab 2013b on an intel core2 2.66 ghz computer with 4 gb ram running windows .
example results of experiments on monoplane and biplane data from one patient are shown in fig .
3 . figure 3a , c , e presents the comparison of projection vector p and ground truth diaphragm position .
linearly rescaling p to the range of ground truth data and overlaying it onto the roi image provides another way to evaluate their correlation qualitatively .
figure 3b , d , f reveals a good agreement between p and diaphragm border .
3retrospective projection on the first principal component for one patient , in comparison with diaphragm position in roi image .
a , b pca was done on one sequence of the biplane data ; c f principal components were derived from the concatenated sequence of both planes .
c , d show the projection in comparison with plane a ; e , f illustrate the comparison with plane b retrospective projection on the first principal component for one patient , in comparison with diaphragm position in roi image .
a , b pca was done on one sequence of the biplane data ; c f principal components were derived from the concatenated sequence of both planes .
c , d show the projection in comparison with plane a ; e , f illustrate the comparison with plane b table 1 provides quantitative measure results on correlation coefficients .
the average correlation coefficient was calculated over all sequences for the various methods . from the table
, it can be seen that all methods give high correlation coefficient ( over 0.85 , close to 1 ) .
the vessel removed method has slightly higher average correlation and lower standard deviation than other methods . for the patient
whose xa sequences contain no diaphragm border , the correlation coefficients for vessel removed method are also high for the monoplane ( 0.89 and 0.88 ) and biplane data ( 0.88 and 0.95 ) .
4 illustrate similar observations : the majority of correlation coefficients are over 0.8 for all methods ; non - mask - based methods ( vessel - removed and downsampled image ) slightly outperform other methods .
. 4
boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for retrospective evaluationtable 1average correlation coefficient of projection vector p and diaphragm positions for various methods for retrospective evaluationmethodsmonoplanebiplane(mean \documentclass[12pt]{minimal }
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boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for retrospective evaluation average correlation coefficient of projection vector p and diaphragm positions for various methods for retrospective evaluation to investigate whether there is a statistically significant difference between the performance of these methods on retrospective respiratory motion surrogates extraction , we used a one - tailed wilcoxon rank - sum test to check the correlation coefficients , as their distribution is not necessarily a normal distribution and most of values are close to 1 .
result ( see table 2 ) shows that the p values for monoplane experiments range from 0.09 to 0.55 ( in upper triangle ) ; p values for biplane experiments range from 0.11 to 0.42 ( in lower triangle ) .
the result means these methods have similar performance on retrospective task in terms of statistical significance .
the numbers in the upper and lower triangle in the table show the results of monoplane and biplane cases respectivelyvessel removeddownsampled imagewith maskinverted maskvessel removed
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statistical significance between various methods for retrospective evaluation ( p values ) .
the numbers in the upper and lower triangle in the table show the results of monoplane and biplane cases respectively figure 5 illustrates the frequency distribution of correlation coefficients for various methods in our retrospective analysis . in both the monoplane and biplane experiments , it can be observed that vessel removed method has the most number of correlation coefficients over 0.9 and no correlation coefficients lower than 0.8 , which outperforms all other methods .
this observation suggests that the vessel removed method is more robust than the other approaches.fig .
5frequency distribution of correlation coefficients for various methods in retrospective analysis frequency distribution of correlation coefficients for various methods in retrospective analysis table 3 compares the average per - frame computation time that is needed to compute the projection vector p. this includes the time for image preprocessing , building statistical model through pca and making projection on the first principal component .
the comparison reveals the advantage of non - mask - based methods to mask - based method that the computation time they need is much shorter , which is favored for real clinical workflow .
table 3average per - frame computation time for various methods for retrospective evaluation ( milliseconds)methodsmonoplane ( ms)biplane ( ms)vessel removed17.234.4downsampled image8.015.5with mask821.62227.8inverted mask854.72920.0 average per - frame computation time for various methods for retrospective evaluation ( milliseconds ) example results of the prospective analysis for the same patient as in fig . 3
figure 6a , c , e present retrospective projection for frames used for learning the statistical model and prospective projection for frames excluded from the learning phase .
it can be seen that the prospective projection vector p still maintains good correlations with ground truth diaphragm position .
6prospective projection on the first principal component for one patient , in comparison with diaphragm position in roi image .
a , b pca was implemented on monoplane data ; c f principal components were derived from the concatenated sequence of biplane .
c , d show the projection in comparison with plane a ; e , f illustrate the comparison with plane b prospective projection on the first principal component for one patient , in comparison with diaphragm position in roi image .
a , b pca was implemented on monoplane data ; c f principal components were derived from the concatenated sequence of biplane .
c , d show the projection in comparison with plane a ; e , f illustrate the comparison with plane b correlation coefficients are shown in table 4 . in general , these numbers are lower than those in the retrospective experiments , while vessel removed method still maintains a high average correlation coefficient over 0.9 .
whose diaphragm can not be seen in the xa sequences , the correlation coefficients for vessel removed method remain good for one of sequences in the monoplane data ( 0.96 and 0.70 ) and both sequences in the biplane data ( 0.91 and 0.87 ) .
7 show that the medians of all methods are quite close to each other , but vessel removed method has fewer correlation coefficients lower than the median value compared to other methods .
we also used wilcoxon rank - sum test to check the statistical significance , and the results are shown in table 5 .
no significant difference is found among these methods ( \documentclass[12pt]{minimal }
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\begin{document}$$p>0.14$$\end{document}p>0.14 ) , which means their performance are similar to each other in terms of statistical significance .
. 7
boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for prospective evaluationtable 4average correlation coefficients of the prospective projection vector and diaphragm positions for various methodsmethodsmonoplanebiplane(mean \documentclass[12pt]{minimal }
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\begin{document}$$\pm $ $ \end{document} 0.2117table 5statistical significance between various methods in prospective evaluation ( p values ) .
the numbers in the upper and lower triangle in the table show the results of monoplane and biplane cases respectivelyvessel removeddownsampled imagewith maskinverted maskvessel removed
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boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for prospective evaluation average correlation coefficients of the prospective projection vector and diaphragm positions for various methods statistical significance between various methods in prospective evaluation ( p values ) .
the numbers in the upper and lower triangle in the table show the results of monoplane and biplane cases respectively frequency distribution of correlation coefficients in fig .
8 reveals that for both monoplane and biplane experiments , more lower value correlation coefficients appear for all methods compared to retrospective analysis .
it is also clear that vessel removed method has the most high correlation coefficients ( cc \documentclass[12pt]{minimal }
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8frequency distribution of correlation coefficients for various methods in prospective analysis frequency distribution of correlation coefficients for various methods in prospective analysis average per - frame prediction time for various methods is listed in table 6 .
the prediction time includes the time for image preprocessing and computing projection on the first principal component .
it is obvious that downsampled image method has the shortest prediction time , while vessel removed method is also quite fast .
mask - based methods are slower than non - mask - based methods , especially when they were run on biplane data .
table 6average per - frame prediction time for various methods ( milliseconds)methodsmonoplane ( ms)biplane ( ms)vessel removed9.620.0downsampled image1.63.8with mask16.3282.6inverted mask31.0792.4 average per - frame prediction time for various methods ( milliseconds )
example results of experiments on monoplane and biplane data from one patient are shown in fig .
3 . figure 3a , c , e presents the comparison of projection vector p and ground truth diaphragm position .
linearly rescaling p to the range of ground truth data and overlaying it onto the roi image provides another way to evaluate their correlation qualitatively .
figure 3b , d , f reveals a good agreement between p and diaphragm border .
3retrospective projection on the first principal component for one patient , in comparison with diaphragm position in roi image .
a , b pca was done on one sequence of the biplane data ; c f principal components were derived from the concatenated sequence of both planes .
c , d show the projection in comparison with plane a ; e , f illustrate the comparison with plane b retrospective projection on the first principal component for one patient , in comparison with diaphragm position in roi image .
a , b pca was done on one sequence of the biplane data ; c f principal components were derived from the concatenated sequence of both planes .
c , d show the projection in comparison with plane a ; e , f illustrate the comparison with plane b table 1 provides quantitative measure results on correlation coefficients .
the average correlation coefficient was calculated over all sequences for the various methods . from the table
, it can be seen that all methods give high correlation coefficient ( over 0.85 , close to 1 ) .
the vessel removed method has slightly higher average correlation and lower standard deviation than other methods . for the patient
whose xa sequences contain no diaphragm border , the correlation coefficients for vessel removed method are also high for the monoplane ( 0.89 and 0.88 ) and biplane data ( 0.88 and 0.95 ) .
4 illustrate similar observations : the majority of correlation coefficients are over 0.8 for all methods ; non - mask - based methods ( vessel - removed and downsampled image ) slightly outperform other methods .
. 4
boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for retrospective evaluationtable 1average correlation coefficient of projection vector p and diaphragm positions for various methods for retrospective evaluationmethodsmonoplanebiplane(mean \documentclass[12pt]{minimal }
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boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for retrospective evaluation average correlation coefficient of projection vector p and diaphragm positions for various methods for retrospective evaluation to investigate whether there is a statistically significant difference between the performance of these methods on retrospective respiratory motion surrogates extraction , we used a one - tailed wilcoxon rank - sum test to check the correlation coefficients , as their distribution is not necessarily a normal distribution and most of values are close to 1 .
result ( see table 2 ) shows that the p values for monoplane experiments range from 0.09 to 0.55 ( in upper triangle ) ; p values for biplane experiments range from 0.11 to 0.42 ( in lower triangle ) .
the result means these methods have similar performance on retrospective task in terms of statistical significance .
the numbers in the upper and lower triangle in the table show the results of monoplane and biplane cases respectivelyvessel removeddownsampled imagewith maskinverted maskvessel removed
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statistical significance between various methods for retrospective evaluation ( p values ) .
the numbers in the upper and lower triangle in the table show the results of monoplane and biplane cases respectively figure 5 illustrates the frequency distribution of correlation coefficients for various methods in our retrospective analysis . in both the monoplane and biplane experiments , it can be observed that vessel removed method has the most number of correlation coefficients over 0.9 and no correlation coefficients lower than 0.8 , which outperforms all other methods .
this observation suggests that the vessel removed method is more robust than the other approaches.fig .
5frequency distribution of correlation coefficients for various methods in retrospective analysis frequency distribution of correlation coefficients for various methods in retrospective analysis table 3 compares the average per - frame computation time that is needed to compute the projection vector p. this includes the time for image preprocessing , building statistical model through pca and making projection on the first principal component .
the comparison reveals the advantage of non - mask - based methods to mask - based method that the computation time they need is much shorter , which is favored for real clinical workflow .
table 3average per - frame computation time for various methods for retrospective evaluation ( milliseconds)methodsmonoplane ( ms)biplane ( ms)vessel removed17.234.4downsampled image8.015.5with mask821.62227.8inverted mask854.72920.0 average per - frame computation time for various methods for retrospective evaluation ( milliseconds )
example results of the prospective analysis for the same patient as in fig . 3 are shown in fig .
6 . figure 6a , c , e present retrospective projection for frames used for learning the statistical model and prospective projection for frames excluded from the learning phase .
it can be seen that the prospective projection vector p still maintains good correlations with ground truth diaphragm position .
6prospective projection on the first principal component for one patient , in comparison with diaphragm position in roi image .
a , b pca was implemented on monoplane data ; c f principal components were derived from the concatenated sequence of biplane .
c , d show the projection in comparison with plane a ; e , f illustrate the comparison with plane b prospective projection on the first principal component for one patient , in comparison with diaphragm position in roi image .
a , b pca was implemented on monoplane data ; c f principal components were derived from the concatenated sequence of biplane .
c , d show the projection in comparison with plane a ; e , f illustrate the comparison with plane b correlation coefficients are shown in table 4 . in general , these numbers are lower than those in the retrospective experiments , while vessel removed method still maintains a high average correlation coefficient over 0.9 .
whose diaphragm can not be seen in the xa sequences , the correlation coefficients for vessel removed method remain good for one of sequences in the monoplane data ( 0.96 and 0.70 ) and both sequences in the biplane data ( 0.91 and 0.87 ) .
7 show that the medians of all methods are quite close to each other , but vessel removed method has fewer correlation coefficients lower than the median value compared to other methods .
we also used wilcoxon rank - sum test to check the statistical significance , and the results are shown in table 5 .
no significant difference is found among these methods ( \documentclass[12pt]{minimal }
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\begin{document}$$p>0.14$$\end{document}p>0.14 ) , which means their performance are similar to each other in terms of statistical significance .
. 7
boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for prospective evaluationtable 4average correlation coefficients of the prospective projection vector and diaphragm positions for various methodsmethodsmonoplanebiplane(mean \documentclass[12pt]{minimal }
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\begin{document}$$\pm $ $ \end{document} 0.2117table 5statistical significance between various methods in prospective evaluation ( p values ) .
the numbers in the upper and lower triangle in the table show the results of monoplane and biplane cases respectivelyvessel removeddownsampled imagewith maskinverted maskvessel removed
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boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for prospective evaluation average correlation coefficients of the prospective projection vector and diaphragm positions for various methods statistical significance between various methods in prospective evaluation ( p values ) .
the numbers in the upper and lower triangle in the table show the results of monoplane and biplane cases respectively frequency distribution of correlation coefficients in fig .
8 reveals that for both monoplane and biplane experiments , more lower value correlation coefficients appear for all methods compared to retrospective analysis .
it is also clear that vessel removed method has the most high correlation coefficients ( cc \documentclass[12pt]{minimal }
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8frequency distribution of correlation coefficients for various methods in prospective analysis frequency distribution of correlation coefficients for various methods in prospective analysis average per - frame prediction time for various methods is listed in table 6 .
the prediction time includes the time for image preprocessing and computing projection on the first principal component .
it is obvious that downsampled image method has the shortest prediction time , while vessel removed method is also quite fast .
mask - based methods are slower than non - mask - based methods , especially when they were run on biplane data .
table 6average per - frame prediction time for various methods ( milliseconds)methodsmonoplane ( ms)biplane ( ms)vessel removed9.620.0downsampled image1.63.8with mask16.3282.6inverted mask31.0792.4 average per - frame prediction time for various methods ( milliseconds )
we developed an automatic method to extract patient - specific respiratory motion surrogate from cardiac interventional x - ray angiograms using principal component analysis .
the method was evaluated on monoplane and biplane data in both retrospective and prospective manner .
our experiments demonstrated that vessel removed method is able to extract breathing information having high correlation with the ground truth diaphragm or lung tissue motion .
the average correlation coefficient is higher than those for other related methods in our experiments .
vessel removed method is also more robust than the other three methods giving that more correlation coefficients for the method are over 0.9 and less are below 0.8 .
it is also observed that the difference between the mentioned methods in this paper is yet not so profound that no statistically significant difference on the correlation coefficients was found .
correlation coefficient is although one of the common ways to measure the similarity of two time series signals , there are other measures which could potentially improve the difference between these algorithms , such as distance correlation .
the limited number of patients ( only eight ) may also be a reason for the lack of statistical significance . in the future
, we will evaluate the method on a much larger set of patients . despite the lack of statistical insignificance , the vessel removed method
performs at least as good as the other three approaches . from the aspect of computation efficiency , the time that vessel removed method needs for building the statistical model and making prediction on our computer are less than 67 ms , corresponding to the 15-hz imaging rate of our data , whereas mask - based methods need longer time to accomplish the same task
image - based respiratory motion surrogates in interventional x - ray angiograms have been studied previously [ 35 ] .
these works either need manually putting a rectangular roi or require specific object being present in images .
the method we have presented is fully automatic and more robust to various image content .
the diaphragm is not necessarily required to be present as long as there is sufficient breathing motion observed , which is true in most of the cases in pci procedures since the lung tissue is usually seen in the background .
the application of dimensional reduction techniques in extraction of respiratory motion information was seen in [ 9 , 10 ] .
presented one example case , and the method in was originally designed for retrospective gating . in comparison with these works ,
the method we have developed is simpler and needs no vessel extraction from images thus is also faster .
in addition , we have evaluated the usability of our method on monoplane and biplane data in retrospective and prospective manner and achieved good correlation in both tasks .
observations on principal components of xa images would help understanding the mechanism of our proposed method .
the first four principal component images , called eigenimages , of two example sequences with and without diaphragm are shown in fig .
it is obvious that in both cases , the cardiac motion pattern is still present in eigenimages of the original images , but significantly suppressed in those of the morphological - closed images .
also in vessel - removed images , for the case with a diaphragm , the diaphragm border is enhanced in the eigenimages showing a white or dark stripe , while in the sequence where diaphragm is not present , white and dark pixels represent background lung tissues .
it can also be observed that the first eigenimage contains strong respiratory motion signals which makes it reasonable of projecting xa sequence on the first principal component to obtain breathing motion feature .
9the first four principal components ( eigenimages ) of images with and without diaphragm being present .
the first two rows of images have diaphragm and the last two rows do not .
the first and third row show the original images and their eigenimages , and the second and fourth row are the same images in the first and third row after morphological closing operation the first four principal components ( eigenimages ) of images with and without diaphragm being present .
the first two rows of images have diaphragm and the last two rows do not .
the first and third row show the original images and their eigenimages , and the second and fourth row are the same images in the first and third row after morphological closing operation in prospective surrogate extraction , the frames that are needed for building the statistical model are required to cover the maximal range of respiratory motion ; therefore , our proposed method might be limited for different breathing patterns .
detector position changes during interventions require model rebuilding , which having been seen to be fast in our experiments .
the method we developed could directly be used for patient - specific coronary motion modeling .
its short modeling and prospective extraction time enables the possibility of running in real time and being used during interventions . due to the robustness of the method to different image contents
, it could also be potentially used for extraction of respiratory motion surrogate for other types of interventions using different imaging modalities . in the future
we will also investigate on cardiac motion surrogates extraction from xa sequences with similar framework and adapting the current approach to varying view angles .
we have presented a fast automatic method that can be used to retrospectively and prospectively extract patient - specific respiratory motion surrogate from cardiac xa sequences .
our experiments demonstrate a high correlation coefficient with manual ground truth : average correlation coefficients are over 0.9 in the retrospective and prospective evaluations .
the method is easy to implement and runs in real time and thus allows to extract respiratory motion surrogates during interventions . | purposeintraoperative coronary motion modeling with motion surrogates enables prospective motion prediction in x - ray angiograms ( xa ) for percutaneous coronary interventions .
the motion of coronary arteries is mainly affected by patients breathing and heartbeat .
purpose of our work is therefore to extract coronary motion surrogates that are related to respiratory and cardiac motion . in particular , we focus on respiratory motion surrogates extraction in this paper.methodswe propose a fast automatic method for extracting patient - specific respiratory motion surrogate from cardiac xa .
the method starts with an image preprocessing step to remove all tubular and curvilinear structures from xa images , such as vessels and guiding catheters , followed by principal component analysis on pixel intensities .
the respiratory motion surrogate of an xa image is then obtained by projecting its vessel - removed image onto the first principal component.resultsthis breathing motion surrogate was demonstrated to get high correlation with ground truth diaphragm motion ( correlation coefficient over 0.9 on average ) . in comparison with other related methods , the method we developed did not show significant difference ( \documentclass[12pt]{minimal }
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\begin{document}$$p>0.05$$\end{document}p>0.05 ) , but did improve robustness and run faster on monoplane and biplane data in retrospective and prospective scenarios.conclusionswe developed and evaluated a method in extraction of respiratory motion surrogate from interventional x - ray images that is easy to implement and runs in real time and thus allows extracting respiratory motion surrogates during interventions . | Introduction
Methods
Preprocessing of XA images
Principal component analysis
Experiments
Image data
Ground truth data
Retrospective evaluation
Prospective evaluation
Results
Retrospective analysis
Prospective analysis
Discussion
Conclusion | for biplane data , we combined information from both planes by simply using the concatenated matrix \documentclass[12pt]{minimal }
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boxplot of correlation coefficients calculated using various methods on monoplane and biplane data for retrospective evaluation average correlation coefficient of projection vector p and diaphragm positions for various methods for retrospective evaluation to investigate whether there is a statistically significant difference between the performance of these methods on retrospective respiratory motion surrogates extraction , we used a one - tailed wilcoxon rank - sum test to check the correlation coefficients , as their distribution is not necessarily a normal distribution and most of values are close to 1 . | [
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associative memories are believed to be stored in specific neuronal assemblies ( reijmers et al . , 2007 ) through long - lasting synaptic and excitability modifications ( disterhoft and oh , 2006 , frick et al . , 2004 ) , which can be localized within dendrites ( frick et al .
, 2004 , losonczy et al . , 2008 , zhang and linden , 2003 ) or seen throughout the cell ( oh et al . , 2010 ) .
activity of the creb ( camp response element - binding protein ) transcription factor was shown to increase excitability and bias the allocation of associative memories into excitable neuronal ensembles ( han et al .
, 2007 , restivo et al . , 2009 , silva et al . , 2009 ) .
based on the dynamics of creb activation , it was proposed that memories learned within short time intervals will be stored in overlapping neuronal populations ( rogerson et al . , 2014 ,
indeed , it was recently shown that two associative memories learned within a period of a few hours interact with each other and are allocated to overlapping populations of neurons in both the hippocampus ( cai et al . , 2016 ) and the amygdala ( rashid et al . , 2016 ) .
these findings show that memory linking across time relies on the overlap of cellular memory engrams .
what remains unclear are the sub - cellular mechanisms that enable this type of memory linking and to what extent they are generic , namely , what underlies the linking of memories of different strengths and numbers across different time intervals .
for example , apart from neuronal excitability , the phenomenon of synaptic tagging and capture ( stc ) provides a mechanistic model for the specificity and co - operativity of synaptic plasticity ( govindarajan et al . , 2011 ,
redondo and morris , 2011 ) , a prerequisite for long - term memory linking .
moreover , the locus of protein synthesis and capture is critical because it determines the distribution ( neuron - wide or spatially restricted ) of synapses that get strengthened or weakened during learning ( rogerson et al . , 2014 ) .
it remains unclear whether the proteins needed to stabilize a synapse are available throughout a neuron or isolated within strongly activated branches , typically equipped with nonlinear mechanisms , or a combination of the two extremes ( redondo and morris , 2011 , steward and schuman , 2007 ) .
together with homeostatic mechanisms ( turrigiano , 2008 ) , these multi - level processes shape the structure ( neuronal , dendritic , and synaptic features ) and interactions of memory engrams in ways that remain largely unexplored .
previous modeling studies have investigated individual memory processes , such as branch strength potentiation ( legenstein and maass , 2011 ) , homeostasis ( wu and mel , 2009 ) , and synaptic capture ( barrett et al . , 2009 ,
, 2008 , odonnell and sejnowski , 2014 ) , and their role in information binding . however , to date , no models integrate these phenomena into a biophysically constrained network model .
given their differential contributions to memory formation , integration of these phenomena is crucial to extract the rules underlying the allocation and linking of memories . toward this goal
we build a biophysically inspired network model of generic neurons with non - linear dendritic subunits that incorporates plasticity of intrinsic excitability , homeostasis , and stc with somatic , dendritic , or combined protein synthesis or capture .
the model is used to examine the cellular and sub - cellular mechanisms underlying memory formation under three different settings : ( 1 ) encoding and recall of a single associative memory , ( 2 ) rescuing of a weak memory by pairing with a strong memory , and ( 3 ) linking of multiple subsequent memories presented at different time intervals . by varying the expression of individual plasticity processes
, we aim to dissect their effects on synaptic and neuronal properties of memory engrams and infer the key mechanisms that enable memory linking across time .
the proposed network model is outlined in the experimental procedures and consists of excitatory neurons with independent dendritic subunits along with feedback inhibitory neurons ( figure 1 ) .
simulations were conducted under the following conditions : ( 1 ) plasticity - related proteins ( prps ) required for synaptic tagging and capture are synthesized at the soma and made available to all dendritic subunits simultaneously ( somatic ) ; ( 2 ) prps are synthesized and made available only to synapses in a dendritic branch ( local ) ; and ( 3 ) a combination of somatic and local ( s&l ) prp synthesis takes place .
the effect of intrinsic neuronal excitability on memory was examined under the following conditions : ( 4 ) enhanced excitability , whereby excitatory neurons recruited in a memory had a reduced afterhyperpolarization ( ahp ) for 12 hr ( zhou et al . , 2009 ) ; and ( 5 ) static excitability , where neuronal excitability did not change ( see supplemental experimental procedures ) .
the model was trained to encode a single associative memory composed of two events or stimuli ( s1 and s2 ) , via the concurrent activation for 4 s of the inputs representing each stimulus .
these events could represent a pair of conditioned and unconditioned stimuli , as in fear conditioning , or any pair of sensory stimuli ( e.g. , a sound and a visual cue ) experienced together as in contextual memories .
successful formation of an associative memory between s1 and s2 was indicated by an enhanced response to the presentation of the first stimulus ( s1 ) during recall , as observed experimentally ( quirk et al . , 1995 ) .
recall was assessed 24 hr post - training , to allow for homeostasis to take place .
model parameters were calibrated so that learning increased the network responsiveness to s1 presentation ( figure 2 ) , to indicate that the two stimuli became associated .
specifically , parameters ( plasticity thresholds , dendritic spike threshold , number of afferent connections , and initial synaptic weight ) were tuned so that the percentage of coding neurons after learning was approximately 30% ( as per quirk et al . , 1995 ,
rumpel et al . , 2005 ) . a sensitivity analysis with respect to model parameters for all prp conditions
is shown in figures s2 and s4a . as a result , both the percentage of coding neurons , excitatory neurons firing above 10 hz in response to s1 presentation , and their average firing rate increased during recall for all prp conditions .
specifically , the percentage of coding neurons ( figure 2a ) increased from 0.7% 0.1% before training ( pre ) to 29.5% 0.9% under somatic , 28.3% 0.7% under local , and 35.7% 0.8% under s&l prp conditions , respectively .
the average firing rate of these coding neurons ( figure 2b ) increased from 10.8 0.4 hz before training to 15.4 0.2 hz under somatic , 12.3 0.2 hz under local , and 14.6 0.3 hz under s&l prp conditions .
note that s&l prps lead to larger engrams , yet somatic prps lead to engrams with largest mean activity .
the difference ( p < 0.01 , one - way anova followed by bonferroni post - test ) between somatic and s&l firing frequencies is due to the steeper sparseness distribution in the former compared with the latter ( figures s1b and s1d ) .
associative fear conditioning was shown to increase population sparseness , so that fewer neurons respond more vigorously to conditioned stimulus ( cs ) presentation after learning ( gdalyahu et al . , 2012 ) .
we assessed the activity sparseness of the excitatory model neurons using the treves - rolls metric ( treves and rolls , 1991 ) , which measures the steepness of the population activity ( firing rate ) distribution ( figures 2c and s1a s1d ) .
we found that learning increased the sparseness of the population response under all prp conditions , albeit to different levels ( figure 2c ) , without any additional parameter tuning .
an interesting prediction is that sparseness will be greatest under somatic and smallest under local prp conditions .
s1d ) : under somatic prp conditions , neurons that cross the calcium threshold for prp synthesis will have all their tagged synapses potentiated , leading to a marked increase in their activity compared with other responsive cells , and thus a steep population activity distribution ( for the same reason , the average firing rate of the coding population is highest in figure 2b ) . in the local prp case ,
only synapses tagged within prp - producing dendrites will be potentiated , thus leading to a broader activity distribution . under s&l
prp conditions , neurons will exhibit variable activity levels , thus leading to an intermediate steepness of the activity distribution .
activity sparseness represents the signal - to - noise ratio or contrast between coding and non - coding neurons during memory recall , with less sparseness potentially resulting in more interference between different memories and/or high noise levels .
overall , model predictions regarding the population response characteristics during recall of a single associative memory are graphically summarized in figure 2d : the neuronal memory engram ( coding neurons ) is expected to be similar in size under conditions of somatic or dendritic ( local ) prps and slightly larger under s&l conditions .
however , it is expected to be highest in contrast under somatic , followed by s&l and local prp conditions .
the model also predicts marked differences in the sub - cellular ( synaptic , dendritic ) properties of the learned memory among prp conditions , which can be used to design experiments that tease out the dominant mode of prp synthesis in vitro and/or in vivo .
first , the percentage of excitatory neurons with 1 potentiated synapses is much smaller under somatic prp conditions ( 29.2% 2.5% ) compared with local and s&l prp conditions ( 80.3% 1.3% ; figure 3a ) .
second , the total number of potentiated synapses after learning is similar in local and somatic conditions , but significantly larger under s&l conditions ( figure 3b ) .
third , within each neuron , more synapses are potentiated on average under somatic ( 32.4 4.5 ) compared with local ( 10.9 6.5 ) and s&l ( 17.4 13.2 ) prp conditions .
importantly , the distributions of potentiated synapses per neuron ( figures s1e s1 g ) are quite different among the three cases ( p < 0.001 , kruskal - wallis test ) , with the former ( somatic ) approximating the sum of the other two .
these differences suggest that it is feasible to infer the mode of prps experimentally with a reasonable sample size , given the assumptions used to configure our model .
specifically , if sampling from a uniform population of neurons and trying to infer which one of the three distributions best describes the data , having just 50 neurons would be sufficient to discriminate between local versus somatic and local versus s&l conditions , whereas 200 samples would be needed to discriminate between somatic and s&l conditions ( alpha : p < 0.01 , power : 1 = 0.9 ; see figure s1 ) .
the average number of potentiated synapses per branch is largest under local conditions ( 3.9 0.9 synapses ; figure 3d ) and smaller under somatic and s&l conditions ( 2.8 1.0 ; figure 3d ) .
the distributions of potentiated synapses per branch ( figures s1h s1j ) reveal that with local prps , synapses are potentiated in groups 3 , reflecting synapse clustering , whereas with somatic or s&l prps , isolated synapses are also potentiated .
again , the distributions of potentiated synapses per branch are different among the three cases ( p < 0.001 , kruskal - wallis test ) , but distinguishing between prp modes based on this feature is more challenging . a sample size of 50 branches would be required to discriminate between local versus s&l and local versus somatic prps , but discriminating between somatic and s&l conditions requires thousands of branches because the two distributions are very similar . in summary ,
model predictions regarding a single associative memory are conceptually depicted in figure 3e and listed later .
first , in line with experimental data , acquisition of a single associative memory increases the activity and sparseness of the population response across all prp conditions ( figure 2 ) .
second , the locus of prp synthesis or capture determines the neuronal , dendritic , and synaptic features of the memory engram : somatic prps result in engrams with high activity and sparsity / contrast ( figures 2c , 2d , and 3e , left ) , whose synaptic trace is restricted to a small neuronal population ( figure 3a ) , containing clusters of two to three potentiated synapses that are widely distributed within dendritic trees ( figure 3d ) . local prps lead to engrams with lower activity and sparsity / contrast ( figures 2c , 2d , and 3e , middle ) , whose synaptic trace is distributed across the majority of the neuronal population ( figure 3a ) , containing clusters of three to five potentiated synapses that are concentrated within a few dendritic branches ( figures 3c and 3d ) . an intermediate version of these characteristics is observed under conditions of combined s&l prps ( figure 3e , right ) .
for example , plasticity markers such as phluorin - tagged glutamate receptors ( zhang et al . , 2015 ) and phosphorylated cofilin ( lynch et al . ,
2015 ) , as well as two - photon in vivo imaging ( fu et al . , 2012 ,
makino and malinow , 2011 ) , could be used to test the distributions of potentiated synapses after learning .
evidence for widespread plasticity across a majority of neurons , with localized clusters of potentiated synapses within a small subset of their dendritic branches , would indicate that local ( as opposed to somatic ) prp mechanisms predominate during associative memory formation . because a key feature of our model is the incorporation of dendritic non - linearities , to understand their contribution , we examined engram formation in an alternative configuration of the network , in which dendrites do not spike .
we found that to have a comparable coding population size , the number of afferent synapses must be increased by a factor of 2.36 to compensate for the loss of calcium influx through dendritic spikes .
a sensitivity analysis of the alternative model is shown in figure s2 , whereby the main difference documented is a smaller size of the coding neuronal population , which is more pronounced under local prp conditions .
interestingly , eliminating dendritic spikes also eliminated the differences in the characteristics of memory engrams formed under somatic versus s&l prp conditions , making them indistinguishable ( see figure s5 ) .
these simulations suggest important roles for dendritic excitability : dendritic spikes not only facilitate engram formation via the use of substantially lower numbers of synapses ( thus saving resources ) but also induce memory engrams whose features are distinguishably different depending on the locus of prps , thus expanding the dynamical range of engram formation . as a consequence , we predict that changes in the biophysics of dendrites that affect dendritic spiking ( e.g. , n - methyl - d - aspartate [ nmda ] receptor deficits ; magnusson , 2012 ) would result in smaller , less diverse memory engrams especially if prps are restricted in dendrites .
we next investigated whether the mechanisms that underlie the formation of a single associative memory can also account for the rescue of a weak memory when paired with a strong one ( so - called behavioral tagging ; ballarini et al . , 2009 ) , which is believed to be a consequence of the synaptic tagging and
capture memory consolidation model ( frey and morris , 1997 , redondo and morris , 2011 ) .
weak learning was simulated by reducing the duration of the s1 and s2 stimuli so that only a small percentage of synapses representing these stimuli were consolidated ( see supplemental experimental procedures ) . because both stc and learning - induced enhancement of intrinsic excitability could affect the two memories , we performed simulations under the three prp conditions whereby the neuronal excitability of recruited neurons was either enhanced or remained static .
we found that rescuing of the weak memory , indicated by an increased percentage of coding neurons compared with baseline at 24 hr , was achieved in all cases and depended on the prp condition and neuronal excitability ( figures 4a4c versus 4d4f ) . with enhanced excitability ,
rescuing was asymmetric and occurred under all prp conditions for pairing intervals between 5 and + 2 hr ( figure 4a ) .
the effect was more pronounced in the somatic condition , followed by the s&l condition , and smallest in the local prp condition .
the latter is due to the lower probability of tagged synapses ( representing the weak memory ) being co - localized in the same dendritic branches with strong synapses that produce prps ( representing the strong memory ) .
the earlier - mentioned asymmetry was abolished if neuronal excitability remained unaltered after learning ( figure 4d ) , in which case the rescuing window was reduced to [ 2 , 2 hr ] .
first , an asymmetry in the pairing window for behavioral tagging is suggestive of learning - induced enhanced neuronal excitability .
second , such asymmetry could act as a mechanism for encoding the order of events : a weak memory learned before a strong one would be smaller ( in size ) than a weak memory learned after a strong one .
third , manipulations that attenuate or block the excitability enhancement , like drugs that enhance the slow afterhyperpolarization , should either blunt or eliminate the ordered encoding of events . according to the stc model ,
synapses coding for the weak memory can be strengthened through the sharing of prp products .
our model predicts that this mechanism also leads to the overlapped storage ( or co - allocation ) of the weak and strong memories to many common neurons ( figures 4b and 4e ) and many common dendrites of these neurons ( figures 4c and 4f ) .
we find that the degree of co - allocation of the strong and weak memories follows the size of the ensemble encoding the weak memory .
similar to neuronal coding populations ( figures 4a and 4d ) , neuronal ( figures 4b and 4e ) as well as dendritic overlaps ( at least two potentiated synapses from each memory ; figures 4c and 4f )
the asymmetry is more pronounced under conditions of somatic and s&l rather than local prps .
moreover , co - allocation within 2 hr is largest under somatic and s&l prp conditions and is significant in the local prp condition in the case of enhanced excitability ( p < 0.01 , one - way anova ) , but not under static excitability ( p > 0.1 ) . finally , rescuing of the weak memory by the strong memory
is associated with increased dendritic co - localization of the two memories ( co - clustering ) in all prp and both excitability conditions ( p < 0.01 , one - way anova ; figures 4c and 4f ) .
the earlier simulations highlight the interactions and dissect the contributions of two distinct plasticity mechanisms on weak - strong memory rescuing : intrinsic excitability and stc , with the latter also depending on the mode of prp synthesis or capture .
first , irrespective of the mode of prp synthesis or capture and neuronal excitability changes , weak - strong memory pairing within a window of 2 hr can lead to rescuing of the weak memory , as shown experimentally ( ballarini et al .
, 2009 , moncada and viola , 2007 ) , with the exception of local prps and static excitability , pairing at 2 hr . second , this rescue is expected to be more effective if learning of a strong memory leads to neuronal excitability increases and if prps are available throughout the neuron ( somatic ) .
third , the rescue could be extended to the [ 5 , 2 hr ] window if the strong memory precedes the weak and leads to increases in somatic excitability .
fourth , under all conditions tested , rescuing of the weak memory is mediated by neuronal co - allocation of the two memories ( figures 4b and 4e ) , as well as respective co - clustering of their synaptic contacts in common dendritic branches ( figures 4c and 4f ) .
it has been proposed that co - allocation of two memories to overlapping populations of neurons links the two memories by increasing the probability of co - recall ( silva et al . , 2009 ) .
indeed , two associative memories learned within a time period of a few hours were shown to be allocated to overlapping populations of neurons and interact during recall , in both the hippocampus ( cai et al . , 2016 ) and the amygdala ( rashid et al . , 2016 ) .
the main hypothesis for co - allocation entails that creb - dependent transcription in the neurons encoding the first memory results in temporary increases in excitability ( stanciu et al . ,
2001 ) that , for a time , bias the allocation of subsequent memories to many of the same neurons that encoded the first memory ( viosca et al . , 2009 , zhou et al . , 2009 ) .
we used our model to test this hypothesis by presenting two strong ( capable of producing prps ) learning events separated by an inter - stimulus interval ( isi ) of 1 , 2 , 5 , or 24 hr . recall was tested 24 hr later by measuring responses to one of the two stimuli associated with each of the memories .
simulations were performed under all three prp conditions , with and without enhanced neuronal excitability .
we found that under conditions of enhanced excitability , the two memories interacted for isis of 15 hr ( figures 5a5d ) , whereas under conditions of static excitability , this interaction was limited to isis of 12 hr ( figures 5e5h ) .
specifically , under all conditions , the coding population size of both memories increased substantially for isis of 12 hr compared with baseline at 24 hr ( 20%60% increase at 1 hr ; figures 5a , 5b , 5e , and 5f ) .
the enhancement of the first memory was independent of the prp condition and neuronal excitability ( figures 5a and 5e ) and was not significant at 5 hr ( p > 0.1 , t test ) .
in contrast , the neuronal size of the second memory was larger under conditions of enhanced excitability and depended on the mode of prps ( somatic prp conditions resulted in the biggest traces , followed by s&l and local conditions ; figures 5b and 5f ) .
moreover , increases in memory size were extended to isis of 5 hr under enhanced excitability conditions ( p < 0.001 ; figure 5b ) , indicating that learning - induced increases in neuronal excitability prime the allocation of the second memory in more neurons , in accordance with experimental studies ( cohen et al . , 1999 ) .
increases in the size of the two memories were accompanied by respective increases in the overlap between the populations coding for the two memories under both enhanced and static excitability conditions ( figures 5c and 5 g ) , except for the case of local prps with static excitability , in which the neuronal co - allocation was not significantly increased beyond an isi of 1 hr ( p > 0.1 , one - way anova followed by bonferroni post - test ; figure 5 g ) . as with the case of weak - strong memory pairing ,
the pairing of strong memories led to co - clustering of synaptic contacts from the two memories in dendritic branches for isis of 12 hr for all cases tested .
co - clustering extended to 5 hr under conditions of enhanced excitability and somatic or s&l but not local , prps ( p < 0.01 , one - way anova followed by bonferroni post - test ; figures 5d and 5h ) .
our results agree with recent experimental data on the overlap of time - linked memory ensembles in ca1 in vivo ( cai et al . , 2016 ) , which indicate an 50% increase of overlap at 5 hr compared with 7 days . in all cases , the restriction of prps to dendritic branches led to a smaller degree of neuronal co - allocation between the two memories for isis of 1 and 2 hr ( p < 0.01 , one - way anova ) .
the effect on dendritic co - clustering , however , was not significant ( p > 0.1 ) , indicating that dendritic co - allocation is not heavily dependent on the levels of neuronal co - allocation .
overall , our model makes the following predictions regarding the interactions between two strong memories and the underlying mechanisms .
first , our simulations support the hypothesis that changes in excitability lead to co - allocation of memories separated by several hours ( silva et al . , 2009 ,
2014 ) and are in line with experimental evidence that linked memories are stored in overlapping neuronal populations ( cai et al . , 2016 , rashid et al . , 2016 ) .
second , our model also supports the hypothesis that stc mechanisms will lead to synapse clustering , in agreement with prior work ( govindarajan et al . , 2011 ,
third , pairing of two strong memories will affect both memories if learned within a couple of hours , but just the second one for longer isis ( i.e. , 5 hr ) .
fourth , the asymmetric enhancement of the second memory is due to increases in neuronal excitability after the acquisition of the first memory and predicts that blocking this increase in excitability should eliminate this asymmetry and possibly restrict memory enhancement to a 2-hr interval .
this prediction is in line with experimental evidence from aged mice ( where neuronal excitability after learning is decreased ) showing impaired memory linking and reduced neuronal overlaps ( cai et al . , 2016 ) .
fifth , for isis shorter than 2 hr , the high overlaps between the neuronal ensembles representing both memories under somatic and s&l prp conditions may lead to memory interference ( robertson , 2012 ) .
restricting prps to dendritic branches ( e.g. , by reducing transcription ) should reduce neuronal co - allocation and , therefore , decrease interference .
finally , we predict that interactions between strong memories are mediated by neuronal co - allocation and dendritic co - clustering of synapses representing the two memories .
the linking of strong memories suggests that overlapping allocation may be a general mechanism for binding together sequences of events to create memory episodes .
the interplay between excitability and stc is likely to favor the creation of memory episodes for the following reasons : ( 1 ) stc links memories according to their temporal proximity , that is , the closer the memories , the higher their co - allocation ; and ( 2 ) learning - induced enhancement of neuronal excitability influences prior and subsequent memories in an asymmetric manner , thus enabling ordered linking of events . to test this hypothesis
, we simulated the encoding of 10 sequentially presented memories separated by inter - stimulus intervals ( isis ) of 1 , 5 , and 24 hr , under conditions of enhanced versus static excitability and somatic versus local prp synthesis and capture . as shown in figure 6a , sequential encoding with an isi of 1 hr leads to high population overlaps between memories that are close in time ( near the diagonal ) .
these overlaps extend to more distant memories in conditions of somatic prps and enhanced neuronal excitability . as shown in figure 6b ( left ) , when memories are presented with 1-hr intervals , neuronal overlaps are largest under conditions of somatic prps and enhanced neuronal excitability compared with the other cases ( p < 0.01 , one - way anova followed by bonferroni post - test ) , and are non - significant under conditions of local prps and static neuronal excitability , for all isis tested .
when memories are separated by 5 hr ( figure 6b , middle ) , the neuronal overlap is significantly smaller compared with 1 hr for all cases ( p < 0.01 ) and remains significantly above baseline only for memories separated by 510 hr under conditions of enhanced ( p < 0.01 ) , but not static , neuronal excitability . this phenomenon can be explained by considering the effect of homeostatic mechanisms , which operate over time periods beyond 24 hr and reduce the overall excitability of the entire neuronal population , as well as the time window of enhanced neuronal excitability that does not extend beyond 12 hr .
this effect is most pronounced when memories are separated by 24 hr , where neuronal overlaps are not different from baseline for both enhanced and static excitability cases and both prp conditions ( figure 6b , right ) .
overall , these simulations predict the overlapping allocation of strings of memories when subsequent events are interleaved with isis up to 5 hr . as with all prior experiments ,
the overlapping allocation at the neuronal level is accompanied by an increased co - clustering of synapses from interacting memories ( figure 6c ) , which follows the pattern of population overlaps : higher under somatic prps and enhanced excitability , and diminished when memories are separated by large intervals .
these simulations suggest that overlapping memory allocation mediated by stc and neuronal excitability mechanisms can be a natural candidate for binding together subsequent events over large timescales to create coherent memory episodes .
our model predicts that manipulations or conditions that disrupt these mechanisms , as , for example , in aging , would lead to diminished ability to bind events , leading to dissociation and partial forgetting of details in memory episodes .
the notion that dendrites may act as semi - independent computational and storage units ( mel , 1992 , poirazi and mel , 2001 ) , together with evidence for dendritically localized synaptic plasticity ( govindarajan et al .
, 2011 , hardie and spruston , 2009 , kang and schuman , 1996 ) , has led to multiple hypotheses regarding the role of dendritic plasticity in the formation of memory engrams ( branco and husser , 2010 , govindarajan et al . , 2006 , kastellakis et al . , 2015 , rogerson et al . , 2014 , zhou et al . , 2009 ) .
modeling studies have only recently begun to investigate this issue , albeit using simplified models and/or focusing on single plasticity rules ( legenstein and maass , 2011 , odonnell and sejnowski , 2014 , wu and mel , 2009 ) .
the model presented here incorporates biologically constrained dendritic compartments , depolarization - dependent plasticity mechanisms , synaptic tagging and capture , and changes in neuronal excitability in neurons embedded in a cortical microcircuit to study memory engram formation .
importantly , we show that each of these mechanisms plays a role in the formation and interaction of memory engrams that would be missed if using simplified models .
thus , the key contribution of this work is the systematic investigation of the biologically relevant parameter space that allows memory linking so as to characterize the underlying sub - cellular mechanisms and the extent to which they are generic , namely underlie information binding across the levels of a single , a pair , or multiple associative memories .
we find that the locus of protein synthesis or capture leads to marked differences in both the neuronal ( e.g. , size / sparsity of the coding population ) and synaptic features ( e.g. , distributions of potentiated synapses within neurons and branches ) of associative memories in our model .
the predicted differences can , through targeted experiments , help identify whether dendritic or somatic protein synthesis is dominant .
for example , experimental evidence for widespread plasticity across a majority of neurons , with localized clusters of potentiated synapses within a small subset of their dendritic branches , would indicate that local ( as opposed to somatic ) prp mechanisms predominate during associative memory formation .
distinguishable characteristics endowed by the locus of prps are also seen in the interaction of associative memories ( weak or strong ) across time . in addition
, we predict a role for intrinsic excitability in weak - strong memory rescuing , which has thus far been attributed to stc mechanisms alone ( ballarini et al . , 2009 ) .
our model also predicts roles for dendritic spikes : they serve as a boosting mechanism for storing and linking memories via the use of fewer synaptic resources , whereas also enhancing the diversity of engram features ( e.g. , sparsity and distributions of potentiated synapses ) , thus increasing their dynamic range .
importantly , our model is in agreement with recent experimental studies , which confirm that there is excitability - dependent overlap of memory engrams learned within time intervals of a few hours .
memory engrams in ca1 are overlapping when they are separated by 5-hr interval , but not at 7 days , and the overlap is linked with behavioral expression ( cai et al . , 2016 ) .
similarly , in the lateral amygdala , memory engrams were allocated in overlapping populations for memories separated up to 6 hr ( rashid et al . , 2016 ) and
our model indicates that this overlapping allocation is also affected by the locus of prp synthesis .
for example , we predict that shorter intervals ( 12 hr ) would lead to higher and qualitatively different co - allocation , primarily because of synaptic tagging and capture mechanisms rather than neuronal excitability ( figure 5 ) .
moreover , we propose that the neuronal overlap will be significantly higher if one of the memories is weak ( figure 4 ) .
this work capitalizes on prior experimental and modeling findings ( barrett et al . , 2009 ,
, 2008 , govindarajan et al . , 2011 , legenstein and maass , 2011 , losonczy et al . , 2008 ,
odonnell and sejnowski , 2014 , smolen et al . , 2006 ) to put together many pieces of a large puzzle .
this is done by identifying a unifying principle for linking information across time : through neuronal and dendritic co - allocation realized via synapse clustering ( schematically illustrated in figure 7 ) .
this simple rule explains memory interactions at the cellular and sub - cellular levels , and is robust under all of the different scenarios tested here , including variations in the locus of prp synthesis and the plasticity of neuronal excitability , different numbers , and strengths of memory , variable time intervals , among others . in all cases
tested , the strength of interactions between memories was proportional to the degree of neuronal and dendritic overlap between those memories .
importantly , our findings are in line with recent studies showing that learning is accompanied by increased synaptic clustering ( fu et al . , 2012 , mcbride et al . , 2008 ) , although evidence is currently missing for or against the co - clustering of afferents representing distinct memories .
prior work has also examined memory associations and contributed important insights with respect to the mechanisms underlying information binding and weak - strong memory interactions ( legenstein and maass , 2011 , odonnell and sejnowski , 2014 ) , several of which are in agreement with this study . through a systematic investigation of a much larger parameter space
, our model predicts that synapse clustering is a universal mechanism that links both weak - strong and strong - strong memories , in the absence of high axonal overlap , irrespective of the location of protein synthesis and for time intervals much longer than the ones explained by prior studies or models .
although most studies have treated memories as independent entities , they are almost always linked to other memories in ways that help us to organize our world into predictive patterns .
our biologically realistic model captured this critical facet of memory and yielded a number of experimental predictions that could guide research on how weak and strong memories are linked across time .
disruptions of the processes that link and organize memories are likely to affect cognitive function and result in psychopathology .
for example , it has been proposed that molecular and cellular disruptions leading to over- or under - clustering may be related to the cognitive symptoms with a number of psychiatric problems , such as schizophrenia ( memory interference caused by over - clustering ) or autism ( difficulty in contextual processing caused by under - clustering ) ( kastellakis et al . , 2015 ) .
future experiments that test predictions proposed here will address the mechanisms underlying these pathologies in animal models , and therefore open new avenues for the understanding and treatment of memory deficits .
dendritic structure ( a branch is a single compartment ) and nonlinearities are incorporated in a simplified manner and the plasticity rules are modeled in a phenomenological way , without modeling the biophysics of membrane mechanisms or the elaborated morphology of dendritic trees .
the model thus provides a general framework for dissecting the contributions of each plasticity mechanism to different memory phenomena . the proposed model ( figure 1 ) consists of 400 excitatory and 100 inhibitory neurons .
excitatory neurons are modeled as two - stage integrators ( poirazi et al . , 2003 ) , consisting of independent , nonlinear dendritic subunits capable of compartmentalized plasticity ( poirazi and mel , 2001 ) .
the somatic spike response was modeled as an adaptive integrate - and - fire unit responsive to the summed dendritic input .
the neurons received feedback inhibition from inhibitory point neurons , as well as excitation from background synapses .
synaptic plasticity conforms to the stc model , which requires both synaptic tagging and the availability of plasticity - related proteins ( prps ) for stable strengthening and weakening of synapses ( redondo and morris , 2011 ) .
calcium acts as the main trigger for the induction of synaptic tags and for the synthesis of prps .
the calcium influx after a presynaptic spike depends on the voltage of the postsynaptic dendritic subunit in an nmda - dependent manner .
the total calcium influx to a synapse during a learning event determines the sign ( long - term potentiation [ ltp ] or long - term depression [ ltd ] ) of synaptic tags according to the calcium control model ( shouval et al . , 2002 ) and triggers the production of prps , which are modeled by alpha functions .
this effect is mediated by the reduction of the spike adaptation at the soma , mimicking a decreased afterhyperpolarization current ( oh et al . , 2009 , zhou et al . , 2009 ) .
the effect of homeostasis on synaptic plasticity is modeled via a synaptic scaling homeostatic rule ( turrigiano , 2008 ) .
the network model consists of a population comprising of the following : ( 1 ) excitatory neurons of a target region , ( 2 ) inhibitory neurons in the same region , ( 3 ) the stimulus - carrying input neurons , and ( 4 ) background noise input neurons .
each memory encoding is performed by the activation of a set of stimulus - carrying neurons , which carry the conditioned and unconditioned stimulus information that represents the memory to be encoded ( figure 1a ) .
stimulus - carrying neurons are grouped in sets of six ( three representing stimulus 1 [ s1 ] and three representing stimulus 2 [ s2 ] ) , which fire with an average firing frequency of 30 hz during the encoding of each memory ( s1+s2 neurons ) , as well as during the recall of each individual event ( s1 neurons only ) .
stimulus - carrying neurons create potential synapses by randomly targeting the dendritic branches of the excitatory neurons .
these stimulus - to - excitatory synapses are the only plastic synapses in the network and are initialized to the same low initial weight .
the background inputs create a number of synaptic contacts to random dendrites and fire with an average frequency of 0.5 hz throughout the simulations .
connectivity parameters among the neuronal populations in the network model are listed in table s2 . when encoding multiple memories , inter - stimulus intervals ( isis ) of 1 hr are introduced between consecutive memories . during isi periods ,
spiking activity is not simulated , but slow consolidation processes ( prp production , synaptic tag consolidation , and homeostasis ) take place . a consolidation period of 36 hr follows the end of all encoding events .
recall takes places after this consolidation period , via the activation of s1 input neurons .
a detailed description of the model and experimental procedures is provided in the supplemental experimental procedures .
the source code of the model is provided under the open source gplv2 license at http://dendrites.gr/ and under modeldb : 206249 . | summarymemories are believed to be stored in distributed neuronal assemblies through activity - induced changes in synaptic and intrinsic properties .
however , the specific mechanisms by which different memories become associated or linked remain a mystery . here , we develop a simplified , biophysically inspired network model that incorporates multiple plasticity processes and explains linking of information at three different levels : ( 1 ) learning of a single associative memory , ( 2 ) rescuing of a weak memory when paired with a strong one , and ( 3 ) linking of multiple memories across time . by dissecting synaptic from intrinsic plasticity and neuron - wide from dendritically restricted protein capture
, the model reveals a simple , unifying principle : linked memories share synaptic clusters within the dendrites of overlapping populations of neurons .
the model generates numerous experimentally testable predictions regarding the cellular and sub - cellular properties of memory engrams as well as their spatiotemporal interactions . | Introduction
Results
Discussion
Experimental Procedures
Author Contributions | associative memories are believed to be stored in specific neuronal assemblies ( reijmers et al . based on the dynamics of creb activation , it was proposed that memories learned within short time intervals will be stored in overlapping neuronal populations ( rogerson et al . , 2014 ,
indeed , it was recently shown that two associative memories learned within a period of a few hours interact with each other and are allocated to overlapping populations of neurons in both the hippocampus ( cai et al . these findings show that memory linking across time relies on the overlap of cellular memory engrams . what remains unclear are the sub - cellular mechanisms that enable this type of memory linking and to what extent they are generic , namely , what underlies the linking of memories of different strengths and numbers across different time intervals . for example , apart from neuronal excitability , the phenomenon of synaptic tagging and capture ( stc ) provides a mechanistic model for the specificity and co - operativity of synaptic plasticity ( govindarajan et al . moreover , the locus of protein synthesis and capture is critical because it determines the distribution ( neuron - wide or spatially restricted ) of synapses that get strengthened or weakened during learning ( rogerson et al . together with homeostatic mechanisms ( turrigiano , 2008 ) , these multi - level processes shape the structure ( neuronal , dendritic , and synaptic features ) and interactions of memory engrams in ways that remain largely unexplored . previous modeling studies have investigated individual memory processes , such as branch strength potentiation ( legenstein and maass , 2011 ) , homeostasis ( wu and mel , 2009 ) , and synaptic capture ( barrett et al . however , to date , no models integrate these phenomena into a biophysically constrained network model . given their differential contributions to memory formation , integration of these phenomena is crucial to extract the rules underlying the allocation and linking of memories . toward this goal
we build a biophysically inspired network model of generic neurons with non - linear dendritic subunits that incorporates plasticity of intrinsic excitability , homeostasis , and stc with somatic , dendritic , or combined protein synthesis or capture . the model is used to examine the cellular and sub - cellular mechanisms underlying memory formation under three different settings : ( 1 ) encoding and recall of a single associative memory , ( 2 ) rescuing of a weak memory by pairing with a strong memory , and ( 3 ) linking of multiple subsequent memories presented at different time intervals . by varying the expression of individual plasticity processes
, we aim to dissect their effects on synaptic and neuronal properties of memory engrams and infer the key mechanisms that enable memory linking across time . the proposed network model is outlined in the experimental procedures and consists of excitatory neurons with independent dendritic subunits along with feedback inhibitory neurons ( figure 1 ) . simulations were conducted under the following conditions : ( 1 ) plasticity - related proteins ( prps ) required for synaptic tagging and capture are synthesized at the soma and made available to all dendritic subunits simultaneously ( somatic ) ; ( 2 ) prps are synthesized and made available only to synapses in a dendritic branch ( local ) ; and ( 3 ) a combination of somatic and local ( s&l ) prp synthesis takes place . the model was trained to encode a single associative memory composed of two events or stimuli ( s1 and s2 ) , via the concurrent activation for 4 s of the inputs representing each stimulus . successful formation of an associative memory between s1 and s2 was indicated by an enhanced response to the presentation of the first stimulus ( s1 ) during recall , as observed experimentally ( quirk et al . model parameters were calibrated so that learning increased the network responsiveness to s1 presentation ( figure 2 ) , to indicate that the two stimuli became associated . specifically , the percentage of coding neurons ( figure 2a ) increased from 0.7% 0.1% before training ( pre ) to 29.5% 0.9% under somatic , 28.3% 0.7% under local , and 35.7% 0.8% under s&l prp conditions , respectively . s1d ) : under somatic prp conditions , neurons that cross the calcium threshold for prp synthesis will have all their tagged synapses potentiated , leading to a marked increase in their activity compared with other responsive cells , and thus a steep population activity distribution ( for the same reason , the average firing rate of the coding population is highest in figure 2b ) . activity sparseness represents the signal - to - noise ratio or contrast between coding and non - coding neurons during memory recall , with less sparseness potentially resulting in more interference between different memories and/or high noise levels . overall , model predictions regarding the population response characteristics during recall of a single associative memory are graphically summarized in figure 2d : the neuronal memory engram ( coding neurons ) is expected to be similar in size under conditions of somatic or dendritic ( local ) prps and slightly larger under s&l conditions . however , it is expected to be highest in contrast under somatic , followed by s&l and local prp conditions . the model also predicts marked differences in the sub - cellular ( synaptic , dendritic ) properties of the learned memory among prp conditions , which can be used to design experiments that tease out the dominant mode of prp synthesis in vitro and/or in vivo . third , within each neuron , more synapses are potentiated on average under somatic ( 32.4 4.5 ) compared with local ( 10.9 6.5 ) and s&l ( 17.4 13.2 ) prp conditions . importantly , the distributions of potentiated synapses per neuron ( figures s1e s1 g ) are quite different among the three cases ( p < 0.001 , kruskal - wallis test ) , with the former ( somatic ) approximating the sum of the other two . these differences suggest that it is feasible to infer the mode of prps experimentally with a reasonable sample size , given the assumptions used to configure our model . the average number of potentiated synapses per branch is largest under local conditions ( 3.9 0.9 synapses ; figure 3d ) and smaller under somatic and s&l conditions ( 2.8 1.0 ; figure 3d ) . in summary ,
model predictions regarding a single associative memory are conceptually depicted in figure 3e and listed later . first , in line with experimental data , acquisition of a single associative memory increases the activity and sparseness of the population response across all prp conditions ( figure 2 ) . second , the locus of prp synthesis or capture determines the neuronal , dendritic , and synaptic features of the memory engram : somatic prps result in engrams with high activity and sparsity / contrast ( figures 2c , 2d , and 3e , left ) , whose synaptic trace is restricted to a small neuronal population ( figure 3a ) , containing clusters of two to three potentiated synapses that are widely distributed within dendritic trees ( figure 3d ) . ,
2015 ) , as well as two - photon in vivo imaging ( fu et al . evidence for widespread plasticity across a majority of neurons , with localized clusters of potentiated synapses within a small subset of their dendritic branches , would indicate that local ( as opposed to somatic ) prp mechanisms predominate during associative memory formation . interestingly , eliminating dendritic spikes also eliminated the differences in the characteristics of memory engrams formed under somatic versus s&l prp conditions , making them indistinguishable ( see figure s5 ) . as a consequence , we predict that changes in the biophysics of dendrites that affect dendritic spiking ( e.g. , n - methyl - d - aspartate [ nmda ] receptor deficits ; magnusson , 2012 ) would result in smaller , less diverse memory engrams especially if prps are restricted in dendrites . we next investigated whether the mechanisms that underlie the formation of a single associative memory can also account for the rescue of a weak memory when paired with a strong one ( so - called behavioral tagging ; ballarini et al . , 2009 ) , which is believed to be a consequence of the synaptic tagging and
capture memory consolidation model ( frey and morris , 1997 , redondo and morris , 2011 ) . because both stc and learning - induced enhancement of intrinsic excitability could affect the two memories , we performed simulations under the three prp conditions whereby the neuronal excitability of recruited neurons was either enhanced or remained static . we found that rescuing of the weak memory , indicated by an increased percentage of coding neurons compared with baseline at 24 hr , was achieved in all cases and depended on the prp condition and neuronal excitability ( figures 4a4c versus 4d4f ) . the latter is due to the lower probability of tagged synapses ( representing the weak memory ) being co - localized in the same dendritic branches with strong synapses that produce prps ( representing the strong memory ) . first , an asymmetry in the pairing window for behavioral tagging is suggestive of learning - induced enhanced neuronal excitability . second , such asymmetry could act as a mechanism for encoding the order of events : a weak memory learned before a strong one would be smaller ( in size ) than a weak memory learned after a strong one . we find that the degree of co - allocation of the strong and weak memories follows the size of the ensemble encoding the weak memory . similar to neuronal coding populations ( figures 4a and 4d ) , neuronal ( figures 4b and 4e ) as well as dendritic overlaps ( at least two potentiated synapses from each memory ; figures 4c and 4f )
the asymmetry is more pronounced under conditions of somatic and s&l rather than local prps . finally , rescuing of the weak memory by the strong memory
is associated with increased dendritic co - localization of the two memories ( co - clustering ) in all prp and both excitability conditions ( p < 0.01 , one - way anova ; figures 4c and 4f ) . first , irrespective of the mode of prp synthesis or capture and neuronal excitability changes , weak - strong memory pairing within a window of 2 hr can lead to rescuing of the weak memory , as shown experimentally ( ballarini et al . second , this rescue is expected to be more effective if learning of a strong memory leads to neuronal excitability increases and if prps are available throughout the neuron ( somatic ) . fourth , under all conditions tested , rescuing of the weak memory is mediated by neuronal co - allocation of the two memories ( figures 4b and 4e ) , as well as respective co - clustering of their synaptic contacts in common dendritic branches ( figures 4c and 4f ) . it has been proposed that co - allocation of two memories to overlapping populations of neurons links the two memories by increasing the probability of co - recall ( silva et al . indeed , two associative memories learned within a time period of a few hours were shown to be allocated to overlapping populations of neurons and interact during recall , in both the hippocampus ( cai et al . we used our model to test this hypothesis by presenting two strong ( capable of producing prps ) learning events separated by an inter - stimulus interval ( isi ) of 1 , 2 , 5 , or 24 hr . we found that under conditions of enhanced excitability , the two memories interacted for isis of 15 hr ( figures 5a5d ) , whereas under conditions of static excitability , this interaction was limited to isis of 12 hr ( figures 5e5h ) . specifically , under all conditions , the coding population size of both memories increased substantially for isis of 12 hr compared with baseline at 24 hr ( 20%60% increase at 1 hr ; figures 5a , 5b , 5e , and 5f ) . as with the case of weak - strong memory pairing ,
the pairing of strong memories led to co - clustering of synaptic contacts from the two memories in dendritic branches for isis of 12 hr for all cases tested . in all cases , the restriction of prps to dendritic branches led to a smaller degree of neuronal co - allocation between the two memories for isis of 1 and 2 hr ( p < 0.01 , one - way anova ) . the effect on dendritic co - clustering , however , was not significant ( p > 0.1 ) , indicating that dendritic co - allocation is not heavily dependent on the levels of neuronal co - allocation . overall , our model makes the following predictions regarding the interactions between two strong memories and the underlying mechanisms . first , our simulations support the hypothesis that changes in excitability lead to co - allocation of memories separated by several hours ( silva et al . , 2009 ,
2014 ) and are in line with experimental evidence that linked memories are stored in overlapping neuronal populations ( cai et al . fourth , the asymmetric enhancement of the second memory is due to increases in neuronal excitability after the acquisition of the first memory and predicts that blocking this increase in excitability should eliminate this asymmetry and possibly restrict memory enhancement to a 2-hr interval . the interplay between excitability and stc is likely to favor the creation of memory episodes for the following reasons : ( 1 ) stc links memories according to their temporal proximity , that is , the closer the memories , the higher their co - allocation ; and ( 2 ) learning - induced enhancement of neuronal excitability influences prior and subsequent memories in an asymmetric manner , thus enabling ordered linking of events . to test this hypothesis
, we simulated the encoding of 10 sequentially presented memories separated by inter - stimulus intervals ( isis ) of 1 , 5 , and 24 hr , under conditions of enhanced versus static excitability and somatic versus local prp synthesis and capture . this phenomenon can be explained by considering the effect of homeostatic mechanisms , which operate over time periods beyond 24 hr and reduce the overall excitability of the entire neuronal population , as well as the time window of enhanced neuronal excitability that does not extend beyond 12 hr . as with all prior experiments ,
the overlapping allocation at the neuronal level is accompanied by an increased co - clustering of synapses from interacting memories ( figure 6c ) , which follows the pattern of population overlaps : higher under somatic prps and enhanced excitability , and diminished when memories are separated by large intervals . , 2011 , hardie and spruston , 2009 , kang and schuman , 1996 ) , has led to multiple hypotheses regarding the role of dendritic plasticity in the formation of memory engrams ( branco and husser , 2010 , govindarajan et al . the model presented here incorporates biologically constrained dendritic compartments , depolarization - dependent plasticity mechanisms , synaptic tagging and capture , and changes in neuronal excitability in neurons embedded in a cortical microcircuit to study memory engram formation . importantly , we show that each of these mechanisms plays a role in the formation and interaction of memory engrams that would be missed if using simplified models . thus , the key contribution of this work is the systematic investigation of the biologically relevant parameter space that allows memory linking so as to characterize the underlying sub - cellular mechanisms and the extent to which they are generic , namely underlie information binding across the levels of a single , a pair , or multiple associative memories . for example , experimental evidence for widespread plasticity across a majority of neurons , with localized clusters of potentiated synapses within a small subset of their dendritic branches , would indicate that local ( as opposed to somatic ) prp mechanisms predominate during associative memory formation . distinguishable characteristics endowed by the locus of prps are also seen in the interaction of associative memories ( weak or strong ) across time . in addition
, we predict a role for intrinsic excitability in weak - strong memory rescuing , which has thus far been attributed to stc mechanisms alone ( ballarini et al . importantly , our model is in agreement with recent experimental studies , which confirm that there is excitability - dependent overlap of memory engrams learned within time intervals of a few hours . memory engrams in ca1 are overlapping when they are separated by 5-hr interval , but not at 7 days , and the overlap is linked with behavioral expression ( cai et al . similarly , in the lateral amygdala , memory engrams were allocated in overlapping populations for memories separated up to 6 hr ( rashid et al . for example , we predict that shorter intervals ( 12 hr ) would lead to higher and qualitatively different co - allocation , primarily because of synaptic tagging and capture mechanisms rather than neuronal excitability ( figure 5 ) . moreover , we propose that the neuronal overlap will be significantly higher if one of the memories is weak ( figure 4 ) . this is done by identifying a unifying principle for linking information across time : through neuronal and dendritic co - allocation realized via synapse clustering ( schematically illustrated in figure 7 ) . this simple rule explains memory interactions at the cellular and sub - cellular levels , and is robust under all of the different scenarios tested here , including variations in the locus of prp synthesis and the plasticity of neuronal excitability , different numbers , and strengths of memory , variable time intervals , among others . our biologically realistic model captured this critical facet of memory and yielded a number of experimental predictions that could guide research on how weak and strong memories are linked across time . future experiments that test predictions proposed here will address the mechanisms underlying these pathologies in animal models , and therefore open new avenues for the understanding and treatment of memory deficits . dendritic structure ( a branch is a single compartment ) and nonlinearities are incorporated in a simplified manner and the plasticity rules are modeled in a phenomenological way , without modeling the biophysics of membrane mechanisms or the elaborated morphology of dendritic trees . the neurons received feedback inhibition from inhibitory point neurons , as well as excitation from background synapses . the network model consists of a population comprising of the following : ( 1 ) excitatory neurons of a target region , ( 2 ) inhibitory neurons in the same region , ( 3 ) the stimulus - carrying input neurons , and ( 4 ) background noise input neurons . each memory encoding is performed by the activation of a set of stimulus - carrying neurons , which carry the conditioned and unconditioned stimulus information that represents the memory to be encoded ( figure 1a ) . stimulus - carrying neurons are grouped in sets of six ( three representing stimulus 1 [ s1 ] and three representing stimulus 2 [ s2 ] ) , which fire with an average firing frequency of 30 hz during the encoding of each memory ( s1+s2 neurons ) , as well as during the recall of each individual event ( s1 neurons only ) . connectivity parameters among the neuronal populations in the network model are listed in table s2 . during isi periods ,
spiking activity is not simulated , but slow consolidation processes ( prp production , synaptic tag consolidation , and homeostasis ) take place . a detailed description of the model and experimental procedures is provided in the supplemental experimental procedures . the source code of the model is provided under the open source gplv2 license at http://dendrites.gr/ and under modeldb : 206249 . | [
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the human stratum corneum ( sc ) is a superficial continuous membrane made of tightly stacked corneocytes parted by multilamellar lipid sheets .
its deep cohesive portion is called the stratum compactum , contrasting with the loose superficial part referred to as the stratum disjunctum .
such a structure undergoes a maturation process while it moves upwardly to the surface of the skin .
there is a progressive reduction in intercorneocyte cohesion , ending with physiologically imperceptible desquamation.1 considering the typical continuous renewal of the epidermis , and the structure of the sc as its end product , the sc corresponds to a recollection of the past history of the recent life of the epidermis.1 alterations in the organization of corneocytes , the presence of parakeratosis , and various other aspects are therefore clues for the intervention of some previous physiopathological disturbances . at most locations on the body surface , the sc
is typically composed of 15 or so layers of ordered flattened corneocytes . at the skin surface , these cells are approximately 1 m thick , and their mean area reaches approximately 1,000 m .
the corneocyte area is influenced by the anatomic location , and a variety of environmental conditions including chemical irritation and ultraviolet light ( uvl ) action modulate the epidermal renewal.1 in addition , the average corneocyte size is assumed to increase with aging .
this feature is probably linked to a prolonged corneocyte transit time during its progression throughout the sc .
some cosmetic compounds act on the sc , and modulate its structure and the functions that it serves .
other compounds target the sc by an indirect route through modulations in the physiology of the underlying stratum spinosum .
no proper pharmacology in the sc exists without adequate and reliable techniques for objective analytical recordings of the effects of the test products . in this review
smears and skin scrapings are simple procedures for getting sc samples . unfortunately , the collected material is obviously torn out and fragmented . the surface topography and the cell organizations
basically , sc stripping is the regular way for collecting the stratum disjunctum.2 using casual adhesive transparent tapes is poorly reliable for quantitative assessments because their adhesion to the sc is uncontrolled and appears variable among different brands .
by contrast , sc stripping using adhesive - coated discs ( sacd ) is conveniently performed.3 an improved method using a liquid cyanoacrylate pressure - sensitive adhesive on a polyester slide was designed for getting a better sampling of the sc structure.410 such a procedure corresponds to the cyanoacrylate skin surface stripping ( csss ) , formerly called skin surface biopsy4 or follicular biopsy.11 sacd uses a crystal clear adhesive - coated disc ( d - squame ; cuderm corporation , dallas , tx , usa ; and corneofix ; ck electronic , cologne , germany ) .
the device provides adequate rigidity and adhesion for uniformly sampling a defined area of the stratum disjunctum . after peeling off the protective seal ,
the disc is applied to the sc surface using a gauge spring dynamometer for ensuring a calibrated and reliable pressure.3 frequently , the chosen pressure is in the range of 100250 g / cm . both the pressure and the application time of the disc influence the amount of collected sc . a short - time ( approximately 5 seconds )
application of the disc removes less stratum disjunctum than an extended duration ( approximately 3060 minutes ) of application .
this is likely due to occlusion modifying both the intrinsic sc moisture and the intercorneocyte cohesion .
any greasy topical agent applied to the skin before sampling impairs the adhesion of the disc to the sc , thus yielding unreliable information .
such sampling failures are limited following delipidization of the skin surface using , for instance , ether : acetone ( 1:1 ) . for the csss method ( 3s - biokit ; ck technology , vis , belgium ) , a droplet of cyanoacrylate glue
this material is pressed against the sc surface for approximately 1530 seconds . in presence of discrete sc moisture , the cyanoacrylate polymerizes and firmly adheres to the sc .
ultimately , the material is gently lifted at one tip , and peeled off the skin .
csss is adequately performed from any site on the body , with two main provisos .
first , csss harvesting from a hairy area is commonly painful due to pulling out hairs .
furthermore , the csss quality is inadequate owing to the erratic contact of the cyanoacrylate bound to the sc .
second , a problem results from the natural strong intercorneocyte cohesion on the palms and soles .
this limitation impairs the collection of a uniform thin layer of corneocytes from these sites .
however , such csss samplings become adequate in some instances when the sc cohesiveness is compromised .
csss from normal skin reveals a regular crisscross network of high - peaked crests corresponding to discrete skin surface creases called the first- , second- , and third - hollow order lines .
the first - order lines correspond to grooves in the latticework papillary relief at the dermo
epidermal interface.7 in young individuals , junctions of the shallow first- and second - order lines border contiguous polyhedral shaped sc plateaus ( figure 1a ) .
on stretching the skin surface , a reorganization of these lines occurs . with aging , such a network progressively alters its original configuration , bringing the lattice along the skin tension lines spontaneously into alignment.8 the process ends with the vanishing of the shallow skin surface lines ( figure 1b ) .
thus , it is possible to assess the overall texture of the superficial dermis on csss . as a result
, conditions such as dermal aging , dermal atrophy ( dermatoporosis ) , striae distensae , sclerosis , scars , and many other skin connective tissue alterations are conveniently observed noninvasively using csss.7,8,10 such morphological assessment of the skin microrelief is possibly quantified combining computerized image analysis and profilometry ( figure 1c ) .
cytologic presentations of corneocytes are hardly visible on unstained csss . for improving their perceived aspect ,
a mixture of toluidine blue and basic fuchsin ( tbbf ) in 30% ethanol is a simple dye that is conveniently handled in an office setting.7,8,10 the regular sc exhibits a rather uniform cohesive pattern of adjacent corneocytes .
each single corneocyte is typically anucleated and contains a water - insoluble protein complex corresponding to a highly organized keratin microfibrillar matrix .
the cell boundaries appear clearly stained by a thin polyhedral tbbf rim.10 the cornified cell envelope exhibits different stages of maturation among corneocytes.12,13 basically , two distinct presentations of cornified cell envelopes are distinguished .
they correspond to the so - called fragile immature envelopes and the rigid mature ones , respectively ( figure 2a ) .
the former type is recognized on csss by a deep tbbf staining contrasting with the rimmed pattern of the mature corneocytes.10 scanning electron microscopy of immature corneocytes commonly exhibits a paving of small protrusions of similar sizes.10 lipid staining such as the nile red stain conveniently reveals sebum - enriched follicular pores and follicular casts collected on csss .
first , the visualized sebum closely represents the direct lipid production by each single follicle .
second , the lipids produced and poured out from other follicles in their vicinity run at the skin surface and are finally collected in the slope of other follicular openings .
skin strippings , including sacd and csss , are used as nearly noninvasive diagnostic methods and are useful in many skin conditions.1,5,7,10 in addition , they are examples of tools that combine analytical morphology and other procedures for the evaluation of the kinetics and efficacy of various cosmetics and toiletries . many cosmetic compounds and cosmeceuticals exert modulating effects , in part influencing the sc presentation . analytical morphology
these procedures include various techniques , and they afford quantitative and statistically evaluable data following image processing .
the challenge of analytical evaluations of skin strippings is to quantify some specific aspects of the structure and functions of the sc.14,15 image analysis of the aspect of samples seen under the microscope , and optical profilometry of the surface of the samples are two classical methods of analytical morphology that are conveniently applied to skin strippings .
other biometrological approaches have been designed , including visual rating evaluations of sampled material placed on a black background .
the weight of collected sc samples on sacd are measured and can further be analyzed for the molecular identification of specific components .
for that purpose , the samples are conveniently deposited in the well of microanalysis plates . in these respects , skin strippings
are used in four main ways :
evaluation of the sc structureevaluation of the sc dynamicsin situ evaluation of the superficial skin bioceneuse of csss as a substrate for a set of ex vivo bioassays evaluation of the sc structure evaluation of the sc dynamics in situ evaluation of the superficial skin biocene use of csss as a substrate for a set of ex vivo bioassays analytical morphology denotes a set of techniques that provide quantitative data and statistically evaluable information from morphological aspects . of note ,
meaningful measurements are dependent upon careful thought given to the sampling process . pressure and time of application of the clear disc in sacd , and the quality of the cyanoacrylate liquid bond in csss
they commonly rely on the combination of optical properties of the samples , reflectance colorimetry , and morphometry - based image analysis .
some aspects of skin conditions , including their severity and any cosmetic improvement , are conveniently assessed on csss following the disclosure of some typical features in the sc .
because sacd and csss are very thin compared to the width of the sampling , both samplings are regarded as nearly two - dimensional structures . under microscopic examination ,
the size of the field of vision is determined by the combination of the magnification and the microscope eyepiece .
usually , the eyepiece field diaphragm opening diameter determines the size of the field of vision .
the proportion of points in an ocular graticule gives a relative value for its area .
such values are converted to absolute values when the area of the sample covered by the graticule is determined .
it is possible to measure the area of the exposed aspect of corneocytes , and to detect any presence of parakeratosis ( figure 2b ) and other cells on such specimens ( figure 2c ) . on healthy skin ,
parakeratotic cells are rare , and those present are not clustered . by contrast , clumps of parakeratotic cells usually suggest a pathologic process .
there is a clinical relevance for the skin roughness parameters ( ra and rz ) gained on csss .
the relationship between xerosis and lack of water in outer layers of the sc is ambiguous.16 most xerotic conditions are in fact a failure of the normal corneocyte shedding process .
these cells remain attached to each other with unequal strength until rafts of cells detach partially from the skin surface ( figure 3 ) .
such crude evaluations suffer from variability by inconsistencies from grade to grade , and from poor reproducibility .
subtle variations in the environmental conditions jeopardize subjective grading systems since ambient hydration swells the outer sc and often camouflages low - grade scaling and dryness . thus , in order to investigate any xerotic process , and to determine the efficacy of a moisturizer , an evaluation program should be encouraged to go beyond usual clinical observations .
normal desquamation takes place imperceptibly as the release of single corneocytes at the skin surface .
when the intracorneal cohesion fails to decline evenly in the stratum disjunctum , clumps of corneocytes remain stuck together , and they come off as scales .
the resulting harsh feel by sensing fingers , and the altered appearance under light reflectance correspond to xerosis , often interpreted as dry skin by laypeople .
in fact , the concept of dry skin remains a controversial matter.16 xerosis refers to various forms of predominantly orthokeratotic hyperkeratosis .
this definition and its limits are idiosyncratic , varying according to different local traditions among laboratories and cosmetic companies . a semiquantitative grading of orthokeratotic xeroses is conveniently achieved on csss stained for 3 minutes with the tbbf staining solution.7,10,16 the samples
are graded7,10 as follows :
type 0 : normal sc without any evidence for hyperkeratosistype 1 : hyperkeratosis of the first- and second - order lines and/or the adnexal openingstype 2 : focal hyperkeratosis covering less than 30% of the skin surface plateaus ( figure 4a)type 3 : hyperkeratosis covering over 30% of the skin surface plateaus ( figure 4b)type 4 : diffuse , confluent , and homogeneous scales with persistence of the first - order linestype 5 : thick , uneven scales covering the entire sc surface , obliterating the shallow lines type 0 : normal sc without any evidence for hyperkeratosis type 1 : hyperkeratosis of the first- and second - order lines and/or the adnexal openings type 2 : focal hyperkeratosis covering less than 30% of the skin surface plateaus ( figure 4a ) type 3 : hyperkeratosis covering over 30% of the skin surface plateaus ( figure 4b ) type 4 : diffuse , confluent , and homogeneous scales with persistence of the first - order lines type 5 : thick , uneven scales covering the entire sc surface , obliterating the shallow lines quantitative assessment of xerosis relies on analytical evaluations of sacd.3,14 the attenuation of optical light transmission has been used to measure the amount of scales collected on the discs .
that method was improved by measuring reflectance colorimetry of the unstained sample deposited onto a colored reference plate .
the parameters of importance are the area ( a ) covered by scales , and their thickness rated on a 5-level gray scale .
the percentage of scales ( tn ) is calculated in relation to each thickness level ( n ) . a desquamation index ( di )
is yielded according to :
di=2a+[tn(n1)]6(1 ) the squamometry index ( sqmi ) is yet another objective evaluation of xerosis and some other sc disturbances.17,18 sacd are stained for 1 minute by dropping the tbbf solution over the sc surface , followed by gentle tap - water rinsing .
the stained sacd is placed over a hole cut out of a slide , which is placed onto a white color reference plate .
the chroma c * value measured by reflectance colorimetry correlates with the amount of removed scales and with the clinical rating of xerosis . a seemingly regular sc of the volar forearm is commonly characterized by sqmi values lower than 20 . by contrast
this technique is conveniently applied for assessing some inflammatory conditions such as sunburn ( figure 5 ) , and to quantify the efficacy of antidandruff shampoos .
xerosis of old age as well as wrinkling are possibly evaluated on csss . in particular ,
the wrinkling process and its correction by some cosmetics is conveniently substantiated by combining image analysis and optical profilometry .
follicular casts and microcomedones are conveniently studied on csss collected from the face or the back .
the horny material sampled from the upper portion of the follicular ducts corresponds to a follicular stripping,11,19 and it reflects the balance between comedo formation and lysis ( figure 6a ) .
comedometry allows for computerized quantification of the number and size of follicular casts collected on csss.11,19,20 the numerical density of follicles is influenced by the body site .
this method finds application in comedogenesis- and comedolysis - related disorders and their treatments.2023 both the number and the size of follicular casts are influenced by some treatments .
comedometry on human skin showing comedogenesis reveals large interindividual differences in the number of horny follicular casts .
csss are used to predict the comedogenic risk linked to products , and , by contrast , to quantify any comedolytic activity of cosmetics .
the concentration in active compounds governs the propensity of modifying the microcomedo density and size .
when an exogenous comedogenic factor is involved , the vast majority of the hair follicles are similarly involved .
by contrast , endogenous comedogenic factors ( androgens , acne , etc ) typically affect to a variable extent a minority of hair follicles.10 the sensitivity of the method is such that it is possible to disclose signs of microcomedolysis after a short time ( a couple of weeks ) of adequate treatment.23 sebum - sensitive foils ( ssf ) , available as sebutape ( cuderm corp ; dallas , tx , usa ) and sebufix ( ck electronic ) , are conveniently used for assessing the sebum output at the skin surface .
it is possible to combine such method with csss.24 in a first step , a non - sticky ssf is applied to the skin for a few seconds .
the outlines of the foil are ink marked on the sc . in a second step , following removal of the ssf , csss is collected from the very same skin site .
the csss and the foil are then exactly superposed using the ink mark as an adjusting mark .
csss samplings are examined under the microscope ( figure 6b ) and submitted to image analysis , considering specifically the darker horny follicular casts and the clear transparent sebum spots .
some correlations are possibly established between the pore sizes , the follicular casts , the microcomedones , and the clear transparent spots of sebum.24 analytical methods for evaluating some features of follicular casts rely on image analysis when illuminating the specimen under the microscope with either white light , polarized light , or fluorescent light .
the assessment of the follicular fluorescence for evaluating the presence of porphyrins produced by propionibacterium acnes in follicles is occasionally important for assessing any impact of cosmetics.25 in fact , some products possibly emit fluorescence by themselves ( figure 6c ) , while others such as sunscreens display a quenching effect by absorption of porphyrin fluorescence .
it is possible to assess the density of hair follicles over a defined surface area , as well as to observe the skin pores and the presence of follicular hyperkeratosis ( kerosis ) , comedones , trichostasis spinulosa , intrafollicular bacteria , and mites ( demodex folliculorum).7,2628 in some instances , other hair follicle structures including hair bulbs and follicular sheaths are visualized on csss .
skin pores distinctly corresponding to follicular or sudoral openings at the skin surface are conveniently explored using csss.27 melanin production in melanocytes is under complex neuroendocrine controls.29 it is identified in regular corneocytes of phototype v and vi individuals .
it is also present in the sc covering various pigmented lesion in clear - skinned people ( figure 7 ) .
it is mandatory to distinguish melanin - laden anucleated corneocytes from neoplastic dendritic nucleated melanocytes as seen after their migration inside the sc covering a malignant melanoma.10 for increasing the sensitivity of the procedure , the aspect of the dusty melanin load is typically increased using an argentaffin stain procedure .
such a method consists of measuring the reduction in light transmission through the csss using a photodensitometer device designed for photomicroscopy.3033 bleaching agent effects are possibly tested and compared using corneomelametry .
melasma and solar lentigines ( aging spots ) are typical lesions explored in that way .
the dansyl chloride ( dc ) test was generally accepted as a noninvasive clinical attempt at evaluating the sc turnover .
the overall time for fluorescence extinction depends on both the rate of transit of corneocytes through the sc and the thickness of that layer .
this test proves to be difficult to interpret on clinical grounds due to the uneven fade - out of fluorescence at the skin surface .
an improved method was introduced by the examination of csss harvested from a dc test area.33 it was further refined by replacing dc with the browning dihydroxyacetone ( dha ) agent.34 corneodynamics is assessed using csss from dc ( figure 8a ) and dha ( figure 8b ) test sites at a predetermined time of a trial .
accordingly , at about day 10 , csss are examined under a fluorescent light microscope because both dc and dha are fluorescent .
image analysis applied to such pictures allows quantification of the nonfluorescent versus fluorescent areas of the sc .
however , both the dc and dha tests are dramatically influenced by cleansing agents and skincare products.35 the extraction of the dyes from the sc by these products is tentatively used to predict irritancy potential.35 an inverse relationship has been suggested between the size of corneocytes and the speed of epidermal turnover . this aspect is possibly studied on skin strippings , particularly csss during corneodynamics .
another facet of the sc dynamics is gained by squamometry , in particular uvl squamometry.3 for instance , the sqmi kinetics after acute photodamage show a complex response of the epidermis with early ( 48 hours ) and late ( 10 days ) changes.36 indeed , a single regular sacd sampling corresponds at the most to the regular corneocyte daily shedding .
normal and moderately dry skin respond to uvl irradiation by an early increase in the sqmi value .
the biocene of resident bacteria and other saprophytic or pathogenic microorganisms is typically confined to the skin surface and the appendages.6,3739 at the surface of the sc , the flora remains largely encased inside the cyanoacrylate bond during csss sampling .
thus , it is not accessible to staining procedures , and it remains invisible at microscopic examination .
therefore , the surface microflora is not adequately disclosed on csss . by contrast , samples of microorganisms entrapped inside the sc and follicular casts are distinctly collected from csss . in particular , the microflora is accessible by scraping out the horny spiky structures appended to the csss .
viability of the intrafollicular bacteria is conveniently assessed using the combination of vital stains ( figure 9a ) , such as neutral red,40 and flow cytometry.39 the load of malassezia spp .
inside squames of dandruff and seborrheic dermatitis is assessed on sacd ( figure 9b ) .
the efficacy of shampoos can be compared by combining squamometry and semiquantitative evaluations of the number of yeasts.41 the adhesion of malassezia yeasts to corneocytes appears to be a prominent feature.42 the presence of various tiny foreign bodies and microscopic parasites , and the impact of various chemical xenobiotics on the sc are conveniently assessed on csss.10,43 in particular , surfactants remove lipids , denature proteins , induce corneocyte swelling , alter the sc barrier function , and produce a feeling of skin roughness and dryness.44 the surest method for testing skin compatibility of surface - active agents was to use large panels of volunteers .
however , such a procedure proved to be costly and time - consuming . over the years , various alternative methods were proposed . among them , corneosurfametry ( csm ) refers specifically to the effects of surfactants and wash solutions on the sc.18,44,45 csm is a noninvasive quantitative test rating the interaction between surfactants and human or animal sc ( figure 10 ) .
it is used as a predictive irritancy test that compares favorably with conventional in vitro tests .
csm entails collection of csss from healthy skin of volunteers followed by a predetermined contact time of the sc samples with neat or diluted surfactants .
a 2-hour experiment is usually convenient to disclose any differences in sc reactivity among products .
the usual test concentrations for proprietary cleansing skincare products ranges from 3% to 10% , but higher dilutions are possibly required for harsh products .
each surfactant solution is sprayed uniformly on about 20 csss placed in a slide tray , which is then covered by a lid . at completion of the predetermined assessment time ,
csss are thoroughly rinsed with tap water , dried , and stained for 3 minutes with the tbbf dye solution .
after removing any excess dye and copious water rinsing , samples are examined under the microscope .
in addition , reflectance colorimetry is applied to the dry samples for recording both l * and chroma c*. to be adequately interpreted , the l * value should be over 45 , and superior to that of chroma c*.18 data out of range suggest an excess surfactant concentration for the test . for instance , the maximum concentration for testing sodium laurylsulfate ( sls ) is approximately 2% .
csm results are expressed by two parameters : namely , the csm index ( csmi ) and the colorimetric index of mildness ( cim ) .
csmi of any test product corresponds to the color difference between water - treated control samples and those exposed to the test product .
thus , csmi is conveniently calculated as follows :
csmi=[(l*)2+(c*)2]0.5(2 ) there is a linear negative correlation between values of l * and chroma c * , and cim of each sample is calculated as follows :
cim = l*c*(3 ) according to such analytical assessments , mild skincare products containing anionic surfactants are characterized by the combination of a low csmi value and a high cim value .
however , a low paradoxical csmi value is experienced with some aggressive products yielding to a cascade of corneocyte swelling , lysis , and loss from the stratum disjunctum . usually , interindividual variations are larger for sqmi than for cim.18 microwave csm is a more rapid procedure than the regular csm.46 in this procedure , csss are immersed in a flask containing the test surfactant solution , and they are placed in a microwave oven containing a 500 ml water load .
responsive csm is a variant of the method where skin to be sampled is preconditioned before harvesting csss.47 the method , based on repeated subclinical injuries by surfactants , is monitored in a controlled forearm immersion test . at completion of the in vivo procedure , csss are harvested for a regular or microwave csm bioassay using the same surfactant as in the preliminary in vivo procedure .
preconditioning the skin in this way increases csm sensitivity to discriminate between different potential irritations from mild surfactants .
shielded csm is used for testing skin - protective products ( spp ) , also called skin barrier formulations , claiming some protection against noxious external agents . in shielded csm ,
csss are first covered by a controlled amount of the test spp before performing regular csm using a reference surfactant .
comparative screenings of various spp are conveniently performed using shielded csm , avoiding exposure of volunteers to any hazardous agents for in vivo testing .
the corneoxenometry ( cxm ) bioassay is offered for testing any chemical xenobiotic other than surfactants.48,49 the compounds consist of any hazardous chemical including organic solvents , acids , bases , toxic agents , etc .
it entails csss collection followed by its controlled immersion in the test solution for 1120 minutes .
one main indication is found in the field of skin irritation while avoiding the risk of in vivo hazards .
skin barrier formulations are conveniently tested using cxm.50 another indication deals with comparative assessments of penetration enhancers commonly used in some topical formulations.51 one of the most important functions of the epidermis is the formation of an efficient barrier between the body and a diversity of xenobiotics .
much research has been undertaken to understand the variability in the skin barrier efficacy , which resides in the sc .
values are kept low when the barrier function is optimal , and they increase with the severity of the barrier defect .
it is generally assumed that the cutaneous barrier is disrupted through intercellular lipid removal and protein denaturations .
for instance , such a condition is reached following applications of various organic solvents to the skin .
since workers are exposed to solvents at many workplaces , methods are needed for the evaluation of the potential solvent toxicity .
such hazards call for ex vivo predictive bioassays on human skin or sc , such as csm and cxm . large interindividual differences in cim
were reported regarding different surfactants and solvents.48 in addition , distinct regional differences are present regarding irritancy and percutaneous absorption . both the dorsal hand and volar forearm appeared to be the least reactive skin locations on the csm bioassay .
by contrast , the same test revealed that the neck , forehead , back , and dorsal foot were more reactive sites . in normal subjects ,
the extent of alterations induced in the human sc by solvents during cxm are more variable than those induced by diluted surfactants at the csm bioassay .
in addition , a heterogeneity in corneocyte alterations exists among solvents and between distinct body sites .
for instance , hexane methanol and chloroform methanol strongly alter the sc structure .
indeed , the chloroform methanol mixture is a potent lipid extractor from biological samples .
however , it is not the top ranked aggressor on the cxm bioassay.48 the intensity of the skin response to irritant xenobiotics is subject to anatomic site variations .
the initial mechanism governing such biological response presumably corresponds to the direct damage to the sc .
subtle regional variations in irritancy are possibly tested using csm and cxm.44 regional variations in skin reactivity presumably correspond in part to an uneven preconditioning by in vivo contacts with a previous irritant stress . a similar , but probably weaker process takes place in the condition of reactive skin.52 this concept illustrates the failure of drawing general conclusions from csm and cxm performed from a given body site .
such a finding contrasts with the frequent variability in cim data from distinct body sites , and with the inconsistency in severity of irritant reactions at different anatomical regions , as assessed by transepidermal water loss measurements at in vivo patch test sites .
the term reactive ( sensitive ) skin covers a broad spectrum of different conditions , sharing in common the occurrence of unwanted changes in response to external environmental factors including personal - care products .
reactive skin is a heterogeneous problem characterized by a reduced cutaneous tolerance to specific environmental factors ( cold , heat , wind , wool , topical products , etc).53 clinical manifestations consist mainly of subjective symptoms linked to sensory irritation including discomfort , itching , stinging , and burning sensations .
other biometrological procedures are possibly useful.54 some individuals complaining of sensitive skin are more readily deranged by some specific chemical and physical irritation .
csm performed with a reference surfactant ( sls 1% ) shows that csss from patients with reactive skin react more severely than those harvested from healthy individuals without sensitive skin .
cim is significantly lower ( approximately 25% reduction ) in patients with reactive skin than in normal nonsensitive individuals , indicating a higher susceptibility to irritation induced by surfactants and some other skincare products.52 csss , using a method similar to csm , is a substrate for the study of the actual bioactivity of emollients and squamolytic ( keratolytic ) agents .
the test product is applied to csss for various periods of time ranging from 15 minutes to 4 hours ( figure 11 ) .
after abundant rinsing and staining with tbbf , reflectance colorimetric values are recorded and csmi and/or cim parameters are calculated in a way similar to csm .
the effect of squamolytic agents and emollients is conveniently assessed on skin strippings collected in clinical trials ( figure 12 ) . in general , it is appropriate to harvest samples at entry into the study , as well as 2 weeks and 4 weeks after treatment . after a 2-week period of treatment corresponding to the regression phase ,
the typical kinetics show first an aggravation in the test values followed by a drop in these values , this latter phase indicating a loss of corneocytes from the csss .
optical profilometry performed on the same samples shows the immediate smoothing effect of the products with reduction in the rz value followed by desquamation and increased rz .
the relationship between xerosis and lack of water in outer layers of the sc is ambiguous.16 most xerotic conditions are in fact a failure of the normal corneocyte shedding process .
these cells remain attached to each other with unequal strength until rafts of cells detach partially from the skin surface ( figure 3 ) .
such crude evaluations suffer from variability by inconsistencies from grade to grade , and from poor reproducibility .
subtle variations in the environmental conditions jeopardize subjective grading systems since ambient hydration swells the outer sc and often camouflages low - grade scaling and dryness .
thus , in order to investigate any xerotic process , and to determine the efficacy of a moisturizer , an evaluation program should be encouraged to go beyond usual clinical observations .
normal desquamation takes place imperceptibly as the release of single corneocytes at the skin surface .
when the intracorneal cohesion fails to decline evenly in the stratum disjunctum , clumps of corneocytes remain stuck together , and they come off as scales .
the resulting harsh feel by sensing fingers , and the altered appearance under light reflectance correspond to xerosis , often interpreted as dry skin by laypeople .
in fact , the concept of dry skin remains a controversial matter.16 xerosis refers to various forms of predominantly orthokeratotic hyperkeratosis .
this definition and its limits are idiosyncratic , varying according to different local traditions among laboratories and cosmetic companies .
a semiquantitative grading of orthokeratotic xeroses is conveniently achieved on csss stained for 3 minutes with the tbbf staining solution.7,10,16 the samples are graded7,10 as follows :
type 0 : normal sc without any evidence for hyperkeratosistype 1 : hyperkeratosis of the first- and second - order lines and/or the adnexal openingstype 2 : focal hyperkeratosis covering less than 30% of the skin surface plateaus ( figure 4a)type 3 : hyperkeratosis covering over 30% of the skin surface plateaus ( figure 4b)type 4 : diffuse , confluent , and homogeneous scales with persistence of the first - order linestype 5 : thick , uneven scales covering the entire sc surface , obliterating the shallow lines type 0 : normal sc without any evidence for hyperkeratosis type 1 : hyperkeratosis of the first- and second - order lines and/or the adnexal openings type 2 : focal hyperkeratosis covering less than 30% of the skin surface plateaus ( figure 4a ) type 3 : hyperkeratosis covering over 30% of the skin surface plateaus ( figure 4b ) type 4 : diffuse , confluent , and homogeneous scales with persistence of the first - order lines type 5 : thick , uneven scales covering the entire sc surface , obliterating the shallow lines quantitative assessment of xerosis relies on analytical evaluations of sacd.3,14 the attenuation of optical light transmission has been used to measure the amount of scales collected on the discs .
that method was improved by measuring reflectance colorimetry of the unstained sample deposited onto a colored reference plate .
the parameters of importance are the area ( a ) covered by scales , and their thickness rated on a 5-level gray scale .
the percentage of scales ( tn ) is calculated in relation to each thickness level ( n ) . a desquamation index ( di )
is yielded according to :
di=2a+[tn(n1)]6(1 ) the squamometry index ( sqmi ) is yet another objective evaluation of xerosis and some other sc disturbances.17,18 sacd are stained for 1 minute by dropping the tbbf solution over the sc surface , followed by gentle tap - water rinsing .
the stained sacd is placed over a hole cut out of a slide , which is placed onto a white color reference plate .
the chroma c * value measured by reflectance colorimetry correlates with the amount of removed scales and with the clinical rating of xerosis . a seemingly regular sc of the volar forearm is commonly characterized by sqmi values lower than 20 . by contrast
this technique is conveniently applied for assessing some inflammatory conditions such as sunburn ( figure 5 ) , and to quantify the efficacy of antidandruff shampoos .
xerosis of old age as well as wrinkling are possibly evaluated on csss . in particular ,
the wrinkling process and its correction by some cosmetics is conveniently substantiated by combining image analysis and optical profilometry .
follicular casts and microcomedones are conveniently studied on csss collected from the face or the back .
the horny material sampled from the upper portion of the follicular ducts corresponds to a follicular stripping,11,19 and it reflects the balance between comedo formation and lysis ( figure 6a ) .
comedometry allows for computerized quantification of the number and size of follicular casts collected on csss.11,19,20 the numerical density of follicles is influenced by the body site .
this method finds application in comedogenesis- and comedolysis - related disorders and their treatments.2023 both the number and the size of follicular casts are influenced by some treatments .
comedometry on human skin showing comedogenesis reveals large interindividual differences in the number of horny follicular casts .
csss are used to predict the comedogenic risk linked to products , and , by contrast , to quantify any comedolytic activity of cosmetics .
the concentration in active compounds governs the propensity of modifying the microcomedo density and size .
when an exogenous comedogenic factor is involved , the vast majority of the hair follicles are similarly involved .
by contrast , endogenous comedogenic factors ( androgens , acne , etc ) typically affect to a variable extent a minority of hair follicles.10 the sensitivity of the method is such that it is possible to disclose signs of microcomedolysis after a short time ( a couple of weeks ) of adequate treatment.23 sebum - sensitive foils ( ssf ) , available as sebutape ( cuderm corp ; dallas , tx , usa ) and sebufix ( ck electronic ) , are conveniently used for assessing the sebum output at the skin surface .
it is possible to combine such method with csss.24 in a first step , a non - sticky ssf is applied to the skin for a few seconds .
the outlines of the foil are ink marked on the sc . in a second step , following removal of the ssf , csss is collected from the very same skin site .
the csss and the foil are then exactly superposed using the ink mark as an adjusting mark .
csss samplings are examined under the microscope ( figure 6b ) and submitted to image analysis , considering specifically the darker horny follicular casts and the clear transparent sebum spots .
some correlations are possibly established between the pore sizes , the follicular casts , the microcomedones , and the clear transparent spots of sebum.24 analytical methods for evaluating some features of follicular casts rely on image analysis when illuminating the specimen under the microscope with either white light , polarized light , or fluorescent light .
the assessment of the follicular fluorescence for evaluating the presence of porphyrins produced by propionibacterium acnes in follicles is occasionally important for assessing any impact of cosmetics.25 in fact , some products possibly emit fluorescence by themselves ( figure 6c ) , while others such as sunscreens display a quenching effect by absorption of porphyrin fluorescence .
it is possible to assess the density of hair follicles over a defined surface area , as well as to observe the skin pores and the presence of follicular hyperkeratosis ( kerosis ) , comedones , trichostasis spinulosa , intrafollicular bacteria , and mites ( demodex folliculorum).7,2628 in some instances , other hair follicle structures including hair bulbs and follicular sheaths are visualized on csss .
skin pores distinctly corresponding to follicular or sudoral openings at the skin surface are conveniently explored using csss.27
melanin production in melanocytes is under complex neuroendocrine controls.29 it is identified in regular corneocytes of phototype v and vi individuals .
it is also present in the sc covering various pigmented lesion in clear - skinned people ( figure 7 ) .
it is mandatory to distinguish melanin - laden anucleated corneocytes from neoplastic dendritic nucleated melanocytes as seen after their migration inside the sc covering a malignant melanoma.10 for increasing the sensitivity of the procedure , the aspect of the dusty melanin load is typically increased using an argentaffin stain procedure . the relative darkness of these csss is conveniently assessed using corneomelametry .
such a method consists of measuring the reduction in light transmission through the csss using a photodensitometer device designed for photomicroscopy.3033 bleaching agent effects are possibly tested and compared using corneomelametry .
melasma and solar lentigines ( aging spots ) are typical lesions explored in that way .
the dansyl chloride ( dc ) test was generally accepted as a noninvasive clinical attempt at evaluating the sc turnover .
the overall time for fluorescence extinction depends on both the rate of transit of corneocytes through the sc and the thickness of that layer .
this test proves to be difficult to interpret on clinical grounds due to the uneven fade - out of fluorescence at the skin surface .
an improved method was introduced by the examination of csss harvested from a dc test area.33 it was further refined by replacing dc with the browning dihydroxyacetone ( dha ) agent.34 corneodynamics is assessed using csss from dc ( figure 8a ) and dha ( figure 8b ) test sites at a predetermined time of a trial .
accordingly , at about day 10 , csss are examined under a fluorescent light microscope because both dc and dha are fluorescent .
image analysis applied to such pictures allows quantification of the nonfluorescent versus fluorescent areas of the sc .
however , both the dc and dha tests are dramatically influenced by cleansing agents and skincare products.35 the extraction of the dyes from the sc by these products is tentatively used to predict irritancy potential.35 an inverse relationship has been suggested between the size of corneocytes and the speed of epidermal turnover . this aspect is possibly studied on skin strippings , particularly csss during corneodynamics .
another facet of the sc dynamics is gained by squamometry , in particular uvl squamometry.3 for instance , the sqmi kinetics after acute photodamage show a complex response of the epidermis with early ( 48 hours ) and late ( 10 days ) changes.36 indeed , a single regular sacd sampling corresponds at the most to the regular corneocyte daily shedding .
normal and moderately dry skin respond to uvl irradiation by an early increase in the sqmi value .
the biocene of resident bacteria and other saprophytic or pathogenic microorganisms is typically confined to the skin surface and the appendages.6,3739 at the surface of the sc , the flora remains largely encased inside the cyanoacrylate bond during csss sampling .
thus , it is not accessible to staining procedures , and it remains invisible at microscopic examination .
therefore , the surface microflora is not adequately disclosed on csss . by contrast , samples of microorganisms entrapped inside the sc and follicular casts are distinctly collected from csss . in particular , the microflora is accessible by scraping out the horny spiky structures appended to the csss .
viability of the intrafollicular bacteria is conveniently assessed using the combination of vital stains ( figure 9a ) , such as neutral red,40 and flow cytometry.39 the load of malassezia spp .
inside squames of dandruff and seborrheic dermatitis is assessed on sacd ( figure 9b ) .
the efficacy of shampoos can be compared by combining squamometry and semiquantitative evaluations of the number of yeasts.41 the adhesion of malassezia yeasts to corneocytes appears to be a prominent feature.42
the presence of various tiny foreign bodies and microscopic parasites , and the impact of various chemical xenobiotics on the sc are conveniently assessed on csss.10,43 in particular , surfactants remove lipids , denature proteins , induce corneocyte swelling , alter the sc barrier function , and produce a feeling of skin roughness and dryness.44 the surest method for testing skin compatibility of surface - active agents was to use large panels of volunteers .
however , such a procedure proved to be costly and time - consuming . over the years , various alternative methods were proposed . among them , corneosurfametry ( csm ) refers specifically to the effects of surfactants and wash solutions on the sc.18,44,45 csm is a noninvasive quantitative test rating the interaction between surfactants and human or animal sc ( figure 10 ) .
it is used as a predictive irritancy test that compares favorably with conventional in vitro tests .
csm entails collection of csss from healthy skin of volunteers followed by a predetermined contact time of the sc samples with neat or diluted surfactants .
a 2-hour experiment is usually convenient to disclose any differences in sc reactivity among products .
the usual test concentrations for proprietary cleansing skincare products ranges from 3% to 10% , but higher dilutions are possibly required for harsh products .
each surfactant solution is sprayed uniformly on about 20 csss placed in a slide tray , which is then covered by a lid . at completion of the predetermined assessment time ,
csss are thoroughly rinsed with tap water , dried , and stained for 3 minutes with the tbbf dye solution .
after removing any excess dye and copious water rinsing , samples are examined under the microscope .
in addition , reflectance colorimetry is applied to the dry samples for recording both l * and chroma c*. to be adequately interpreted , the l * value should be over 45 , and superior to that of chroma c*.18 data out of range suggest an excess surfactant concentration for the test . for instance , the maximum concentration for testing sodium laurylsulfate ( sls ) is approximately 2% .
csm results are expressed by two parameters : namely , the csm index ( csmi ) and the colorimetric index of mildness ( cim ) .
csmi of any test product corresponds to the color difference between water - treated control samples and those exposed to the test product .
thus , csmi is conveniently calculated as follows :
csmi=[(l*)2+(c*)2]0.5(2 ) there is a linear negative correlation between values of l * and chroma c * , and cim of each sample is calculated as follows :
cim = l*c*(3 ) according to such analytical assessments , mild skincare products containing anionic surfactants are characterized by the combination of a low csmi value and a high cim value .
however , a low paradoxical csmi value is experienced with some aggressive products yielding to a cascade of corneocyte swelling , lysis , and loss from the stratum disjunctum .
usually , interindividual variations are larger for sqmi than for cim.18 microwave csm is a more rapid procedure than the regular csm.46 in this procedure , csss are immersed in a flask containing the test surfactant solution , and they are placed in a microwave oven containing a 500 ml water load .
responsive csm is a variant of the method where skin to be sampled is preconditioned before harvesting csss.47 the method , based on repeated subclinical injuries by surfactants , is monitored in a controlled forearm immersion test . at completion of the in vivo procedure , csss are harvested for a regular or microwave csm bioassay using the same surfactant as in the preliminary in vivo procedure .
preconditioning the skin in this way increases csm sensitivity to discriminate between different potential irritations from mild surfactants .
shielded csm is used for testing skin - protective products ( spp ) , also called skin barrier formulations , claiming some protection against noxious external agents . in shielded csm ,
csss are first covered by a controlled amount of the test spp before performing regular csm using a reference surfactant .
comparative screenings of various spp are conveniently performed using shielded csm , avoiding exposure of volunteers to any hazardous agents for in vivo testing .
the corneoxenometry ( cxm ) bioassay is offered for testing any chemical xenobiotic other than surfactants.48,49 the compounds consist of any hazardous chemical including organic solvents , acids , bases , toxic agents , etc .
it entails csss collection followed by its controlled immersion in the test solution for 1120 minutes .
one main indication is found in the field of skin irritation while avoiding the risk of in vivo hazards .
skin barrier formulations are conveniently tested using cxm.50 another indication deals with comparative assessments of penetration enhancers commonly used in some topical formulations.51
one of the most important functions of the epidermis is the formation of an efficient barrier between the body and a diversity of xenobiotics .
much research has been undertaken to understand the variability in the skin barrier efficacy , which resides in the sc .
values are kept low when the barrier function is optimal , and they increase with the severity of the barrier defect . it is generally assumed that the cutaneous barrier is disrupted through intercellular lipid removal and protein denaturations .
for instance , such a condition is reached following applications of various organic solvents to the skin .
since workers are exposed to solvents at many workplaces , methods are needed for the evaluation of the potential solvent toxicity .
such hazards call for ex vivo predictive bioassays on human skin or sc , such as csm and cxm .
large interindividual differences in cim were reported regarding different surfactants and solvents.48 in addition , distinct regional differences are present regarding irritancy and percutaneous absorption . both the dorsal hand and volar forearm appeared to be the least reactive skin locations on the csm bioassay .
by contrast , the same test revealed that the neck , forehead , back , and dorsal foot were more reactive sites . in normal subjects ,
the extent of alterations induced in the human sc by solvents during cxm are more variable than those induced by diluted surfactants at the csm bioassay .
in addition , a heterogeneity in corneocyte alterations exists among solvents and between distinct body sites .
for instance , hexane methanol and chloroform methanol strongly alter the sc structure .
indeed , the chloroform methanol mixture is a potent lipid extractor from biological samples .
however , it is not the top ranked aggressor on the cxm bioassay.48 the intensity of the skin response to irritant xenobiotics is subject to anatomic site variations .
the initial mechanism governing such biological response presumably corresponds to the direct damage to the sc .
subtle regional variations in irritancy are possibly tested using csm and cxm.44 regional variations in skin reactivity presumably correspond in part to an uneven preconditioning by in vivo contacts with a previous irritant stress . a similar , but probably weaker process takes place in the condition of reactive skin.52 this concept illustrates the failure of drawing general conclusions from csm and cxm performed from a given body site .
such a finding contrasts with the frequent variability in cim data from distinct body sites , and with the inconsistency in severity of irritant reactions at different anatomical regions , as assessed by transepidermal water loss measurements at in vivo patch test sites .
the term reactive ( sensitive ) skin covers a broad spectrum of different conditions , sharing in common the occurrence of unwanted changes in response to external environmental factors including personal - care products .
reactive skin is a heterogeneous problem characterized by a reduced cutaneous tolerance to specific environmental factors ( cold , heat , wind , wool , topical products , etc).53 clinical manifestations consist mainly of subjective symptoms linked to sensory irritation including discomfort , itching , stinging , and burning sensations .
other biometrological procedures are possibly useful.54 some individuals complaining of sensitive skin are more readily deranged by some specific chemical and physical irritation .
csm performed with a reference surfactant ( sls 1% ) shows that csss from patients with reactive skin react more severely than those harvested from healthy individuals without sensitive skin .
cim is significantly lower ( approximately 25% reduction ) in patients with reactive skin than in normal nonsensitive individuals , indicating a higher susceptibility to irritation induced by surfactants and some other skincare products.52
csss , using a method similar to csm , is a substrate for the study of the actual bioactivity of emollients and squamolytic ( keratolytic ) agents .
the test product is applied to csss for various periods of time ranging from 15 minutes to 4 hours ( figure 11 ) .
after abundant rinsing and staining with tbbf , reflectance colorimetric values are recorded and csmi and/or cim parameters are calculated in a way similar to csm .
the effect of squamolytic agents and emollients is conveniently assessed on skin strippings collected in clinical trials ( figure 12 ) . in general , it is appropriate to harvest samples at entry into the study , as well as 2 weeks and 4 weeks after treatment . after a 2-week period of treatment corresponding to the regression phase ,
the typical kinetics show first an aggravation in the test values followed by a drop in these values , this latter phase indicating a loss of corneocytes from the csss .
optical profilometry performed on the same samples shows the immediate smoothing effect of the products with reduction in the rz value followed by desquamation and increased rz .
the emergence of new analytical methods increases the validity of the information yielded by sacd and csss . with regulations avoiding animal experiments and ethical considerations in human testing ,
there is a need and a real possibility to develop new predictive bioassays using controlled skin strippings as substrate .
fortunately , sound mathematical relationships are perceived between the original structure and the images , and many of these relationships appear quite obvious . in general , multivariate statistical analysis is required to validate a meaningful conclusion . when testing xenobiotic effects on sc , the stratum disjunctum response as assessed by sqmi on sacd is different from csmi on the thicker sc collected by csss . nevertheless , both methods appear valuable and complementary . beyond sc scrapings , sacd and csss
these simple , low - cost , and minimally invasive methods allow the clinician , the cosmetologist , and the experimentalist to avoid invasive procedures within limits of well - defined indications .
there are obvious features and subtle characteristics discernible in the csss structure for establishing key points in a variety of skin conditions .
it is important to stress that no single criterion should usually be relied upon for a definitive feature on csss .
a range of quantifications are made possible on csss using computer - assisted image analysis .
the sc holds a large amount of information about the skin itself , and the internal milieu as well . in many instances
, csss appears to represent a convenient way that is currently available for exploring many facets of cosmetic science .
the main advantages of csss are as follows :
swift and safe method for sc samplingsmultiple diagnostic possibilitiescheap procedurehygienic procedureminimally invasiveeasy handlingstable samples over timedefined skin stripping for scientific applicationsestablished method , known for more than 30 yearssupported by a number of scientific contributions swift and safe method for sc samplings multiple diagnostic possibilities stable samples over time defined skin stripping for scientific applications established method , known for more than 30 years supported by a number of scientific contributions | backgroundin cosmetic science , noninvasive sampling of the upper part of the stratum corneum is conveniently performed using strippings with adhesive - coated discs ( sacd ) and cyanoacrylate skin surface strippings ( cssss).methodsunder controlled conditions , it is possible to scrutinize sacd and csss with objectivity using appropriate methods of analytical morphology .
these procedures apply to a series of clinical conditions including xerosis grading , comedometry , corneodynamics , corneomelametry , corneosurfametry , corneoxenometry , and dandruff assessment.resultswith any of the analytical evaluations , sacd and csss provide specific salient information that is useful in the field of cosmetology . in particular , both methods appear valuable and complementary in assessing the human skin compatibility of personal skincare products.conclusiona set of quantitative analytical methods applicable to the minimally invasive and low - cost sacd and csss procedures allow for a sound assessment of cosmetic effects on the stratum corneum . under regular conditions ,
both methods are painless and do not induce adverse events .
globally , csss appears more precise and informative than the regular sacd stripping . | Introduction
SC collection
Overall aspect of normal CSSS
Analytical morphology of skin strippings
Grading and analytical morphology of xerosis
Comedometry
Corneomelametry
Corneodynamics
In vivo evaluation of the skin surface biocene
Corneosurfametry
Corneoxenometry
Regional SC reactivity to irritant xenobiotics
Reactive (sensitive) skin
Potency of squamolytic agents
Conclusion | there is a progressive reduction in intercorneocyte cohesion , ending with physiologically imperceptible desquamation.1 considering the typical continuous renewal of the epidermis , and the structure of the sc as its end product , the sc corresponds to a recollection of the past history of the recent life of the epidermis.1 alterations in the organization of corneocytes , the presence of parakeratosis , and various other aspects are therefore clues for the intervention of some previous physiopathological disturbances . the surface topography and the cell organizations
basically , sc stripping is the regular way for collecting the stratum disjunctum.2 using casual adhesive transparent tapes is poorly reliable for quantitative assessments because their adhesion to the sc is uncontrolled and appears variable among different brands . by contrast , sc stripping using adhesive - coated discs ( sacd ) is conveniently performed.3 an improved method using a liquid cyanoacrylate pressure - sensitive adhesive on a polyester slide was designed for getting a better sampling of the sc structure.410 such a procedure corresponds to the cyanoacrylate skin surface stripping ( csss ) , formerly called skin surface biopsy4 or follicular biopsy.11 sacd uses a crystal clear adhesive - coated disc ( d - squame ; cuderm corporation , dallas , tx , usa ; and corneofix ; ck electronic , cologne , germany ) . thus , it is possible to assess the overall texture of the superficial dermis on csss . as a result
, conditions such as dermal aging , dermal atrophy ( dermatoporosis ) , striae distensae , sclerosis , scars , and many other skin connective tissue alterations are conveniently observed noninvasively using csss.7,8,10 such morphological assessment of the skin microrelief is possibly quantified combining computerized image analysis and profilometry ( figure 1c ) . for improving their perceived aspect ,
a mixture of toluidine blue and basic fuchsin ( tbbf ) in 30% ethanol is a simple dye that is conveniently handled in an office setting.7,8,10 the regular sc exhibits a rather uniform cohesive pattern of adjacent corneocytes . skin strippings , including sacd and csss , are used as nearly noninvasive diagnostic methods and are useful in many skin conditions.1,5,7,10 in addition , they are examples of tools that combine analytical morphology and other procedures for the evaluation of the kinetics and efficacy of various cosmetics and toiletries . analytical morphology
these procedures include various techniques , and they afford quantitative and statistically evaluable data following image processing . the challenge of analytical evaluations of skin strippings is to quantify some specific aspects of the structure and functions of the sc.14,15 image analysis of the aspect of samples seen under the microscope , and optical profilometry of the surface of the samples are two classical methods of analytical morphology that are conveniently applied to skin strippings . in these respects , skin strippings
are used in four main ways :
evaluation of the sc structureevaluation of the sc dynamicsin situ evaluation of the superficial skin bioceneuse of csss as a substrate for a set of ex vivo bioassays evaluation of the sc structure evaluation of the sc dynamics in situ evaluation of the superficial skin biocene use of csss as a substrate for a set of ex vivo bioassays analytical morphology denotes a set of techniques that provide quantitative data and statistically evaluable information from morphological aspects . pressure and time of application of the clear disc in sacd , and the quality of the cyanoacrylate liquid bond in csss
they commonly rely on the combination of optical properties of the samples , reflectance colorimetry , and morphometry - based image analysis . because sacd and csss are very thin compared to the width of the sampling , both samplings are regarded as nearly two - dimensional structures . under microscopic examination ,
the size of the field of vision is determined by the combination of the magnification and the microscope eyepiece . usually , the eyepiece field diaphragm opening diameter determines the size of the field of vision . it is possible to measure the area of the exposed aspect of corneocytes , and to detect any presence of parakeratosis ( figure 2b ) and other cells on such specimens ( figure 2c ) . when the intracorneal cohesion fails to decline evenly in the stratum disjunctum , clumps of corneocytes remain stuck together , and they come off as scales . a semiquantitative grading of orthokeratotic xeroses is conveniently achieved on csss stained for 3 minutes with the tbbf staining solution.7,10,16 the samples
are graded7,10 as follows :
type 0 : normal sc without any evidence for hyperkeratosistype 1 : hyperkeratosis of the first- and second - order lines and/or the adnexal openingstype 2 : focal hyperkeratosis covering less than 30% of the skin surface plateaus ( figure 4a)type 3 : hyperkeratosis covering over 30% of the skin surface plateaus ( figure 4b)type 4 : diffuse , confluent , and homogeneous scales with persistence of the first - order linestype 5 : thick , uneven scales covering the entire sc surface , obliterating the shallow lines type 0 : normal sc without any evidence for hyperkeratosis type 1 : hyperkeratosis of the first- and second - order lines and/or the adnexal openings type 2 : focal hyperkeratosis covering less than 30% of the skin surface plateaus ( figure 4a ) type 3 : hyperkeratosis covering over 30% of the skin surface plateaus ( figure 4b ) type 4 : diffuse , confluent , and homogeneous scales with persistence of the first - order lines type 5 : thick , uneven scales covering the entire sc surface , obliterating the shallow lines quantitative assessment of xerosis relies on analytical evaluations of sacd.3,14 the attenuation of optical light transmission has been used to measure the amount of scales collected on the discs . in particular ,
the wrinkling process and its correction by some cosmetics is conveniently substantiated by combining image analysis and optical profilometry . the horny material sampled from the upper portion of the follicular ducts corresponds to a follicular stripping,11,19 and it reflects the balance between comedo formation and lysis ( figure 6a ) . by contrast , endogenous comedogenic factors ( androgens , acne , etc ) typically affect to a variable extent a minority of hair follicles.10 the sensitivity of the method is such that it is possible to disclose signs of microcomedolysis after a short time ( a couple of weeks ) of adequate treatment.23 sebum - sensitive foils ( ssf ) , available as sebutape ( cuderm corp ; dallas , tx , usa ) and sebufix ( ck electronic ) , are conveniently used for assessing the sebum output at the skin surface . it is possible to combine such method with csss.24 in a first step , a non - sticky ssf is applied to the skin for a few seconds . it is possible to assess the density of hair follicles over a defined surface area , as well as to observe the skin pores and the presence of follicular hyperkeratosis ( kerosis ) , comedones , trichostasis spinulosa , intrafollicular bacteria , and mites ( demodex folliculorum).7,2628 in some instances , other hair follicle structures including hair bulbs and follicular sheaths are visualized on csss . however , both the dc and dha tests are dramatically influenced by cleansing agents and skincare products.35 the extraction of the dyes from the sc by these products is tentatively used to predict irritancy potential.35 an inverse relationship has been suggested between the size of corneocytes and the speed of epidermal turnover . another facet of the sc dynamics is gained by squamometry , in particular uvl squamometry.3 for instance , the sqmi kinetics after acute photodamage show a complex response of the epidermis with early ( 48 hours ) and late ( 10 days ) changes.36 indeed , a single regular sacd sampling corresponds at the most to the regular corneocyte daily shedding . the biocene of resident bacteria and other saprophytic or pathogenic microorganisms is typically confined to the skin surface and the appendages.6,3739 at the surface of the sc , the flora remains largely encased inside the cyanoacrylate bond during csss sampling . thus , it is not accessible to staining procedures , and it remains invisible at microscopic examination . in particular , the microflora is accessible by scraping out the horny spiky structures appended to the csss . the efficacy of shampoos can be compared by combining squamometry and semiquantitative evaluations of the number of yeasts.41 the adhesion of malassezia yeasts to corneocytes appears to be a prominent feature.42 the presence of various tiny foreign bodies and microscopic parasites , and the impact of various chemical xenobiotics on the sc are conveniently assessed on csss.10,43 in particular , surfactants remove lipids , denature proteins , induce corneocyte swelling , alter the sc barrier function , and produce a feeling of skin roughness and dryness.44 the surest method for testing skin compatibility of surface - active agents was to use large panels of volunteers . among them , corneosurfametry ( csm ) refers specifically to the effects of surfactants and wash solutions on the sc.18,44,45 csm is a noninvasive quantitative test rating the interaction between surfactants and human or animal sc ( figure 10 ) . at completion of the predetermined assessment time ,
csss are thoroughly rinsed with tap water , dried , and stained for 3 minutes with the tbbf dye solution . thus , csmi is conveniently calculated as follows :
csmi=[(l*)2+(c*)2]0.5(2 ) there is a linear negative correlation between values of l * and chroma c * , and cim of each sample is calculated as follows :
cim = l*c*(3 ) according to such analytical assessments , mild skincare products containing anionic surfactants are characterized by the combination of a low csmi value and a high cim value . however , a low paradoxical csmi value is experienced with some aggressive products yielding to a cascade of corneocyte swelling , lysis , and loss from the stratum disjunctum . usually , interindividual variations are larger for sqmi than for cim.18 microwave csm is a more rapid procedure than the regular csm.46 in this procedure , csss are immersed in a flask containing the test surfactant solution , and they are placed in a microwave oven containing a 500 ml water load . at completion of the in vivo procedure , csss are harvested for a regular or microwave csm bioassay using the same surfactant as in the preliminary in vivo procedure . one main indication is found in the field of skin irritation while avoiding the risk of in vivo hazards . however , it is not the top ranked aggressor on the cxm bioassay.48 the intensity of the skin response to irritant xenobiotics is subject to anatomic site variations . optical profilometry performed on the same samples shows the immediate smoothing effect of the products with reduction in the rz value followed by desquamation and increased rz . when the intracorneal cohesion fails to decline evenly in the stratum disjunctum , clumps of corneocytes remain stuck together , and they come off as scales . a semiquantitative grading of orthokeratotic xeroses is conveniently achieved on csss stained for 3 minutes with the tbbf staining solution.7,10,16 the samples are graded7,10 as follows :
type 0 : normal sc without any evidence for hyperkeratosistype 1 : hyperkeratosis of the first- and second - order lines and/or the adnexal openingstype 2 : focal hyperkeratosis covering less than 30% of the skin surface plateaus ( figure 4a)type 3 : hyperkeratosis covering over 30% of the skin surface plateaus ( figure 4b)type 4 : diffuse , confluent , and homogeneous scales with persistence of the first - order linestype 5 : thick , uneven scales covering the entire sc surface , obliterating the shallow lines type 0 : normal sc without any evidence for hyperkeratosis type 1 : hyperkeratosis of the first- and second - order lines and/or the adnexal openings type 2 : focal hyperkeratosis covering less than 30% of the skin surface plateaus ( figure 4a ) type 3 : hyperkeratosis covering over 30% of the skin surface plateaus ( figure 4b ) type 4 : diffuse , confluent , and homogeneous scales with persistence of the first - order lines type 5 : thick , uneven scales covering the entire sc surface , obliterating the shallow lines quantitative assessment of xerosis relies on analytical evaluations of sacd.3,14 the attenuation of optical light transmission has been used to measure the amount of scales collected on the discs . in particular ,
the wrinkling process and its correction by some cosmetics is conveniently substantiated by combining image analysis and optical profilometry . the horny material sampled from the upper portion of the follicular ducts corresponds to a follicular stripping,11,19 and it reflects the balance between comedo formation and lysis ( figure 6a ) . by contrast , endogenous comedogenic factors ( androgens , acne , etc ) typically affect to a variable extent a minority of hair follicles.10 the sensitivity of the method is such that it is possible to disclose signs of microcomedolysis after a short time ( a couple of weeks ) of adequate treatment.23 sebum - sensitive foils ( ssf ) , available as sebutape ( cuderm corp ; dallas , tx , usa ) and sebufix ( ck electronic ) , are conveniently used for assessing the sebum output at the skin surface . it is possible to combine such method with csss.24 in a first step , a non - sticky ssf is applied to the skin for a few seconds . it is possible to assess the density of hair follicles over a defined surface area , as well as to observe the skin pores and the presence of follicular hyperkeratosis ( kerosis ) , comedones , trichostasis spinulosa , intrafollicular bacteria , and mites ( demodex folliculorum).7,2628 in some instances , other hair follicle structures including hair bulbs and follicular sheaths are visualized on csss . however , both the dc and dha tests are dramatically influenced by cleansing agents and skincare products.35 the extraction of the dyes from the sc by these products is tentatively used to predict irritancy potential.35 an inverse relationship has been suggested between the size of corneocytes and the speed of epidermal turnover . another facet of the sc dynamics is gained by squamometry , in particular uvl squamometry.3 for instance , the sqmi kinetics after acute photodamage show a complex response of the epidermis with early ( 48 hours ) and late ( 10 days ) changes.36 indeed , a single regular sacd sampling corresponds at the most to the regular corneocyte daily shedding . the biocene of resident bacteria and other saprophytic or pathogenic microorganisms is typically confined to the skin surface and the appendages.6,3739 at the surface of the sc , the flora remains largely encased inside the cyanoacrylate bond during csss sampling . thus , it is not accessible to staining procedures , and it remains invisible at microscopic examination . in particular , the microflora is accessible by scraping out the horny spiky structures appended to the csss . the efficacy of shampoos can be compared by combining squamometry and semiquantitative evaluations of the number of yeasts.41 the adhesion of malassezia yeasts to corneocytes appears to be a prominent feature.42
the presence of various tiny foreign bodies and microscopic parasites , and the impact of various chemical xenobiotics on the sc are conveniently assessed on csss.10,43 in particular , surfactants remove lipids , denature proteins , induce corneocyte swelling , alter the sc barrier function , and produce a feeling of skin roughness and dryness.44 the surest method for testing skin compatibility of surface - active agents was to use large panels of volunteers . among them , corneosurfametry ( csm ) refers specifically to the effects of surfactants and wash solutions on the sc.18,44,45 csm is a noninvasive quantitative test rating the interaction between surfactants and human or animal sc ( figure 10 ) . at completion of the predetermined assessment time ,
csss are thoroughly rinsed with tap water , dried , and stained for 3 minutes with the tbbf dye solution . thus , csmi is conveniently calculated as follows :
csmi=[(l*)2+(c*)2]0.5(2 ) there is a linear negative correlation between values of l * and chroma c * , and cim of each sample is calculated as follows :
cim = l*c*(3 ) according to such analytical assessments , mild skincare products containing anionic surfactants are characterized by the combination of a low csmi value and a high cim value . however , a low paradoxical csmi value is experienced with some aggressive products yielding to a cascade of corneocyte swelling , lysis , and loss from the stratum disjunctum . usually , interindividual variations are larger for sqmi than for cim.18 microwave csm is a more rapid procedure than the regular csm.46 in this procedure , csss are immersed in a flask containing the test surfactant solution , and they are placed in a microwave oven containing a 500 ml water load . at completion of the in vivo procedure , csss are harvested for a regular or microwave csm bioassay using the same surfactant as in the preliminary in vivo procedure . one main indication is found in the field of skin irritation while avoiding the risk of in vivo hazards . however , it is not the top ranked aggressor on the cxm bioassay.48 the intensity of the skin response to irritant xenobiotics is subject to anatomic site variations . optical profilometry performed on the same samples shows the immediate smoothing effect of the products with reduction in the rz value followed by desquamation and increased rz . the emergence of new analytical methods increases the validity of the information yielded by sacd and csss . when testing xenobiotic effects on sc , the stratum disjunctum response as assessed by sqmi on sacd is different from csmi on the thicker sc collected by csss . nevertheless , both methods appear valuable and complementary . beyond sc scrapings , sacd and csss
these simple , low - cost , and minimally invasive methods allow the clinician , the cosmetologist , and the experimentalist to avoid invasive procedures within limits of well - defined indications . in many instances
, csss appears to represent a convenient way that is currently available for exploring many facets of cosmetic science . | [
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it has been shown that interictal epileptiform discharges ( ieds ) observed from eeg constitute useful markers of epileptogenesis in patients with focal epilepsy .
the regions inside the brain from which these ieds originate , named irritative zones , can in practice be localized based on eeg recordings .
preclinical models are essential to find correlates between these irritative zones and the actual regions underlying seizure initiation . however ,
recording eeg from small animals is challenging because of the small surface area of the head compared to the human scalp .
although invasive methods for chronic recording in rats can be used , techniques are not available at this moment to acquire traditional eeg recordings on rodents under acute conditions without the need of anesthesia . to solve this problem ,
we apply a patented eeg mini - cap , which allows us to record 32-channel eeg data from rodents noninvasively . in this study
, we also provide evidence about the need of an analgesic to preserve ied frequency . therefore , although fixation of eeg mini - cap was performed under isoflurane , eeg recordings were obtained with rats only under sedation ( dexdomitor ) .
the method proposed in this study can be used in any preclinical rat model of focal epilepsy . to illustrate the capabilities of this methodology
, we apply it to understand the correlates between irritative and seizure - onset zones in focal cortical dysplasia ( fcd ) . to that end
double - hit model of fcd in wistar rats . to perform brain source analysis
, it is required to : a ) accurately extract ieds from eeg raw data and b ) obtain a volume conductor model for the individual animal head . to generate a practical volume conductor model , we use an in vivo rat mri atlas , comprising average images of intensity / shape and obtained via non - linear registration of t2 images of 31 wistar rats .
the forward model for the generated volume conductor was computed by boundary element method ( bem ) . as in the case of humans ,
two typical patterns of ieds ( sharp - waves and spikes ) were detected and sub - classified into different clusters through an intelligent feature extraction , feature selection , and classification process .
these sub - classified signals are used to estimate the brain source localizations associated with different types of irritative zones .
we present the source analysis steps using a well - known public software called brainstorm .
the eeg source localizations for each ied sub - type and the seizure onset time frames were performed using standardized low - resolution brain electromagnetic tomography ( sloreta ) , which is available in brainstorm .
ethics statement : all experiments are performed following the policies established by the institutional animal care and use committee ( iacuc ) at florida international university ( iacuc 13 - 004 ) . preparation of the eeg mini - cap immerse the electrode tips of the eeg mini - cap at least 12 hrs in distilled water with 0.2% chloride . rinse the eeg mini - cap gently in distilled water .
dry the cap and the electrodes in the air.mix eeg electrode paste with 0.9% nacl solution in the volume proportion of 2 : 1 .
add a drop of methylene blue , which will help to visualize the electrode paste inside the electrodes and on the skin .
inject the gel into each of the 32 electrodes , filling them without introducing any air bubbles .
this provides better access to each electrode and reduces the possibility of the gel spilling over.turn on eeg and physiological recording system , and open the corresponding recording software on the computer in use .
immerse the electrode tips of the eeg mini - cap at least 12 hrs in distilled water with 0.2% chloride .
mix eeg electrode paste with 0.9% nacl solution in the volume proportion of 2 : 1 .
add a drop of methylene blue , which will help to visualize the electrode paste inside the electrodes and on the skin .
inject the gel into each of the 32 electrodes , filling them without introducing any air bubbles .
this provides better access to each electrode and reduces the possibility of the gel spilling over .
turn on eeg and physiological recording system , and open the corresponding recording software on the computer in use .
animal preparation and anesthesia note : chronic epilepsy was created using a protocol for fcd in wistar rats .
the eeg recording were conducted in adult wistar rats ( 8 weeks old , 300 - 400 g ) .
record the rat s weight in an experiment sheet . use this information to calculate sedative dose ( dexdomitor 0.25 mg / kg ) .
induce anesthesia in the rat with 5% isoflurane and 100% oxygen ( 1 l / min at 14.7 psi).after trim the rat s head , reduce isoflurane to 2% and maintain it during the entire setting of the eeg mini - cap .
place the rat on a heating pad in the stereotaxic apparatus by fixing the ear canals using ear bars .
make sure anesthesia nose cone is secure.apply lubricant ophthalmic ointment to each eye.shave the extra hair on the rat head and ears using a razor .
note : any hair left on the skin will produce noise in the eeg recordings . rub the rat s skin with 90% isopropyl alcohol to stimulate the blood vessels and degrease the skin.place a saline swab on the scalp and cover it completely to keep good skin conductance until the eeg mini - cap is ready to be placed.connect temperature , respiration , and three lead electrocardiogram probes .
ensure that the normal temperature is 37 c , respiration range is 30 - 60 breaths per minute , and heart rate is around 350 - 450 beats per minute .
induce anesthesia in the rat with 5% isoflurane and 100% oxygen ( 1 l / min at 14.7 psi ) .
after trim the rat s head , reduce isoflurane to 2% and maintain it during the entire setting of the eeg mini - cap . check rat reflexes
place the rat on a heating pad in the stereotaxic apparatus by fixing the ear canals using ear bars .
note : any hair left on the skin will produce noise in the eeg recordings . rub the rat s skin with 90% isopropyl alcohol to stimulate the blood vessels and degrease the skin .
place a saline swab on the scalp and cover it completely to keep good skin conductance until the eeg mini - cap is ready to be placed .
ensure that the normal temperature is 37 c , respiration range is 30 - 60 breaths per minute , and heart rate is around 350 - 450 beats per minute .
recording procedures remove the saline swab on the rat s scalp and place the prepared eeg mini - cap on its skin .
place one rubber band on the front side of the scalp , usually in front of the eyes , and another band at the back of the scalp between the ears and neck .
use a plastic protector under the neck to facilitate normal respiration.put a layer of high conductance electrode paste on both the ground and reference electrodes . place them on the respective ear .
note : the reference electrode can be possibly placed in other locations.connect the eeg mini - cap to the amplifiers and observe a preview of the workbench for electrode impedance .
ensure that the impedance value is in the range of 5 - 30 k. if there are any noisy electrodes , provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode.administer dexdomitor ( 0.25 mg / kg ) intraperitoneally and immediately reduce isoflurane rate to 0% . if the respiration rate is not within 30 - 60 breaths per minute range , start increasing the isoflurane rate gently .
monitor this step carefully because the mixture of isoflurane and dexdomitor could prompt the animals to a critical condition .
note : on the preclinical model of focal epilepsy , isoflurane affects ieds , whereas dexdomitor does not .
subjects under isoflurane have weaker epileptogenic property , i.e. , relatively fewer ieds can be detected compared to other conditions .
thus , to save the time for its effect , the preparation was carried out under isoflurane.conduct eeg recordings .
after the recording , mark the positions of the three jutting circles of the eeg mini - cap on top of the skin by inserting a color pen inside them before the eeg mini - cap is removed .
use them as landmarks for mri co - registration . take a picture of the rat head with the landmarks .
place the rat back inside the cage and monitor it until complete recovery from dexdomitor s effect . note : in this experiment , red color ( opponent color to green ) was used to distinguish from the electrode positions ( green ) .
however , it is recommended to use other colors ( purple / green ) if small bleeding spots are observed in the skin .
remove the saline swab on the rat s scalp and place the prepared eeg mini - cap on its skin .
place one rubber band on the front side of the scalp , usually in front of the eyes , and another band at the back of the scalp between the ears and neck .
put a layer of high conductance electrode paste on both the ground and reference electrodes .
note : the reference electrode can be possibly placed in other locations . connect the eeg mini - cap to the amplifiers and observe a preview of the workbench for electrode impedance .
ensure that the impedance value is in the range of 5 - 30 k. if there are any noisy electrodes , provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode .
administer dexdomitor ( 0.25 mg / kg ) intraperitoneally and immediately reduce isoflurane rate to 0% .
if the respiration rate is not within 30 - 60 breaths per minute range , start increasing the isoflurane rate gently .
monitor this step carefully because the mixture of isoflurane and dexdomitor could prompt the animals to a critical condition .
note : on the preclinical model of focal epilepsy , isoflurane affects ieds , whereas dexdomitor does not .
subjects under isoflurane have weaker epileptogenic property , i.e. , relatively fewer ieds can be detected compared to other conditions .
thus , to save the time for its effect , the preparation was carried out under isoflurane .
after the recording , mark the positions of the three jutting circles of the eeg mini - cap on top of the skin by inserting a color pen inside them before the eeg mini - cap is removed . use them as landmarks for mri co - registration .
place the rat back inside the cage and monitor it until complete recovery from dexdomitor s effect .
note : in this experiment , red color ( opponent color to green ) was used to distinguish from the electrode positions ( green ) . however , it is recommended to use other colors ( purple / green ) if small bleeding spots are observed in the skin .
ied classification note : the ied detection and classification is performed using self - developed codes in matlab based on the previous study .
remove ekg artifacts using an automatic method for periodic waveform subtraction , which is based on a template and a correlation analysis .
note : usually , the experimenter who recorded the eeg shares the written experimental sheet for the observed bad channel information based on the impedance values .
apply a band - pass filter with cutoff frequencies of 3 - 150 hz and a notch filter to remove the line frequency ( 60 hz in general and 50 hz in some countries ) component offline.detect two types of ieds ( spikes and sharp - waves ) .
spikes and sharp - waves constitute large electrical events of 20 - 70 ms and 70 - 200 ms in duration respectively .
therefore , after applying a respective band - pass filter ( cutoff frequencies of 15 - 50 hz for spikes and 5 - 15 hz for sharp - waves ) , the ieds are detected based on amplitude thresholds .
note : the thresholds are automatically set to 4 as suggested in the previous study for multiunit activity .
here , is an estimated standard deviation of the bandpass - filtered signal , = median{|filtered signal| 0.6745}.sub - classify spikes and sharp - waves into different clusters .
the distinctive features of different spikes and sharp - waves are extracted using wavelet transform .
they are sub - classified into multiple clusters using k - means , and the optimal cluster number k is determined using silhouette.average the sub - classified signals within the same cluster .
the average eeg signals for each ied sub - type will be used for brain source analysis .
remove ekg artifacts using an automatic method for periodic waveform subtraction , which is based on a template and a correlation analysis .
note : usually , the experimenter who recorded the eeg shares the written experimental sheet for the observed bad channel information based on the impedance values .
apply a band - pass filter with cutoff frequencies of 3 - 150 hz and a notch filter to remove the line frequency ( 60 hz in general and 50 hz in some countries ) component offline .
spikes and sharp - waves constitute large electrical events of 20 - 70 ms and 70 - 200 ms in duration respectively .
therefore , after applying a respective band - pass filter ( cutoff frequencies of 15 - 50 hz for spikes and 5 - 15 hz for sharp - waves ) , the ieds are detected based on amplitude thresholds .
note : the thresholds are automatically set to 4 as suggested in the previous study for multiunit activity .
here , is an estimated standard deviation of the bandpass - filtered signal , = median{|filtered signal| 0.6745}. sub - classify spikes and sharp - waves into different clusters .
the distinctive features of different spikes and sharp - waves are extracted using wavelet transform .
they are sub - classified into multiple clusters using k - means , and the optimal cluster number k is determined using silhouette .
the average eeg signals for each ied sub - type will be used for brain source analysis .
for the following sections , the open source software , brainstorm , will be used with the mri atlas for wistar rats . however
, individual rat s mri can also be used to generate the volume conductor model if available .
the mri atlas can be downloaded at http://www.idac.tohoku.ac.jp / bir / en/. this website provides the atlas as nifti format under
please click here to view a larger version of this figure.generate head surface with default setting .
please click here to view a larger version of this figure.generate scalp and inner / outer skull surfaces based on mri for lead field computation .
note : the resolution of the vertices influences the accuracy of the estimated source , but large number of vertices results in high computational complexity . recommended number of vertices of each layer is 642 for acceptable accuracy with fair computational complexity .
the thickness of the skull can be checked from the mri , and in the case of the mri atlas , it is approximately 1 mm . after inserting above values in the software , corresponding triangle
please click here to view a larger version of this figure.check the orientation and location of each surface with respect to the mri using the visualization option .
please click here to view a larger version of this figure.using the rat head picture acquired in 1.3.5 .
co - register the positions of the 3 landmarks ( r1 , r2 , and r3 ) in the mri .
use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold ( figure 3b ) .
( a ) rat head picture used to obtain electrode positions and ( b ) the eeg mini - cap diagram with the coordinate system .
red dots in ( a ) indicate the landmarks mentioned in 1.3.5 . which correspond to the red numbers in ( b ) .
also , the green marks in ( a ) depict the 32 electrode positions , and they correspond to the blue numbers in ( b ) .
generate n 3 electrode position matrix based on the 3 landmarks . here , n is the number of channels ( n = 32 ) and the column represents the corresponding x , y , and z coordinate values . note : the eeg mini - cap is a rigid scaffold .
therefore , once the 3 reference grids ( r1 , r2 , and r3 ) are obtained , the position of the electrodes are automatically set .
the user will only need to redefine the z - values on a way that the mini - cap is appropriately projected on the scalp .
the n point grids can be numbered sequentially as depicted in figure 3b blue numbers .
the standard scaffold for the eeg mini - cap is commercially available ( table of materials ) .
the software for co - registration is also available to the community.input the generated channel file .
please click here to view a larger version of this figure.display and confirm the location of all electrodes .
modify any misplaced electrodes . the final coordinate system for the electrode positions should coincide with the coordinate system used for the above - mentioned surfaces .
note : the created surfaces can be visually inspected on an mri by using the visualization option , mri registration\check mri / surface registration .
and then , a selected surface will be displayed as yellow line on the mri .
in addition , the 3 landmarks and the 32 electrode positions can be displayed on the mri by selecting the toolbox s option ,
the locations can be visually inspected by comparing the distributions based on the rat s eye and ear locations ( figure 4 ) .
( a ) mri atlas with co - registered brain surface ( yellow line ) , ( b ) the created volume conductor model with the aligned 32 electrodes and 3 landmarks ( red dots ) , and ( c ) mri atlas with co - registered reference grid r1 .
generate scalp and inner / outer skull surfaces based on mri for lead field computation .
note : the resolution of the vertices influences the accuracy of the estimated source , but large number of vertices results in high computational complexity . recommended
number of vertices of each layer is 642 for acceptable accuracy with fair computational complexity .
the thickness of the skull can be checked from the mri , and in the case of the mri atlas , it is approximately 1 mm . after inserting above values in the software , corresponding triangle
check the orientation and location of each surface with respect to the mri using the visualization option .
please click here to view a larger version of this figure . using the rat head picture acquired in 1.3.5
co - register the positions of the 3 landmarks ( r1 , r2 , and r3 ) in the mri .
use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold ( figure 3b ) .
( a ) rat head picture used to obtain electrode positions and ( b ) the eeg mini - cap diagram with the coordinate system .
red dots in ( a ) indicate the landmarks mentioned in 1.3.5 . which correspond to the red numbers in ( b ) .
also , the green marks in ( a ) depict the 32 electrode positions , and they correspond to the blue numbers in ( b ) .
generate n 3 electrode position matrix based on the 3 landmarks . here , n is the number of channels ( n = 32 ) and the column represents the corresponding x , y , and z coordinate values . note : the eeg mini - cap is a rigid scaffold
. therefore , once the 3 reference grids ( r1 , r2 , and r3 ) are obtained , the position of the electrodes are automatically set .
the user will only need to redefine the z - values on a way that the mini - cap is appropriately projected on the scalp .
the n point grids can be numbered sequentially as depicted in figure 3b blue numbers .
the standard scaffold for the eeg mini - cap is commercially available ( table of materials ) .
display and confirm the location of all electrodes . modify any misplaced electrodes . the final coordinate system for the electrode positions should coincide with the coordinate system used for the above - mentioned surfaces .
note : the created surfaces can be visually inspected on an mri by using the visualization option , mri registration\check mri / surface registration .
and then , a selected surface will be displayed as yellow line on the mri .
in addition , the 3 landmarks and the 32 electrode positions can be displayed on the mri by selecting the toolbox s option ,
can be visually inspected by comparing the distributions based on the rat s eye and ear locations ( figure 4 ) .
( a ) mri atlas with co - registered brain surface ( yellow line ) , ( b ) the created volume conductor model with the aligned 32 electrodes and 3 landmarks ( red dots ) , and ( c ) mri atlas with co - registered reference grid r1 .
input the conductivity values which satisfy the ratio of skin , skull , and brain as 1 : 1/80 : 1 .
obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2 .
.
please click here to view a larger version of this figure.input the average eeg signals for each ied sub - type stored in 2.1.4 .
please click here to view a larger version of this figure.obtain sloreta solution based on the computed lead field matrix and the input eeg signals . by selecting the source estimation method option , the inverse solution can be obtained .
please click here to view a larger version of this figure.plot the estimated sources .
input the conductivity values which satisfy the ratio of skin , skull , and brain as 1 : 1/80 : 1 .
obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2 .
input the average eeg signals for each ied sub - type stored in 2.1.4 .
obtain sloreta solution based on the computed lead field matrix and the input eeg signals . by selecting the source estimation method option , the inverse solution can be obtained .
please click here to view a larger version of this figure . plot the estimated sources .
preparation of the eeg mini - cap immerse the electrode tips of the eeg mini - cap at least 12 hrs in distilled water with 0.2% chloride .
dry the cap and the electrodes in the air.mix eeg electrode paste with 0.9% nacl solution in the volume proportion of 2 : 1 . add a drop of methylene blue , which will help to visualize the electrode paste inside the electrodes and on the skin .
inject the gel into each of the 32 electrodes , filling them without introducing any air bubbles .
this provides better access to each electrode and reduces the possibility of the gel spilling over.turn on eeg and physiological recording system , and open the corresponding recording software on the computer in use .
immerse the electrode tips of the eeg mini - cap at least 12 hrs in distilled water with 0.2% chloride .
mix eeg electrode paste with 0.9% nacl solution in the volume proportion of 2 : 1 .
add a drop of methylene blue , which will help to visualize the electrode paste inside the electrodes and on the skin .
inject the gel into each of the 32 electrodes , filling them without introducing any air bubbles .
this provides better access to each electrode and reduces the possibility of the gel spilling over .
turn on eeg and physiological recording system , and open the corresponding recording software on the computer in use .
animal preparation and anesthesia note : chronic epilepsy was created using a protocol for fcd in wistar rats .
the eeg recording were conducted in adult wistar rats ( 8 weeks old , 300 - 400 g ) .
record the rat s weight in an experiment sheet . use this information to calculate sedative dose ( dexdomitor 0.25 mg / kg ) .
induce anesthesia in the rat with 5% isoflurane and 100% oxygen ( 1 l / min at 14.7 psi).after trim the rat s head , reduce isoflurane to 2% and maintain it during the entire setting of the eeg mini - cap .
place the rat on a heating pad in the stereotaxic apparatus by fixing the ear canals using ear bars .
make sure anesthesia nose cone is secure.apply lubricant ophthalmic ointment to each eye.shave the extra hair on the rat head and ears using a razor .
note : any hair left on the skin will produce noise in the eeg recordings . rub the rat s skin with 90% isopropyl alcohol to stimulate the blood vessels and degrease the skin.place a saline swab on the scalp and cover it completely to keep good skin conductance until the eeg mini - cap is ready to be placed.connect temperature , respiration , and three lead electrocardiogram probes .
ensure that the normal temperature is 37 c , respiration range is 30 - 60 breaths per minute , and heart rate is around 350 - 450 beats per minute .
induce anesthesia in the rat with 5% isoflurane and 100% oxygen ( 1 l / min at 14.7 psi ) .
after trim the rat s head , reduce isoflurane to 2% and maintain it during the entire setting of the eeg mini - cap . check rat reflexes are absent ( toe - pinch ) .
place the rat on a heating pad in the stereotaxic apparatus by fixing the ear canals using ear bars .
note : any hair left on the skin will produce noise in the eeg recordings . rub the rat s skin with 90% isopropyl alcohol to stimulate the blood vessels and degrease the skin .
place a saline swab on the scalp and cover it completely to keep good skin conductance until the eeg mini - cap is ready to be placed .
ensure that the normal temperature is 37 c , respiration range is 30 - 60 breaths per minute , and heart rate is around 350 - 450 beats per minute .
recording procedures remove the saline swab on the rat s scalp and place the prepared eeg mini - cap on its skin .
place one rubber band on the front side of the scalp , usually in front of the eyes , and another band at the back of the scalp between the ears and neck .
use a plastic protector under the neck to facilitate normal respiration.put a layer of high conductance electrode paste on both the ground and reference electrodes . place them on the respective ear .
note : the reference electrode can be possibly placed in other locations.connect the eeg mini - cap to the amplifiers and observe a preview of the workbench for electrode impedance . check the performance of all electrodes . for a high - quality recording ,
ensure that the impedance value is in the range of 5 - 30 k. if there are any noisy electrodes , provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode.administer dexdomitor ( 0.25 mg / kg ) intraperitoneally and immediately reduce isoflurane rate to 0% .
if the respiration rate is not within 30 - 60 breaths per minute range , start increasing the isoflurane rate gently .
monitor this step carefully because the mixture of isoflurane and dexdomitor could prompt the animals to a critical condition .
note : on the preclinical model of focal epilepsy , isoflurane affects ieds , whereas dexdomitor does not .
subjects under isoflurane have weaker epileptogenic property , i.e. , relatively fewer ieds can be detected compared to other conditions .
thus , to save the time for its effect , the preparation was carried out under isoflurane.conduct eeg recordings .
after the recording , mark the positions of the three jutting circles of the eeg mini - cap on top of the skin by inserting a color pen inside them before the eeg mini - cap is removed . use them as landmarks for mri co - registration .
place the rat back inside the cage and monitor it until complete recovery from dexdomitor s effect . note : in this experiment , red color ( opponent color to green ) was used to distinguish from the electrode positions ( green ) .
however , it is recommended to use other colors ( purple / green ) if small bleeding spots are observed in the skin .
remove the saline swab on the rat s scalp and place the prepared eeg mini - cap on its skin .
place one rubber band on the front side of the scalp , usually in front of the eyes , and another band at the back of the scalp between the ears and neck .
put a layer of high conductance electrode paste on both the ground and reference electrodes .
note : the reference electrode can be possibly placed in other locations . connect the eeg mini - cap to the amplifiers and observe a preview of the workbench for electrode impedance .
ensure that the impedance value is in the range of 5 - 30 k. if there are any noisy electrodes , provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode .
administer dexdomitor ( 0.25 mg / kg ) intraperitoneally and immediately reduce isoflurane rate to 0% . if the respiration rate is not within 30 - 60 breaths per minute range , start increasing the isoflurane rate gently
monitor this step carefully because the mixture of isoflurane and dexdomitor could prompt the animals to a critical condition .
note : on the preclinical model of focal epilepsy , isoflurane affects ieds , whereas dexdomitor does not .
subjects under isoflurane have weaker epileptogenic property , i.e. , relatively fewer ieds can be detected compared to other conditions .
thus , to save the time for its effect , the preparation was carried out under isoflurane .
after the recording , mark the positions of the three jutting circles of the eeg mini - cap on top of the skin by inserting a color pen inside them before the eeg mini - cap is removed . use them as landmarks for mri co - registration . take a picture of the rat head with the landmarks .
place the rat back inside the cage and monitor it until complete recovery from dexdomitor s effect .
note : in this experiment , red color ( opponent color to green ) was used to distinguish from the electrode positions ( green ) .
however , it is recommended to use other colors ( purple / green ) if small bleeding spots are observed in the skin .
figure 1 . a picture of the eeg mini - cap placed on a particular rat .
ied classification note : the ied detection and classification is performed using self - developed codes in matlab based on the previous study .
remove ekg artifacts using an automatic method for periodic waveform subtraction , which is based on a template and a correlation analysis .
note : usually , the experimenter who recorded the eeg shares the written experimental sheet for the observed bad channel information based on the impedance values .
apply a band - pass filter with cutoff frequencies of 3 - 150 hz and a notch filter to remove the line frequency ( 60 hz in general and 50 hz in some countries ) component offline.detect two types of ieds ( spikes and sharp - waves ) . spikes and sharp - waves constitute large electrical events of 20 - 70 ms and 70 - 200 ms in duration respectively .
therefore , after applying a respective band - pass filter ( cutoff frequencies of 15 - 50 hz for spikes and 5 - 15 hz for sharp - waves ) , the ieds are detected based on amplitude thresholds .
note : the thresholds are automatically set to 4 as suggested in the previous study for multiunit activity .
here , is an estimated standard deviation of the bandpass - filtered signal , = median{|filtered signal| 0.6745}.sub - classify spikes and sharp - waves into different clusters .
the distinctive features of different spikes and sharp - waves are extracted using wavelet transform .
they are sub - classified into multiple clusters using k - means , and the optimal cluster number k is determined using silhouette.average the sub - classified signals within the same cluster .
the average eeg signals for each ied sub - type will be used for brain source analysis .
remove ekg artifacts using an automatic method for periodic waveform subtraction , which is based on a template and a correlation analysis .
note : usually , the experimenter who recorded the eeg shares the written experimental sheet for the observed bad channel information based on the impedance values .
apply a band - pass filter with cutoff frequencies of 3 - 150 hz and a notch filter to remove the line frequency ( 60 hz in general and 50 hz in some countries ) component offline .
spikes and sharp - waves constitute large electrical events of 20 - 70 ms and 70 - 200 ms in duration respectively .
therefore , after applying a respective band - pass filter ( cutoff frequencies of 15 - 50 hz for spikes and 5 - 15 hz for sharp - waves ) , the ieds are detected based on amplitude thresholds .
note : the thresholds are automatically set to 4 as suggested in the previous study for multiunit activity .
here , is an estimated standard deviation of the bandpass - filtered signal , = median{|filtered signal| 0.6745}. sub - classify spikes and sharp - waves into different clusters .
the distinctive features of different spikes and sharp - waves are extracted using wavelet transform .
they are sub - classified into multiple clusters using k - means , and the optimal cluster number k is determined using silhouette .
the average eeg signals for each ied sub - type will be used for brain source analysis . volume conductor model note : for the following sections , the open source software , brainstorm , will be used with the mri atlas for wistar rats . however
, individual rat s mri can also be used to generate the volume conductor model if available .
the mri atlas can be downloaded at http://www.idac.tohoku.ac.jp / bir / en/. this website provides the atlas as nifti format under
please click here to view a larger version of this figure.generate head surface with default setting .
please click here to view a larger version of this figure.generate scalp and inner / outer skull surfaces based on mri for lead field computation .
note : the resolution of the vertices influences the accuracy of the estimated source , but large number of vertices results in high computational complexity . recommended number of vertices of each layer is 642 for acceptable accuracy with fair computational complexity .
the thickness of the skull can be checked from the mri , and in the case of the mri atlas , it is approximately 1 mm . after inserting above values in the software , corresponding triangle
please click here to view a larger version of this figure.check the orientation and location of each surface with respect to the mri using the visualization option .
please click here to view a larger version of this figure.using the rat head picture acquired in 1.3.5 .
co - register the positions of the 3 landmarks ( r1 , r2 , and r3 ) in the mri .
use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold ( figure 3b ) .
( a ) rat head picture used to obtain electrode positions and ( b ) the eeg mini - cap diagram with the coordinate system .
red dots in ( a ) indicate the landmarks mentioned in 1.3.5 . which correspond to the red numbers in ( b ) .
also , the green marks in ( a ) depict the 32 electrode positions , and they correspond to the blue numbers in ( b ) .
generate n 3 electrode position matrix based on the 3 landmarks . here , n is the number of channels ( n = 32 ) and the column represents the corresponding x , y , and z coordinate values . note : the eeg mini - cap is a rigid scaffold .
therefore , once the 3 reference grids ( r1 , r2 , and r3 ) are obtained , the position of the electrodes are automatically set .
the user will only need to redefine the z - values on a way that the mini - cap is appropriately projected on the scalp .
the n point grids can be numbered sequentially as depicted in figure 3b blue numbers .
the standard scaffold for the eeg mini - cap is commercially available ( table of materials ) .
the software for co - registration is also available to the community.input the generated channel file .
please click here to view a larger version of this figure.display and confirm the location of all electrodes .
the final coordinate system for the electrode positions should coincide with the coordinate system used for the above - mentioned surfaces . note : the created surfaces can be visually inspected on an mri by using the visualization option , mri registration\check mri / surface registration . and then , a selected surface will be displayed as yellow line on the mri .
in addition , the 3 landmarks and the 32 electrode positions can be displayed on the mri by selecting the toolbox s option ,
the locations can be visually inspected by comparing the distributions based on the rat s eye and ear locations ( figure 4 ) .
( a ) mri atlas with co - registered brain surface ( yellow line ) , ( b ) the created volume conductor model with the aligned 32 electrodes and 3 landmarks ( red dots ) , and ( c ) mri atlas with co - registered reference grid r1 .
generate scalp and inner / outer skull surfaces based on mri for lead field computation .
note : the resolution of the vertices influences the accuracy of the estimated source , but large number of vertices results in high computational complexity . recommended number of vertices of each layer is 642 for acceptable accuracy with fair computational complexity .
the thickness of the skull can be checked from the mri , and in the case of the mri atlas , it is approximately 1 mm . after inserting above values in the software , corresponding triangle
check the orientation and location of each surface with respect to the mri using the visualization option .
please click here to view a larger version of this figure . using the rat head picture acquired in 1.3.5 .
co - register the positions of the 3 landmarks ( r1 , r2 , and r3 ) in the mri .
use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold ( figure 3b ) .
( a ) rat head picture used to obtain electrode positions and ( b ) the eeg mini - cap diagram with the coordinate system .
red dots in ( a ) indicate the landmarks mentioned in 1.3.5 . which correspond to the red numbers in ( b ) .
also , the green marks in ( a ) depict the 32 electrode positions , and they correspond to the blue numbers in ( b ) .
generate n 3 electrode position matrix based on the 3 landmarks . here , n is the number of channels ( n = 32 ) and the column represents the corresponding x , y , and z coordinate values . note : the eeg mini - cap is a rigid scaffold .
therefore , once the 3 reference grids ( r1 , r2 , and r3 ) are obtained , the position of the electrodes are automatically set .
the user will only need to redefine the z - values on a way that the mini - cap is appropriately projected on the scalp .
the n point grids can be numbered sequentially as depicted in figure 3b blue numbers .
the standard scaffold for the eeg mini - cap is commercially available ( table of materials ) .
display and confirm the location of all electrodes . modify any misplaced electrodes . the final coordinate system for the electrode positions should coincide with the coordinate system used for the above - mentioned surfaces .
note : the created surfaces can be visually inspected on an mri by using the visualization option , mri registration\check mri / surface registration .
and then , a selected surface will be displayed as yellow line on the mri .
in addition , the 3 landmarks and the 32 electrode positions can be displayed on the mri by selecting the toolbox s option ,
the locations can be visually inspected by comparing the distributions based on the rat s eye and ear locations ( figure 4 ) .
( a ) mri atlas with co - registered brain surface ( yellow line ) , ( b ) the created volume conductor model with the aligned 32 electrodes and 3 landmarks ( red dots ) , and ( c ) mri atlas with co - registered reference grid r1 .
input the conductivity values which satisfy the ratio of skin , skull , and brain as 1 : 1/80 : 1 .
obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2 .
please click here to view a larger version of this figure.input the average eeg signals for each ied sub - type stored in 2.1.4 .
please click here to view a larger version of this figure.obtain sloreta solution based on the computed lead field matrix and the input eeg signals . by selecting the source estimation method option , the inverse solution can be obtained .
please click here to view a larger version of this figure.plot the estimated sources .
input the conductivity values which satisfy the ratio of skin , skull , and brain as 1 : 1/80 : 1 .
obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2 .
input the average eeg signals for each ied sub - type stored in 2.1.4 .
obtain sloreta solution based on the computed lead field matrix and the input eeg signals . by selecting the source estimation method option , the inverse solution can be obtained .
please click here to view a larger version of this figure . plot the estimated sources .
once all procedures are properly completed , estimated sources can be visualized in the brain surface of the pre - clinical model .
figure 5 shows the estimated sources from one particular sub - type of spikes ( top ) and sharp - waves ( bottom ) from ieds .
in addition , figure 6 displays how the source distribution changes in sequential time frames during a seizure establishment .
these results support the capability of the proposed methodologies to record high - resolution eeg on rats with focal epilepsy and to conduct source analysis using the recorded eeg .
estimated brain source locations of ieds with respect to different clusters in spikes ( top ) and sharp - waves ( bottom).(a ) time series , ( b ) eeg topography , and ( c ) cortical current sources .
the evaluation is performed at a specific time marked with a red vertical line in ( a ) .
a new methodology to non - invasively record multichannel eeg in a particular preclinical model of focal epilepsy is described .
the particulars for the recording and analysis procedures , with specific experimental tips , are provided .
proper viscosity of the eeg paste should be applied to each electrode during the mini - cap preparation , and the rat s head and ear hair should be completely removed during shaving .
also , the generated electrode positions should have minimum distance error from the actual electrode locations on the rat s scalp .
even though this manuscript introduces source analysis procedures using brainstorm , they can be conducted using other open softwares and commercial products . also , besides sloreta , other inverse solutions such as multiple dipole models and beamformer can be applied .
one limitation of this approach is that behavior analysis can not be conducted since the eeg recording is carried out under sedation .
however , compared to the other methods for eeg recording in rats , this approach is noninvasive .
our preliminary results support the importance for a precise classification of ied markers from eeg recordings to determine the irritative zones in a rat with focal epilepsy , as well as to evaluate their relationship with the underlying mechanisms for seizure initiation .
in addition , it has been shown that eeg source localization for such specific ieds showed a good correspondence with the respective bold activation and deactivation regions .
our study will stimulate the use of preclinical models to evaluate bed - bench - bed strategies developed by biomedical engineers .
for example , ied extraction is nowadays performed in hospitals manually , which required a considerable human effort .
we hypothesize that the use of this methodology will produce similar results when applied to patients with fcd .
we are preparing irb protocols for the evaluation of this and other aspects of the methodology in human dataset .
moreover , the use of preclinical models will help us understand the capabilities and limitations of eeg source localization in epilepsy .
accurate estimation of brain sources underling epileptogenesis is crucial for therapeutic strategies and surgical planning .
also , having a standard platform for eeg recording in rats will be useful for the evaluation of the effectiveness of several anti - epileptic drugs in preclinical trials .
this is the first study in which epileptic rats are recorded non - invasively under sedation , which will open new doors for the evaluation of eeg biomarkers for epilepsy .
however , the entire methodology presented in this study is extendable to other experimental conditions and brain disorders .
the eeg mini - cap can be also used in other rodent s types . in the past
, a forepaw stimulation paradigm in wistar rats has been used to evaluate the quality and reproducibility of data recorded with the eeg mini - cap .
moreover , validations for the brain source reconstruction have been performed from high - resolution skull eeg concurrently recorded with laminar local field potentials from wistar rats under a whisker stimulation paradigm .
this methodology has been developed for wistar rats because of the existence of an mri atlas for this particular rat strain .
however , it can be applied to other rodent types with their standard format of atlas including mouse , sprague - dawley rats , and paxinos and watson rats .
in addition , the fundamental procedures of our proposed methodology could be used in any rodent preclinical models for which eeg is an important modality . however , many aspects of this methodology are particularly for epilepsy , especially those related to eeg preprocessing ( ied detection and classification ) .
also , researchers must be aware of proper drugs used for sedation in different cases .
the use of isoflurane and dexdomitor in our study has been carefully considered due to the reduced impact on ieds .
regarding eeg recordings , in the case of mouse , the relatively small scalp surface area would reduce the number of channels considerably . | electroencephalogram ( eeg ) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy .
this methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges ( ieds ) emerge .
preclinical models are very useful to find correlates between ied locations and the actual regions underlying seizure initiation in focal epilepsy .
rats have been commonly used in preclinical studies of epilepsy1 ; hence , there exist a large variety of models for focal epilepsy in this particular species . however
, it is challenging to record multichannel eeg and to perform brain source imaging in such a small animal . to overcome this issue , we combine a patented - technology to obtain 32-channel eeg recordings from rodents2 and an mri probabilistic atlas for brain anatomical structures in wistar rats to perform brain source imaging . in this video ,
we introduce the procedures to acquire multichannel eeg from wistar rats with focal cortical dysplasia , and describe the steps both to define the volume conductor model from the mri atlas and to uniquely determine the ieds .
finally , we validate the whole methodology by obtaining brain source images of ieds and compare them with those obtained at different time frames during the seizure onset . | Introduction
Protocol
1. EEG Recordings
2. Brain Source Imaging
Representative Results
Discussion
Disclosures | it has been shown that interictal epileptiform discharges ( ieds ) observed from eeg constitute useful markers of epileptogenesis in patients with focal epilepsy . the regions inside the brain from which these ieds originate , named irritative zones , can in practice be localized based on eeg recordings . preclinical models are essential to find correlates between these irritative zones and the actual regions underlying seizure initiation . however ,
recording eeg from small animals is challenging because of the small surface area of the head compared to the human scalp . although invasive methods for chronic recording in rats can be used , techniques are not available at this moment to acquire traditional eeg recordings on rodents under acute conditions without the need of anesthesia . to solve this problem ,
we apply a patented eeg mini - cap , which allows us to record 32-channel eeg data from rodents noninvasively . in this study
, we also provide evidence about the need of an analgesic to preserve ied frequency . the method proposed in this study can be used in any preclinical rat model of focal epilepsy . to illustrate the capabilities of this methodology
, we apply it to understand the correlates between irritative and seizure - onset zones in focal cortical dysplasia ( fcd ) . to that end
double - hit model of fcd in wistar rats . to perform brain source analysis
, it is required to : a ) accurately extract ieds from eeg raw data and b ) obtain a volume conductor model for the individual animal head . to generate a practical volume conductor model , we use an in vivo rat mri atlas , comprising average images of intensity / shape and obtained via non - linear registration of t2 images of 31 wistar rats . as in the case of humans ,
two typical patterns of ieds ( sharp - waves and spikes ) were detected and sub - classified into different clusters through an intelligent feature extraction , feature selection , and classification process . these sub - classified signals are used to estimate the brain source localizations associated with different types of irritative zones . the eeg source localizations for each ied sub - type and the seizure onset time frames were performed using standardized low - resolution brain electromagnetic tomography ( sloreta ) , which is available in brainstorm . dry the cap and the electrodes in the air.mix eeg electrode paste with 0.9% nacl solution in the volume proportion of 2 : 1 . this provides better access to each electrode and reduces the possibility of the gel spilling over.turn on eeg and physiological recording system , and open the corresponding recording software on the computer in use . turn on eeg and physiological recording system , and open the corresponding recording software on the computer in use . animal preparation and anesthesia note : chronic epilepsy was created using a protocol for fcd in wistar rats . induce anesthesia in the rat with 5% isoflurane and 100% oxygen ( 1 l / min at 14.7 psi).after trim the rat s head , reduce isoflurane to 2% and maintain it during the entire setting of the eeg mini - cap . note : any hair left on the skin will produce noise in the eeg recordings . rub the rat s skin with 90% isopropyl alcohol to stimulate the blood vessels and degrease the skin.place a saline swab on the scalp and cover it completely to keep good skin conductance until the eeg mini - cap is ready to be placed.connect temperature , respiration , and three lead electrocardiogram probes . after trim the rat s head , reduce isoflurane to 2% and maintain it during the entire setting of the eeg mini - cap . note : any hair left on the skin will produce noise in the eeg recordings . place one rubber band on the front side of the scalp , usually in front of the eyes , and another band at the back of the scalp between the ears and neck . ensure that the impedance value is in the range of 5 - 30 k. if there are any noisy electrodes , provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode.administer dexdomitor ( 0.25 mg / kg ) intraperitoneally and immediately reduce isoflurane rate to 0% . note : on the preclinical model of focal epilepsy , isoflurane affects ieds , whereas dexdomitor does not . note : in this experiment , red color ( opponent color to green ) was used to distinguish from the electrode positions ( green ) . however , it is recommended to use other colors ( purple / green ) if small bleeding spots are observed in the skin . place one rubber band on the front side of the scalp , usually in front of the eyes , and another band at the back of the scalp between the ears and neck . ensure that the impedance value is in the range of 5 - 30 k. if there are any noisy electrodes , provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode . note : on the preclinical model of focal epilepsy , isoflurane affects ieds , whereas dexdomitor does not . note : in this experiment , red color ( opponent color to green ) was used to distinguish from the electrode positions ( green ) . however , it is recommended to use other colors ( purple / green ) if small bleeding spots are observed in the skin . they are sub - classified into multiple clusters using k - means , and the optimal cluster number k is determined using silhouette.average the sub - classified signals within the same cluster . the average eeg signals for each ied sub - type will be used for brain source analysis . they are sub - classified into multiple clusters using k - means , and the optimal cluster number k is determined using silhouette . the average eeg signals for each ied sub - type will be used for brain source analysis . for the following sections , the open source software , brainstorm , will be used with the mri atlas for wistar rats . however
, individual rat s mri can also be used to generate the volume conductor model if available . the mri atlas can be downloaded at http://www.idac.tohoku.ac.jp / bir / en/. the thickness of the skull can be checked from the mri , and in the case of the mri atlas , it is approximately 1 mm . co - register the positions of the 3 landmarks ( r1 , r2 , and r3 ) in the mri . use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold ( figure 3b ) . ( a ) rat head picture used to obtain electrode positions and ( b ) the eeg mini - cap diagram with the coordinate system . here , n is the number of channels ( n = 32 ) and the column represents the corresponding x , y , and z coordinate values . therefore , once the 3 reference grids ( r1 , r2 , and r3 ) are obtained , the position of the electrodes are automatically set . and then , a selected surface will be displayed as yellow line on the mri . in addition , the 3 landmarks and the 32 electrode positions can be displayed on the mri by selecting the toolbox s option ,
the locations can be visually inspected by comparing the distributions based on the rat s eye and ear locations ( figure 4 ) . ( a ) mri atlas with co - registered brain surface ( yellow line ) , ( b ) the created volume conductor model with the aligned 32 electrodes and 3 landmarks ( red dots ) , and ( c ) mri atlas with co - registered reference grid r1 . the thickness of the skull can be checked from the mri , and in the case of the mri atlas , it is approximately 1 mm . using the rat head picture acquired in 1.3.5
co - register the positions of the 3 landmarks ( r1 , r2 , and r3 ) in the mri . use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold ( figure 3b ) . ( a ) rat head picture used to obtain electrode positions and ( b ) the eeg mini - cap diagram with the coordinate system . here , n is the number of channels ( n = 32 ) and the column represents the corresponding x , y , and z coordinate values . therefore , once the 3 reference grids ( r1 , r2 , and r3 ) are obtained , the position of the electrodes are automatically set . the user will only need to redefine the z - values on a way that the mini - cap is appropriately projected on the scalp . and then , a selected surface will be displayed as yellow line on the mri . in addition , the 3 landmarks and the 32 electrode positions can be displayed on the mri by selecting the toolbox s option ,
can be visually inspected by comparing the distributions based on the rat s eye and ear locations ( figure 4 ) . ( a ) mri atlas with co - registered brain surface ( yellow line ) , ( b ) the created volume conductor model with the aligned 32 electrodes and 3 landmarks ( red dots ) , and ( c ) mri atlas with co - registered reference grid r1 . obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2 . please click here to view a larger version of this figure.obtain sloreta solution based on the computed lead field matrix and the input eeg signals . obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2 . obtain sloreta solution based on the computed lead field matrix and the input eeg signals . dry the cap and the electrodes in the air.mix eeg electrode paste with 0.9% nacl solution in the volume proportion of 2 : 1 . this provides better access to each electrode and reduces the possibility of the gel spilling over.turn on eeg and physiological recording system , and open the corresponding recording software on the computer in use . turn on eeg and physiological recording system , and open the corresponding recording software on the computer in use . animal preparation and anesthesia note : chronic epilepsy was created using a protocol for fcd in wistar rats . induce anesthesia in the rat with 5% isoflurane and 100% oxygen ( 1 l / min at 14.7 psi).after trim the rat s head , reduce isoflurane to 2% and maintain it during the entire setting of the eeg mini - cap . note : any hair left on the skin will produce noise in the eeg recordings . rub the rat s skin with 90% isopropyl alcohol to stimulate the blood vessels and degrease the skin.place a saline swab on the scalp and cover it completely to keep good skin conductance until the eeg mini - cap is ready to be placed.connect temperature , respiration , and three lead electrocardiogram probes . after trim the rat s head , reduce isoflurane to 2% and maintain it during the entire setting of the eeg mini - cap . note : any hair left on the skin will produce noise in the eeg recordings . place one rubber band on the front side of the scalp , usually in front of the eyes , and another band at the back of the scalp between the ears and neck . for a high - quality recording ,
ensure that the impedance value is in the range of 5 - 30 k. if there are any noisy electrodes , provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode.administer dexdomitor ( 0.25 mg / kg ) intraperitoneally and immediately reduce isoflurane rate to 0% . note : on the preclinical model of focal epilepsy , isoflurane affects ieds , whereas dexdomitor does not . note : in this experiment , red color ( opponent color to green ) was used to distinguish from the electrode positions ( green ) . however , it is recommended to use other colors ( purple / green ) if small bleeding spots are observed in the skin . place one rubber band on the front side of the scalp , usually in front of the eyes , and another band at the back of the scalp between the ears and neck . ensure that the impedance value is in the range of 5 - 30 k. if there are any noisy electrodes , provide better contact with the scalp by either moving them inside the scaffold toward the scalp or gently injecting more gel from the top of the electrode . note : on the preclinical model of focal epilepsy , isoflurane affects ieds , whereas dexdomitor does not . note : in this experiment , red color ( opponent color to green ) was used to distinguish from the electrode positions ( green ) . however , it is recommended to use other colors ( purple / green ) if small bleeding spots are observed in the skin . they are sub - classified into multiple clusters using k - means , and the optimal cluster number k is determined using silhouette.average the sub - classified signals within the same cluster . the average eeg signals for each ied sub - type will be used for brain source analysis . they are sub - classified into multiple clusters using k - means , and the optimal cluster number k is determined using silhouette . the average eeg signals for each ied sub - type will be used for brain source analysis . volume conductor model note : for the following sections , the open source software , brainstorm , will be used with the mri atlas for wistar rats . however
, individual rat s mri can also be used to generate the volume conductor model if available . the mri atlas can be downloaded at http://www.idac.tohoku.ac.jp / bir / en/. the thickness of the skull can be checked from the mri , and in the case of the mri atlas , it is approximately 1 mm . co - register the positions of the 3 landmarks ( r1 , r2 , and r3 ) in the mri . use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold ( figure 3b ) . ( a ) rat head picture used to obtain electrode positions and ( b ) the eeg mini - cap diagram with the coordinate system . here , n is the number of channels ( n = 32 ) and the column represents the corresponding x , y , and z coordinate values . therefore , once the 3 reference grids ( r1 , r2 , and r3 ) are obtained , the position of the electrodes are automatically set . and then , a selected surface will be displayed as yellow line on the mri . in addition , the 3 landmarks and the 32 electrode positions can be displayed on the mri by selecting the toolbox s option ,
the locations can be visually inspected by comparing the distributions based on the rat s eye and ear locations ( figure 4 ) . ( a ) mri atlas with co - registered brain surface ( yellow line ) , ( b ) the created volume conductor model with the aligned 32 electrodes and 3 landmarks ( red dots ) , and ( c ) mri atlas with co - registered reference grid r1 . the thickness of the skull can be checked from the mri , and in the case of the mri atlas , it is approximately 1 mm . co - register the positions of the 3 landmarks ( r1 , r2 , and r3 ) in the mri . use the grid points of the landmarks as references to generate the electrode positions as the electrodes are fixed on the scaffold ( figure 3b ) . ( a ) rat head picture used to obtain electrode positions and ( b ) the eeg mini - cap diagram with the coordinate system . here , n is the number of channels ( n = 32 ) and the column represents the corresponding x , y , and z coordinate values . therefore , once the 3 reference grids ( r1 , r2 , and r3 ) are obtained , the position of the electrodes are automatically set . and then , a selected surface will be displayed as yellow line on the mri . in addition , the 3 landmarks and the 32 electrode positions can be displayed on the mri by selecting the toolbox s option ,
the locations can be visually inspected by comparing the distributions based on the rat s eye and ear locations ( figure 4 ) . ( a ) mri atlas with co - registered brain surface ( yellow line ) , ( b ) the created volume conductor model with the aligned 32 electrodes and 3 landmarks ( red dots ) , and ( c ) mri atlas with co - registered reference grid r1 . obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2 . please click here to view a larger version of this figure.obtain sloreta solution based on the computed lead field matrix and the input eeg signals . obtain the lead field matrix based on the volume conductor model and the electrode positions created in 2.2 . obtain sloreta solution based on the computed lead field matrix and the input eeg signals . once all procedures are properly completed , estimated sources can be visualized in the brain surface of the pre - clinical model . in addition , figure 6 displays how the source distribution changes in sequential time frames during a seizure establishment . these results support the capability of the proposed methodologies to record high - resolution eeg on rats with focal epilepsy and to conduct source analysis using the recorded eeg . estimated brain source locations of ieds with respect to different clusters in spikes ( top ) and sharp - waves ( bottom). a new methodology to non - invasively record multichannel eeg in a particular preclinical model of focal epilepsy is described . proper viscosity of the eeg paste should be applied to each electrode during the mini - cap preparation , and the rat s head and ear hair should be completely removed during shaving . also , the generated electrode positions should have minimum distance error from the actual electrode locations on the rat s scalp . our preliminary results support the importance for a precise classification of ied markers from eeg recordings to determine the irritative zones in a rat with focal epilepsy , as well as to evaluate their relationship with the underlying mechanisms for seizure initiation . in addition , it has been shown that eeg source localization for such specific ieds showed a good correspondence with the respective bold activation and deactivation regions . we hypothesize that the use of this methodology will produce similar results when applied to patients with fcd . also , having a standard platform for eeg recording in rats will be useful for the evaluation of the effectiveness of several anti - epileptic drugs in preclinical trials . however , the entire methodology presented in this study is extendable to other experimental conditions and brain disorders . in the past
, a forepaw stimulation paradigm in wistar rats has been used to evaluate the quality and reproducibility of data recorded with the eeg mini - cap . moreover , validations for the brain source reconstruction have been performed from high - resolution skull eeg concurrently recorded with laminar local field potentials from wistar rats under a whisker stimulation paradigm . this methodology has been developed for wistar rats because of the existence of an mri atlas for this particular rat strain . however , it can be applied to other rodent types with their standard format of atlas including mouse , sprague - dawley rats , and paxinos and watson rats . in addition , the fundamental procedures of our proposed methodology could be used in any rodent preclinical models for which eeg is an important modality . however , many aspects of this methodology are particularly for epilepsy , especially those related to eeg preprocessing ( ied detection and classification ) . | [
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] |
this was a cross - sectional study including both eyes of 191 participants from the diagnostic innovations in glaucoma study ( digs ) , a previously described prospective longitudinal study .
the study was conducted at the hamilton glaucoma center of the department of ophthalmology , university of california san diego ( ucsd ) . written informed consent
was obtained from all participants , and the institutional review board and human subjects committee at the university of california san diego prospectively approved all methods .
all study methods adhered to the tenets of the declaration of helsinki for research involving human subjects and the study was conducted in accordance with the regulations of the health insurance portability and accountability act . at each visit
subjects underwent comprehensive ophthalmologic examination , including best - corrected visual acuity , slit - lamp biomicroscopy , iop measurement , gonioscopy , dilated funduscopic examination , simultaneous stereoscopic optic disc photography ( kowa wx3d ; kowa optimed , inc . ,
torrance , ca , usa ) , and sap using the swedish interactive threshold algorithm ( sita standard 24 - 2 ) .
subjects were excluded if they had a best - corrected visual acuity less than 20/40 or any other ocular or systemic disease that could affect the optic nerve or the visual field .
patients using systemic or topical cholinergic or anticholinergic medications ( including pilocarpine ) that might affect pupil responses were excluded .
eyes were classified as glaucomatous if they had repeatable ( 3 consecutive ) abnormal sap test results on the 24 - 2 humphrey visual field analyzer test ( carl zeiss meditec , inc . , dublin , ca , usa ) .
an abnormal sap result was defined by a pattern standard deviation outside the 95% confidence limits ( ci ) or a glaucoma hemifield test result outside the reference range .
healthy subjects were recruited from the general population and required iop 21 mm hg with no history of increased iop and normal sap in both eyes .
each subject had cirrus sdoct ( software v. 5.2 , model 4000 ; carl zeiss meditec , inc . ) , sap , and automated pupillometry within a 4-month interval .
the cirrus sdoct was used to acquire cprnfl , mgcipl , and total macular thickness measurements .
the cprnfl thickness measurements were acquired using the optic disc cube 200 200 protocol using a 3-dimensional scan of a 6 6mm area centered on the optic disc .
average cprnfl thickness was calculated automatically from a 3.46-mm diameter circular scan ( 10.87 mm length ) around the optic disc .
this protocol is based on a 3-dimensional scan centered on the macula in which information from a 1024 ( depth ) 200 200 point parallelepiped is collected .
the ganglion cell analysis algorithm automatically segmented the mgcipl , defined by the outer boundary of rnfl and outer boundary of inner plexiform layer .
cirrus sdoct images were reviewed and included if well centered , signal strength > 7 and movement artifacts and segmentation errors were absent .
all visual fields were evaluated by the ucsd visual field assessment center ( visfact ) .
visual fields with more than 33% fixation losses or more than 15% false - positive errors were excluded .
visual fields were reviewed further for the following artifacts : eyelid and rim artifacts , fatigue effects , inappropriate fixation , evidence that the visual field results were caused by a disease other than glaucoma and inattention .
pupil responses were studied using the rapdx ( konan medical usa , inc . , irvine , ca , usa ) , a binocular infrared pupillometer . the device measures bilateral pupil responses to monocularly presented visual stimuli .
stimuli are created using a single lcd screen with a central physical barrier creating two optical channels .
the screen displays a target for fixation and during testing each portion of the screen can be enabled selectively to achieve separate stimulation of each eye .
the screen is viewed at infinity through a pair of 50-mm objective lenses providing an approximate 25 field of view in each eye .
eyes also are illuminated by a pair of infrared emitting diodes , with peak emission at 880 nm , mounted at a 35 angle . under infrared conditions information regarding the dark pupil diameter
is captured as camera pixels and this measurement is converted to millimeters using a scaling factor .
the stimulus then is presented as a series of trials , either to the full field of each eye or limited to predetermined regions .
the size , color , intensity , and length of time of each stimulus were controlled automatically via proprietary software .
each trial consisted of a period of stimulation followed by a period of darkness during which the cameras record continuously .
the total time of each trial was 2.0 seconds plus a 100-ms post - trial rest period during which no images were acquired .
the full field white stimulus was presented for 200 ms of the 2.1-second duty cycle and 18 trials ( nine for each eye ) used this stimulus for a total test time of 37.8 seconds .
the right eye was stimulated first , followed by the left , then the right , with continued stimulation alternating between eyes .
the full - field stimuli had luminances of 384 ( white stimulus ) , 88 ( red stimulus ; 605 nm ) , 27 ( green stimulus ; 555 nm ) , 23 ( blue stimulus ; 440 nm ) , and 380 ( yellow stimulus ; 576 nm ) cd / m , and during all tests , there was a nominal background luminance of 0.01 cd / m .
the pupillometer includes proprietary analysis software , which was used to parse the generated pupil diameter waveforms into specific metrics .
the repetitions from each eye were averaged ( median ) before analysis to minimize noise inherent in the pupil responses .
parameters measured by the pupillometer include prestimulus pupil diameter ( in millimeters ) , minimum pupil diameter following the stimulus ( in millimeters ) , response amplitude ( i.e. , maximal contraction of the pupil as a percentage of the prestimulation size , that is , the prestimulus pupil diameter minus the minimum pupil diameter , divided by the prestimulus pupil diameter ) , response latency ( time in milliseconds between stimulus onset and time when pupil velocity has reached 50% of the peak velocity of constriction ) , and time to peak constriction ( in milliseconds ) . an rapd is defined as a difference in average pupillary constriction ( response amplitude ) when each eye is stimulated monocularly .
an index of the direction and magnitude of pupil response asymmetry , known as the rapd score , is generated automatically by the rapdx device .
the rapd score is calculated as the difference in the amplitude of pupil constriction between stimulation of the two eyes using the following formula :
,
where od is the mean response amplitude in both eyes , in response to right eye stimulation , and os is the mean response amplitude in both eyes in response to left eye stimulation .
a positive value indicates a relative abnormality of the left afferent system and a negative value indicates a relative abnormality of the right afferent system .
the rapd score is useful as it confers information regarding the direction as well as the magnitude of an rapd .
however , to investigate the effect of potential confounders , such as average disease severity , the absolute rapd score also was calculated as an overall measure of asymmetry of the afferent visual pathways , regardless of which eye was affected .
the arithmetic difference in response amplitude on stimulation of the better eye and on stimulation of the worse eye also was calculated .
descriptive statistics included mean and standard deviation ( sd ) and t - tests for normally distributed variables ; and median , interquartile range , and wilcoxon rank - sum for nonparametrically distributed variables .
the relationship between rapd scores and intereye differences ( right eye minus left eye ) in structural parameters was examined using scatter plots and linear regression .
the average cprnfl thickness , mgcipl , macular thickness , and sap mean deviation ( md ) were included in the analyses to evaluate the effect of disease severity on asymmetry needed for an rapd .
all statistical analyses were performed with commercially available software ( stata , version 13 ; statacorp lp , college station , tx , usa ) . the level ( type i error )
this was a cross - sectional study including both eyes of 191 participants from the diagnostic innovations in glaucoma study ( digs ) , a previously described prospective longitudinal study .
the study was conducted at the hamilton glaucoma center of the department of ophthalmology , university of california san diego ( ucsd ) . written informed consent
was obtained from all participants , and the institutional review board and human subjects committee at the university of california san diego prospectively approved all methods .
all study methods adhered to the tenets of the declaration of helsinki for research involving human subjects and the study was conducted in accordance with the regulations of the health insurance portability and accountability act . at each visit
subjects underwent comprehensive ophthalmologic examination , including best - corrected visual acuity , slit - lamp biomicroscopy , iop measurement , gonioscopy , dilated funduscopic examination , simultaneous stereoscopic optic disc photography ( kowa wx3d ; kowa optimed , inc . ,
torrance , ca , usa ) , and sap using the swedish interactive threshold algorithm ( sita standard 24 - 2 ) .
subjects were excluded if they had a best - corrected visual acuity less than 20/40 or any other ocular or systemic disease that could affect the optic nerve or the visual field .
patients using systemic or topical cholinergic or anticholinergic medications ( including pilocarpine ) that might affect pupil responses were excluded .
eyes were classified as glaucomatous if they had repeatable ( 3 consecutive ) abnormal sap test results on the 24 - 2 humphrey visual field analyzer test ( carl zeiss meditec , inc . , dublin , ca , usa ) .
an abnormal sap result was defined by a pattern standard deviation outside the 95% confidence limits ( ci ) or a glaucoma hemifield test result outside the reference range .
healthy subjects were recruited from the general population and required iop 21 mm hg with no history of increased iop and normal sap in both eyes .
each subject had cirrus sdoct ( software v. 5.2 , model 4000 ; carl zeiss meditec , inc . ) , sap , and automated pupillometry within a 4-month interval .
the cirrus sdoct was used to acquire cprnfl , mgcipl , and total macular thickness measurements .
the cprnfl thickness measurements were acquired using the optic disc cube 200 200 protocol using a 3-dimensional scan of a 6 6mm area centered on the optic disc .
average cprnfl thickness was calculated automatically from a 3.46-mm diameter circular scan ( 10.87 mm length ) around the optic disc .
this protocol is based on a 3-dimensional scan centered on the macula in which information from a 1024 ( depth ) 200 200 point parallelepiped is collected .
the ganglion cell analysis algorithm automatically segmented the mgcipl , defined by the outer boundary of rnfl and outer boundary of inner plexiform layer .
cirrus sdoct images were reviewed and included if well centered , signal strength > 7 and movement artifacts and segmentation errors were absent .
all visual fields were evaluated by the ucsd visual field assessment center ( visfact ) .
visual fields with more than 33% fixation losses or more than 15% false - positive errors were excluded .
visual fields were reviewed further for the following artifacts : eyelid and rim artifacts , fatigue effects , inappropriate fixation , evidence that the visual field results were caused by a disease other than glaucoma and inattention .
pupil responses were studied using the rapdx ( konan medical usa , inc . , irvine , ca , usa ) , a binocular infrared pupillometer . the device measures bilateral pupil responses to monocularly presented visual stimuli .
stimuli are created using a single lcd screen with a central physical barrier creating two optical channels .
the screen displays a target for fixation and during testing each portion of the screen can be enabled selectively to achieve separate stimulation of each eye .
the screen is viewed at infinity through a pair of 50-mm objective lenses providing an approximate 25 field of view in each eye .
eyes also are illuminated by a pair of infrared emitting diodes , with peak emission at 880 nm , mounted at a 35 angle . under infrared conditions information regarding the dark pupil diameter
is captured as camera pixels and this measurement is converted to millimeters using a scaling factor .
the stimulus then is presented as a series of trials , either to the full field of each eye or limited to predetermined regions .
the size , color , intensity , and length of time of each stimulus were controlled automatically via proprietary software .
each trial consisted of a period of stimulation followed by a period of darkness during which the cameras record continuously .
the total time of each trial was 2.0 seconds plus a 100-ms post - trial rest period during which no images were acquired .
the full field white stimulus was presented for 200 ms of the 2.1-second duty cycle and 18 trials ( nine for each eye ) used this stimulus for a total test time of 37.8 seconds .
the right eye was stimulated first , followed by the left , then the right , with continued stimulation alternating between eyes .
the full - field stimuli had luminances of 384 ( white stimulus ) , 88 ( red stimulus ; 605 nm ) , 27 ( green stimulus ; 555 nm ) , 23 ( blue stimulus ; 440 nm ) , and 380 ( yellow stimulus ; 576 nm ) cd / m , and during all tests , there was a nominal background luminance of 0.01 cd / m .
the pupillometer includes proprietary analysis software , which was used to parse the generated pupil diameter waveforms into specific metrics .
the repetitions from each eye were averaged ( median ) before analysis to minimize noise inherent in the pupil responses .
parameters measured by the pupillometer include prestimulus pupil diameter ( in millimeters ) , minimum pupil diameter following the stimulus ( in millimeters ) , response amplitude ( i.e. , maximal contraction of the pupil as a percentage of the prestimulation size , that is , the prestimulus pupil diameter minus the minimum pupil diameter , divided by the prestimulus pupil diameter ) , response latency ( time in milliseconds between stimulus onset and time when pupil velocity has reached 50% of the peak velocity of constriction ) , and time to peak constriction ( in milliseconds ) . an rapd is defined as a difference in average pupillary constriction ( response amplitude ) when each eye is stimulated monocularly .
an index of the direction and magnitude of pupil response asymmetry , known as the rapd score , is generated automatically by the rapdx device .
the rapd score is calculated as the difference in the amplitude of pupil constriction between stimulation of the two eyes using the following formula :
,
where od is the mean response amplitude in both eyes , in response to right eye stimulation , and os is the mean response amplitude in both eyes in response to left eye stimulation . an rapd score of 0
a positive value indicates a relative abnormality of the left afferent system and a negative value indicates a relative abnormality of the right afferent system .
the rapd score is useful as it confers information regarding the direction as well as the magnitude of an rapd .
however , to investigate the effect of potential confounders , such as average disease severity , the absolute rapd score also was calculated as an overall measure of asymmetry of the afferent visual pathways , regardless of which eye was affected .
the arithmetic difference in response amplitude on stimulation of the better eye and on stimulation of the worse eye also was calculated .
descriptive statistics included mean and standard deviation ( sd ) and t - tests for normally distributed variables ; and median , interquartile range , and wilcoxon rank - sum for nonparametrically distributed variables .
the relationship between rapd scores and intereye differences ( right eye minus left eye ) in structural parameters was examined using scatter plots and linear regression .
the average cprnfl thickness , mgcipl , macular thickness , and sap mean deviation ( md ) were included in the analyses to evaluate the effect of disease severity on asymmetry needed for an rapd .
all statistical analyses were performed with commercially available software ( stata , version 13 ; statacorp lp , college station , tx , usa ) . the level ( type i error )
the study included 106 patients with glaucoma and 85 healthy controls . of those with glaucoma , 67 had glaucoma in both eyes , 33 had glaucoma in one eye and suspected glaucoma in the other , and 6 had glaucoma in one eye and a putative healthy fellow eye .
baseline demographic and clinical characteristics of study patients ( mean sd ) subjects with glaucoma had an average md of 6.9 7.3 db in the worse eye and 2.9 4.4 db in the better eye , which was significantly worse than in healthy subjects ( p < 0.001 for both comparisons ) .
the cprnfl , mgcipl , and macular thickness also were significantly thinner in the worse and better eyes of those with glaucoma compared to controls ( table 1 ) .
those with glaucoma also had greater asymmetry in sap and sdoct measurements between eyes compared to healthy subjects .
the mean ( sd ) intereye difference in md was 4.0 4.9 db in those with glaucoma compared to only 0.6 0.4 db in healthy subjects .
intereye difference in cprnfl thickness was 9.2 7.6 m in patients with glaucoma , compared to only 3.8 3.6 m in healthy participants .
corresponding intereye differences in mgcipl thickness were 6.8 6.8 and 1.7 1.8 m , respectively .
the absolute rapd score averaged 0.30 0.35 in those with glaucoma compared to 0.15 0.10 in healthy subjects ( p = 0.006 ; table 2 ) .
only 12 ( 14% ) healthy subjects had an absolute rapd score higher than 0.25 and none had higher than 0.50 .
however , in the glaucomatous group , 42 of 106 subjects ( 40% ) had an absolute rapd score higher than 0.25 , 31 of 106 ( 29% ) had an absolute rapd score higher than 0.30 , and 22 ( 21% ) had an absolute rapd score higher than 0.50 .
subjects with glaucoma also had significantly greater intereye differences in pupil response latency and time to peak pupil constriction compared to healthy subjects ( p = 0.031 and p = 0.006 , respectively ) .
those with glaucoma also had , on average , a greater absolute rapd score for blue ( p = 0.013 ) , green ( p < 0.001 ) , yellow ( p < 0.001 ) , and red ( p <
pupillary reflex characteristics of healthy subjects compared to glaucoma patients ( mean sd ) histogram showing the distribution of absolute rapd scores in healthy eyes and those with glaucoma in at least one eye .
there was a significant association between rapd scores and intereye differences in macular structural damage , as measured by intereye difference in mgcipl thickness ( r = 0.285 , p < 0.001 ) .
the relationship between rapd score and intereye difference in macular thickness was weaker ( r = 0.167 , p < 0.001 ) than that with mgcipl . however , intereye differences in cprnfl thickness ( r = 0.350 , p < 0.001 ) and sap md ( r = 0.594 , p < 0.001 ) had stronger associations with rapd score than macular parameters ( table 3 , fig .
results of univariable regression analysis of rapd score investigating the relationship between intereye difference ( , right minus left eye ) in cprnfl thickness , mgcipl thickness , macular thickness , and md from sap scatter plot showing rapd score association with intereye differences in cprnfl thickness ( a ) , mgcipl thickness ( b ) , macular thickness ( c ) , and sap md ( d ) .
intereye asymmetry of glaucoma damage may be influenced by disease severity ; for example , patients with bilateral end - stage glaucoma are likely to have less asymmetry than those with early glaucoma due to a floor effect in structural and functional measurements .
in contrast , those with moderate glaucoma may have greater asymmetry than those with very early disease . therefore ,
greater intereye difference in mgcipl thickness remained associated with greater absolute rapd score ( table 4 ) but worse average md also was associated with greater absolute rapd scores ( p < 0.001 ) .
similar effects of disease severity also were noted on the association between rapd score with asymmetries in the other structural and functional variables , that is , intereye cprnfl thickness ( p < 0.001 ) ; absolute intereye sap md difference ( p < 0.001 ) , and absolute intereye macular thickness difference ( p < 0.001 ) .
in multivariable analyses , age had no significant influence on the relationship between absolute rapd score and absolute intereye mgcipl thickness difference ( p = 0.560 ) , intereye differences in cprnfl thickness , macular thickness , or sap md ( p = 0.378 , p = 0.156 , and p = 0.888 , respectively ) .
results of multivariable regression analysis of absolute rapd score and absolute intereye difference ( , right minus left eye ) in mgcipl thickness , including average md from sap as a covariable although the association between rapd scores obtained using colored stimuli and intereye differences in structural and functional parameters were significant , the associations with rapd score to the white stimulus generally were stronger ( table 5 , fig .
figure 4 is an example of a patient with advanced glaucoma in the right eye and mild glaucoma in the left eye showing a rapd score of 1.21 , while figure 5 shows a patient with advanced glaucoma in both eyes and a rapd score of only 0.12 .
figure 6 shows a patient with mild glaucoma in both eyes and a rapd score of only 0.09 .
results of univariable regression analysis of rapd score in different colors compared to intereye difference ( , right minus left eye ) in mgcipl , macular , cprnfl thickness , and md from sap scatter plot showing intereye difference of mgcipl thickness association with different colors for rapd score : red ( a ) , blue ( b ) , green ( c ) , and yellow ( md ) ( d ) .
optical coherence tomography measurements of cprnfl ( a ) , mgcipl ( b ) , and total macular thickness ( c ) are shown .
automated pupillography showing the average pupil response on right and left eye stimulation ( d ) revealed reduced response amplitude ( denoted by the arrow and letter a ) on stimulation of the right eye .
average response amplitude on right eye stimulation was 0.20 compared to 0.27 on left eye stimulation , giving an rapd score of 10*log10 ( 0.20/0.27 ) = 1.21 , indicating a relative abnormality of the right afferent pathway .
optical coherence tomography measurements of cprnfl ( a ) , mgcipl ( b ) , and total macular thickness ( c ) are shown .
automated pupillography showing the average pupil response on right and left eye stimulation ( d ) revealed reduced response amplitude ( denoted by the arrow and letter a ) on stimulation of the right eye .
the average response amplitude on right eye stimulation was 0.33 compared to 0.34 on left eye stimulation , giving an rapd score of 10*log10 ( 0.33/0.34 ) = 0.12 , indicating a small relative abnormality of the right afferent pathway .
standard automated perimetry md in the right and left eyes was 21.36 and 16.6 db , respectively ( e ) .
optical coherence tomography measurements of cprnfl ( a ) , mgcipl ( b ) , and total macular thickness ( c ) are shown .
automated pupillography showing the average pupil response on right and left eye stimulation ( d ) revealed reduced response amplitude ( denoted by the arrow and letter a ) on stimulation of the right eye .
the average response amplitude on right eye stimulation was 0.32 compared to 0.33 on left eye stimulation , giving an rapd score of 10*log10 ( 0.32/0.33 ) = 0.09 , indicating a small relative abnormality of the right afferent pathway .
standard automated perimetry md in the right and left eyes was 3.97 and 3.76 db , respectively ( e ) .
this study has shown that the magnitude of rapd measured using an automated pupillometer is correlated with measures of macular structural asymmetry in glaucoma .
subjects with larger rapds had greater intereye differences in macular thickness and mgcipl thickness , in addition to greater intereye differences in cprnfl and sap md . to our knowledge , this is the first study to evaluate the relationship between magnitude of rapd and macular structural damage in glaucoma .
patients with glaucoma were more likely to have intereye asymmetry in structural and functional measurements than healthy controls , likely because , although glaucoma typically is a bilateral disease , changes often are worse in one eye than the other .
patients with glaucoma also had larger rapd scores than controls , with average rapd scores of 0.30 0.35 and 0.15 0.10 , respectively .
a significant association was seen between rapd and intereye difference in mgcipl ( r = 0.285 ; p < 0.001 ) . for each 10-m increase in mgcipl asymmetry
such association was stronger than that for total macular thickness , indicating that the mgcipl parameter was a more useful indicator of the degree of pupillary defect in glaucoma .
the stronger association between rapd score and mgcipl asymmetry is likely to be due to total macular thickness including nonneuronal support tissues , not directly involved in the pupillary light response . in contrast , mgcipl thickness is a measurement of the inner layers containing rgcs . although asymmetries in macular measurements were associated with rapd , we found that the relationships between rapd score and asymmetry in cprnfl and sap md were stronger still , with r of 0.350 and 0.594 , respectively .
this is likely explained by the fact that although the macula accounts for a large proportion of rgcs , rgcs outside this area are likely also to contribute to the pupillary reflex . as cprnfl thickness measurements and
sap md depend on the integrity of all rgc axons throughout the retina , this probably explains why these parameters showed higher association with pupillometry results .
we found an rapd score of 0.50 is likely to correspond to intereye differences in cprnfl and sap md of approximately 23 m and 8 db , respectively , which are similar results to those reported previously using the swinging flashlight test , suggesting that an intereye difference in cprnfl thickness of 17% to 27% , or an intereye difference in sap md of 9.5 to 12 db , would correlate with an rapd of 0.3 to 0.6 .
as one might expect , we also found that the relationship between magnitude of rapd and intereye difference in mgcipl thickness depends on disease severity , with worse average md associated with greater absolute rapd scores .
our study included patients with a range of disease severities ; however , there were few with very severe disease .
only 19 patients had a md in the worse of eye of worse than 15 db .
it is important to note that in patients with advanced but symmetrical glaucoma in both eyes there is not likely to be an rapd .
although the swinging flashlight test is a simple and widely available test of rapd , automated pupillometry is likely to be a more sensitive method of quantifying abnormalities of the pupillary light reflex .
examined the ability of pupillometry to detect glaucoma and found an rapd in 56% in 70 patients with glaucoma , compared to only 29% using swinging flashlight test .
a recent systematic review , including 12 studies of pupillography , concluded that automated pupillometers were able to detect smaller intereye differences in pupil reactions than detectable using the swinging flashlight test .
further , although the swinging flashlight test is quick and easy to perform , it requires considerable practice to perform reliably and can be challenging in patients with dark irides or small or poorly reactive pupils .
we did not use the swinging flashlight test in our study ; however , previous studies have suggested an rapd of more than 0.3 is likely to be abnormal . in the present study ,
we evaluated pupil responses to a range of colored stimuli but the rapd score to the white stimulus had the strongest association with all structural and functional parameters .
however , patients with glaucoma did have greater rapd scores for blue , green , yellow , and red colored stimuli compared to healthy subjects and there was an association between rapd scores obtained using colored stimuli , and asymmetric structural and functional damage .
these results are in agreement with previous studies that showed rapd scores using different wavelength stimuli were greater in glaucoma patients and correlated with intereye sap md difference .
the magnitude of rapd can be influenced by factors other than glaucoma , such as brightness , location of the stimulus or time between stimuli .
however , to minimize these effects , only the large full field flash stimulus was used . as another limitation of our study
however , we did not find any significant influence of age on the magnitude of the rapd score .
in addition , although the pupillometer used different colored stimuli , it is important to emphasize that these stimuli do not selectively target specific rgc subtypes , such as intrinsically photosensitive rgcs ( iprgcs ) .
this would be an interesting topic for further study , since targeting specific subgroups of rgcs may allow earlier detection of damage . in conclusion , we found a significant association between the magnitude of an rapd and intereye difference in mgcipl and macular thickness .
however , rnfl thickness parameters and the degree of visual field loss asymmetry had stronger relationships with pupillometry - measured rapd and may be better indictors of the degree of expected pupillary afferent defect . | purposewe examined the relationship between relative afferent pupillary defects ( rapds ) and macular structural damage measured by macular thickness and macular ganglion cell - inner plexiform layer ( mgcipl ) thickness in patients with glaucoma.methodsa cross - sectional study was done of 106 glaucoma patients and 85 healthy individuals from the diagnostic innovations in glaucoma study .
all subjects underwent standard automated perimetry ( sap ) and optic nerve and macular imaging using cirrus spectral domain optical coherence tomography ( sdoct ) .
glaucoma was defined as repeatable abnormal sap or progressive glaucomatous changes on stereo photographs .
pupil responses were assessed using an automated pupillometer , which records the magnitude of rapd ( rapd score ) , with additional rapd scores recorded for each of a series of colored stimuli ( blue , red , green , and yellow ) .
the relationship between rapd score and intereye differences ( right minus left eye ) in circumpapillary retinal nerve fiber layer ( cprnfl ) thickness , mgcipl , macular thickness , and sap mean deviation ( md ) , was examined using linear regression.resultsthere was fair correlation between rapd score and asymmetric macular structural damage measured by intereye difference in mgcipl thickness ( r2 = 0.285 , p < 0.001 ) .
the relationship between rapd score and intereye difference in macular thickness was weaker ( r2 = 0.167 , p < 0.001 ) .
intereye difference in cprnfl thickness ( r2 = 0.350 , p < 0.001 ) and sap md ( r2 = 0.594 , p < 0.001 ) had stronger association with rapd scores compared to intereye difference in mgcipl and macular thickness.conclusionsobjective assessment of pupillary responses using a pupillometer was associated with asymmetric macular structural damage in patients with glaucoma . | Methods
Study Sample
Optical Coherence Tomography
Standard Automated Perimetry
Pupillometry Stimuli
Procedure
Statistical Analysis
Results
Discussion | this was a cross - sectional study including both eyes of 191 participants from the diagnostic innovations in glaucoma study ( digs ) , a previously described prospective longitudinal study . ,
torrance , ca , usa ) , and sap using the swedish interactive threshold algorithm ( sita standard 24 - 2 ) . patients using systemic or topical cholinergic or anticholinergic medications ( including pilocarpine ) that might affect pupil responses were excluded . an abnormal sap result was defined by a pattern standard deviation outside the 95% confidence limits ( ci ) or a glaucoma hemifield test result outside the reference range . the cirrus sdoct was used to acquire cprnfl , mgcipl , and total macular thickness measurements . the cprnfl thickness measurements were acquired using the optic disc cube 200 200 protocol using a 3-dimensional scan of a 6 6mm area centered on the optic disc . average cprnfl thickness was calculated automatically from a 3.46-mm diameter circular scan ( 10.87 mm length ) around the optic disc . the ganglion cell analysis algorithm automatically segmented the mgcipl , defined by the outer boundary of rnfl and outer boundary of inner plexiform layer . pupil responses were studied using the rapdx ( konan medical usa , inc . the stimulus then is presented as a series of trials , either to the full field of each eye or limited to predetermined regions . the full field white stimulus was presented for 200 ms of the 2.1-second duty cycle and 18 trials ( nine for each eye ) used this stimulus for a total test time of 37.8 seconds . the full - field stimuli had luminances of 384 ( white stimulus ) , 88 ( red stimulus ; 605 nm ) , 27 ( green stimulus ; 555 nm ) , 23 ( blue stimulus ; 440 nm ) , and 380 ( yellow stimulus ; 576 nm ) cd / m , and during all tests , there was a nominal background luminance of 0.01 cd / m . parameters measured by the pupillometer include prestimulus pupil diameter ( in millimeters ) , minimum pupil diameter following the stimulus ( in millimeters ) , response amplitude ( i.e. , maximal contraction of the pupil as a percentage of the prestimulation size , that is , the prestimulus pupil diameter minus the minimum pupil diameter , divided by the prestimulus pupil diameter ) , response latency ( time in milliseconds between stimulus onset and time when pupil velocity has reached 50% of the peak velocity of constriction ) , and time to peak constriction ( in milliseconds ) . an rapd is defined as a difference in average pupillary constriction ( response amplitude ) when each eye is stimulated monocularly . an index of the direction and magnitude of pupil response asymmetry , known as the rapd score , is generated automatically by the rapdx device . the rapd score is calculated as the difference in the amplitude of pupil constriction between stimulation of the two eyes using the following formula :
,
where od is the mean response amplitude in both eyes , in response to right eye stimulation , and os is the mean response amplitude in both eyes in response to left eye stimulation . the rapd score is useful as it confers information regarding the direction as well as the magnitude of an rapd . descriptive statistics included mean and standard deviation ( sd ) and t - tests for normally distributed variables ; and median , interquartile range , and wilcoxon rank - sum for nonparametrically distributed variables . the relationship between rapd scores and intereye differences ( right eye minus left eye ) in structural parameters was examined using scatter plots and linear regression . the average cprnfl thickness , mgcipl , macular thickness , and sap mean deviation ( md ) were included in the analyses to evaluate the effect of disease severity on asymmetry needed for an rapd . the level ( type i error )
this was a cross - sectional study including both eyes of 191 participants from the diagnostic innovations in glaucoma study ( digs ) , a previously described prospective longitudinal study . ,
torrance , ca , usa ) , and sap using the swedish interactive threshold algorithm ( sita standard 24 - 2 ) . patients using systemic or topical cholinergic or anticholinergic medications ( including pilocarpine ) that might affect pupil responses were excluded . eyes were classified as glaucomatous if they had repeatable ( 3 consecutive ) abnormal sap test results on the 24 - 2 humphrey visual field analyzer test ( carl zeiss meditec , inc . an abnormal sap result was defined by a pattern standard deviation outside the 95% confidence limits ( ci ) or a glaucoma hemifield test result outside the reference range . the cirrus sdoct was used to acquire cprnfl , mgcipl , and total macular thickness measurements . the cprnfl thickness measurements were acquired using the optic disc cube 200 200 protocol using a 3-dimensional scan of a 6 6mm area centered on the optic disc . average cprnfl thickness was calculated automatically from a 3.46-mm diameter circular scan ( 10.87 mm length ) around the optic disc . the ganglion cell analysis algorithm automatically segmented the mgcipl , defined by the outer boundary of rnfl and outer boundary of inner plexiform layer . pupil responses were studied using the rapdx ( konan medical usa , inc . the stimulus then is presented as a series of trials , either to the full field of each eye or limited to predetermined regions . the full field white stimulus was presented for 200 ms of the 2.1-second duty cycle and 18 trials ( nine for each eye ) used this stimulus for a total test time of 37.8 seconds . the full - field stimuli had luminances of 384 ( white stimulus ) , 88 ( red stimulus ; 605 nm ) , 27 ( green stimulus ; 555 nm ) , 23 ( blue stimulus ; 440 nm ) , and 380 ( yellow stimulus ; 576 nm ) cd / m , and during all tests , there was a nominal background luminance of 0.01 cd / m . parameters measured by the pupillometer include prestimulus pupil diameter ( in millimeters ) , minimum pupil diameter following the stimulus ( in millimeters ) , response amplitude ( i.e. , maximal contraction of the pupil as a percentage of the prestimulation size , that is , the prestimulus pupil diameter minus the minimum pupil diameter , divided by the prestimulus pupil diameter ) , response latency ( time in milliseconds between stimulus onset and time when pupil velocity has reached 50% of the peak velocity of constriction ) , and time to peak constriction ( in milliseconds ) . an rapd is defined as a difference in average pupillary constriction ( response amplitude ) when each eye is stimulated monocularly . an index of the direction and magnitude of pupil response asymmetry , known as the rapd score , is generated automatically by the rapdx device . the rapd score is calculated as the difference in the amplitude of pupil constriction between stimulation of the two eyes using the following formula :
,
where od is the mean response amplitude in both eyes , in response to right eye stimulation , and os is the mean response amplitude in both eyes in response to left eye stimulation . the rapd score is useful as it confers information regarding the direction as well as the magnitude of an rapd . descriptive statistics included mean and standard deviation ( sd ) and t - tests for normally distributed variables ; and median , interquartile range , and wilcoxon rank - sum for nonparametrically distributed variables . the relationship between rapd scores and intereye differences ( right eye minus left eye ) in structural parameters was examined using scatter plots and linear regression . the average cprnfl thickness , mgcipl , macular thickness , and sap mean deviation ( md ) were included in the analyses to evaluate the effect of disease severity on asymmetry needed for an rapd . the level ( type i error )
the study included 106 patients with glaucoma and 85 healthy controls . of those with glaucoma , 67 had glaucoma in both eyes , 33 had glaucoma in one eye and suspected glaucoma in the other , and 6 had glaucoma in one eye and a putative healthy fellow eye . baseline demographic and clinical characteristics of study patients ( mean sd ) subjects with glaucoma had an average md of 6.9 7.3 db in the worse eye and 2.9 4.4 db in the better eye , which was significantly worse than in healthy subjects ( p < 0.001 for both comparisons ) . the cprnfl , mgcipl , and macular thickness also were significantly thinner in the worse and better eyes of those with glaucoma compared to controls ( table 1 ) . those with glaucoma also had greater asymmetry in sap and sdoct measurements between eyes compared to healthy subjects . the mean ( sd ) intereye difference in md was 4.0 4.9 db in those with glaucoma compared to only 0.6 0.4 db in healthy subjects . intereye difference in cprnfl thickness was 9.2 7.6 m in patients with glaucoma , compared to only 3.8 3.6 m in healthy participants . corresponding intereye differences in mgcipl thickness were 6.8 6.8 and 1.7 1.8 m , respectively . the absolute rapd score averaged 0.30 0.35 in those with glaucoma compared to 0.15 0.10 in healthy subjects ( p = 0.006 ; table 2 ) . however , in the glaucomatous group , 42 of 106 subjects ( 40% ) had an absolute rapd score higher than 0.25 , 31 of 106 ( 29% ) had an absolute rapd score higher than 0.30 , and 22 ( 21% ) had an absolute rapd score higher than 0.50 . subjects with glaucoma also had significantly greater intereye differences in pupil response latency and time to peak pupil constriction compared to healthy subjects ( p = 0.031 and p = 0.006 , respectively ) . those with glaucoma also had , on average , a greater absolute rapd score for blue ( p = 0.013 ) , green ( p < 0.001 ) , yellow ( p < 0.001 ) , and red ( p <
pupillary reflex characteristics of healthy subjects compared to glaucoma patients ( mean sd ) histogram showing the distribution of absolute rapd scores in healthy eyes and those with glaucoma in at least one eye . there was a significant association between rapd scores and intereye differences in macular structural damage , as measured by intereye difference in mgcipl thickness ( r = 0.285 , p < 0.001 ) . the relationship between rapd score and intereye difference in macular thickness was weaker ( r = 0.167 , p < 0.001 ) than that with mgcipl . however , intereye differences in cprnfl thickness ( r = 0.350 , p < 0.001 ) and sap md ( r = 0.594 , p < 0.001 ) had stronger associations with rapd score than macular parameters ( table 3 , fig . results of univariable regression analysis of rapd score investigating the relationship between intereye difference ( , right minus left eye ) in cprnfl thickness , mgcipl thickness , macular thickness , and md from sap scatter plot showing rapd score association with intereye differences in cprnfl thickness ( a ) , mgcipl thickness ( b ) , macular thickness ( c ) , and sap md ( d ) . therefore ,
greater intereye difference in mgcipl thickness remained associated with greater absolute rapd score ( table 4 ) but worse average md also was associated with greater absolute rapd scores ( p < 0.001 ) . similar effects of disease severity also were noted on the association between rapd score with asymmetries in the other structural and functional variables , that is , intereye cprnfl thickness ( p < 0.001 ) ; absolute intereye sap md difference ( p < 0.001 ) , and absolute intereye macular thickness difference ( p < 0.001 ) . in multivariable analyses , age had no significant influence on the relationship between absolute rapd score and absolute intereye mgcipl thickness difference ( p = 0.560 ) , intereye differences in cprnfl thickness , macular thickness , or sap md ( p = 0.378 , p = 0.156 , and p = 0.888 , respectively ) . results of multivariable regression analysis of absolute rapd score and absolute intereye difference ( , right minus left eye ) in mgcipl thickness , including average md from sap as a covariable although the association between rapd scores obtained using colored stimuli and intereye differences in structural and functional parameters were significant , the associations with rapd score to the white stimulus generally were stronger ( table 5 , fig . figure 4 is an example of a patient with advanced glaucoma in the right eye and mild glaucoma in the left eye showing a rapd score of 1.21 , while figure 5 shows a patient with advanced glaucoma in both eyes and a rapd score of only 0.12 . results of univariable regression analysis of rapd score in different colors compared to intereye difference ( , right minus left eye ) in mgcipl , macular , cprnfl thickness , and md from sap scatter plot showing intereye difference of mgcipl thickness association with different colors for rapd score : red ( a ) , blue ( b ) , green ( c ) , and yellow ( md ) ( d ) . optical coherence tomography measurements of cprnfl ( a ) , mgcipl ( b ) , and total macular thickness ( c ) are shown . average response amplitude on right eye stimulation was 0.20 compared to 0.27 on left eye stimulation , giving an rapd score of 10*log10 ( 0.20/0.27 ) = 1.21 , indicating a relative abnormality of the right afferent pathway . optical coherence tomography measurements of cprnfl ( a ) , mgcipl ( b ) , and total macular thickness ( c ) are shown . the average response amplitude on right eye stimulation was 0.33 compared to 0.34 on left eye stimulation , giving an rapd score of 10*log10 ( 0.33/0.34 ) = 0.12 , indicating a small relative abnormality of the right afferent pathway . standard automated perimetry md in the right and left eyes was 21.36 and 16.6 db , respectively ( e ) . optical coherence tomography measurements of cprnfl ( a ) , mgcipl ( b ) , and total macular thickness ( c ) are shown . the average response amplitude on right eye stimulation was 0.32 compared to 0.33 on left eye stimulation , giving an rapd score of 10*log10 ( 0.32/0.33 ) = 0.09 , indicating a small relative abnormality of the right afferent pathway . standard automated perimetry md in the right and left eyes was 3.97 and 3.76 db , respectively ( e ) . this study has shown that the magnitude of rapd measured using an automated pupillometer is correlated with measures of macular structural asymmetry in glaucoma . subjects with larger rapds had greater intereye differences in macular thickness and mgcipl thickness , in addition to greater intereye differences in cprnfl and sap md . to our knowledge , this is the first study to evaluate the relationship between magnitude of rapd and macular structural damage in glaucoma . patients with glaucoma were more likely to have intereye asymmetry in structural and functional measurements than healthy controls , likely because , although glaucoma typically is a bilateral disease , changes often are worse in one eye than the other . patients with glaucoma also had larger rapd scores than controls , with average rapd scores of 0.30 0.35 and 0.15 0.10 , respectively . a significant association was seen between rapd and intereye difference in mgcipl ( r = 0.285 ; p < 0.001 ) . for each 10-m increase in mgcipl asymmetry
such association was stronger than that for total macular thickness , indicating that the mgcipl parameter was a more useful indicator of the degree of pupillary defect in glaucoma . the stronger association between rapd score and mgcipl asymmetry is likely to be due to total macular thickness including nonneuronal support tissues , not directly involved in the pupillary light response . in contrast , mgcipl thickness is a measurement of the inner layers containing rgcs . although asymmetries in macular measurements were associated with rapd , we found that the relationships between rapd score and asymmetry in cprnfl and sap md were stronger still , with r of 0.350 and 0.594 , respectively . as cprnfl thickness measurements and
sap md depend on the integrity of all rgc axons throughout the retina , this probably explains why these parameters showed higher association with pupillometry results . we found an rapd score of 0.50 is likely to correspond to intereye differences in cprnfl and sap md of approximately 23 m and 8 db , respectively , which are similar results to those reported previously using the swinging flashlight test , suggesting that an intereye difference in cprnfl thickness of 17% to 27% , or an intereye difference in sap md of 9.5 to 12 db , would correlate with an rapd of 0.3 to 0.6 . as one might expect , we also found that the relationship between magnitude of rapd and intereye difference in mgcipl thickness depends on disease severity , with worse average md associated with greater absolute rapd scores . it is important to note that in patients with advanced but symmetrical glaucoma in both eyes there is not likely to be an rapd . examined the ability of pupillometry to detect glaucoma and found an rapd in 56% in 70 patients with glaucoma , compared to only 29% using swinging flashlight test . further , although the swinging flashlight test is quick and easy to perform , it requires considerable practice to perform reliably and can be challenging in patients with dark irides or small or poorly reactive pupils . in the present study ,
we evaluated pupil responses to a range of colored stimuli but the rapd score to the white stimulus had the strongest association with all structural and functional parameters . however , patients with glaucoma did have greater rapd scores for blue , green , yellow , and red colored stimuli compared to healthy subjects and there was an association between rapd scores obtained using colored stimuli , and asymmetric structural and functional damage . these results are in agreement with previous studies that showed rapd scores using different wavelength stimuli were greater in glaucoma patients and correlated with intereye sap md difference . the magnitude of rapd can be influenced by factors other than glaucoma , such as brightness , location of the stimulus or time between stimuli . as another limitation of our study
however , we did not find any significant influence of age on the magnitude of the rapd score . in conclusion , we found a significant association between the magnitude of an rapd and intereye difference in mgcipl and macular thickness . | [
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] |
ischemic stroke is
a leading cause of mortality and morbidity in
the world . each year , 100000 people experience a new or recurrent
stroke . although the pathologic mechanisms
of the life - threatening disease are still not clear , it has been well
accepted that neuroinflammation is a key component of the pathogenic
cascade after ischemic damage in the brain . although the activated glia cells can encapsulate the infarct or
enhance neuronal plasticity , lymphocyte infiltration
is detrimental because of the hampering of axonal growth .
indeed , the inflammatory response during the
brain remodeling period after injury is considered to be a negative
factor for the regenerative process .
the long - lasting neuroinflammation
induced by wallerian degeneration in local infarction , the remote
area , and the connecting fibers could further lead to the loss of
brain function .
chemokine receptor 4 ( cxcr4 ) and stromal - cell - derived factor
1
( sdf-1 ) are constitutively expressed by microglia and astrocytes in
both developing and mature central neural systems ( cnss ) .
after experimental stroke , the level of expression
of sdf-1 is elevated for several days and closely associated with
the infiltration of cxcr4-expressing cells .
when ischemic injury happens , the level of expression of sdf-1 is
elevated in the peri - infarct and infarct region .
this phenomenon appears to be related to the initiation
of a cascade of events leading to neuroinflammation , such as local
microglia activation and peripheral monocyte and leukocyte migration
to the central neural system ( cns ) against a sdf-1 concentration gradient . in view of this
, the sdf-1 pathway has been proposed to be an attractive
pharmacological target for modulating the recruitment of immune cells
into the ischemic territory and improving functional recovery after
stroke . indeed , ruscher et al .
found
that inhibition of the sdf-1/cxcr4 pathway with amd3100 , a specific
antagonist of cxcr4 , suppresses the invasion of the ischemic territory
by peripheral immune cells , which promotes the recovery of function .
in another study ,
amd3100 significantly inhibited the inflammatory
response and reduced the extent of blood brain barrier disruption
after mcao by attenuating ischemia - induced acute inflammation by suppressing
leukocyte migration and infiltration .
the acute inflammatory responses consist of local microglia activation ,
peripheral immune cell migration , and pro - inflammatory cytokine and
chemokine secretion , while chronic inflammation in cns is mainly characterized
by angiogenesis , glial scar formation , and nutritious cytokine secretion .
therefore , a better understanding of these complicated
processes will be the starting point for developing curative treatments .
recently , pet imaging of translocator protein ( tspo ) has been investigated
as a noninvasive strategy for evaluating neuroinflammation .
tspo is an 18 kda protein that is localized at the outer mitochondrial
membrane of different cell types .
it is expressed
at low levels in healthy brain but is robustly upregulated in response
to injury and inflammation . in cerebral ischemia , the level of expression
of tspo is increased in activated microglia as well as in macrophages
and neutrophils entering the brain from the peripheral blood within
the first days after the insult . among
the various radiolabeled tspo ligands , [ f]dpa-714 shows
a comparatively high specificity and binding potential in different
disease models such as cerebral ischemia , herpes encephalitis , and
chemically induced acute neuroinflammation .
studies of
[ f]dpa-714 in healthy volunteers further confirm its
potential use in the diagnosis of cns diseases and perhaps therapeutic
efficacy monitoring . in this study
,
we developed a mouse mcao model and treated the
mice that had suffered strokes with amd3100 to alleviate injury - induced
neuroinflammation .
we expected to identify
the causal relationship between amd3100 treatment and the change in
tspo expression level after stroke and investigate its potential anti - inflammation
property .
the long - term goal of this study is to apply tspo pet imaging
for optimizing the anti - inflammatory intervention of ischemic brain
injuries .
[ f]f radionuclide was obtained from the national institutes of health
clinical center s cyclotron facility by proton irradiation
of o - enriched water .
[ f]fdg was purchased
from the nuclear pharmacy of cardinal health and diluted , as necessary ,
with sterile saline . automated synthesis of [ f]dpa-714
was conducted using a slightly modified tracerlab fx - fn module ( ge
medical systems ) .
all animal
studies were approved by the institutional animal care and use committee
of the clinical center of the national institutes of health .
all the
experiments were conducted in accordance with the principles and procedures
outlined in the guide for the care and use of laboratory animals .
transient mcao was induced in male balb / c
mice weighing 2530 g as previously reported with modifications .
briefly , mice were anesthetized in a ventilated
chamber with isoflurane ( 3.5% for induction and 2% for maintenance )
and then placed under a stereomicroscope in a supine position .
a warming
pad was used to keep the body temperature at 37 c during the
surgery .
a midline skin incision was made at the neck region followed
by careful exposure of the external , internal , and common carotid
arteries ( eca , ica , and cca , respectively ) .
one silicone - coated 6 - 0
mcao suture ( 602356pk10 , doccol mcao suture ) was gently advanced into
the ica to block the blood flow of the middle cerebral artery for
60 min .
after that , the suture was moved out for reperfusion of the
ischemic brain area .
sham - treated controls underwent artery exposure
without suture insertion . for treatment , amd3100 ( santa cruz
biotechnology ) was injected
intraperitoneally for three consecutive days with the first injection
performed immediately after the surgery with a dose of 1 mg / kg / day .
the mice in the control group were injected with
the same volume of saline .
all mri was performed
on a high - field micro - mr scanner ( 7.0 t , bruker , pharmascan ) with
a body coil .
mice were anesthetized with isofluorane ( 3.5% for induction
and 1.5% for maintenance ) and kept warm with a circulating water pad
that covered the whole coil .
magnetic t2-weighted images
were obtained by a rare sequence with a respiratory - gating technology .
the parameters were as follows : tr , 3000 ms ; effective te , 70 ms ;
nex , 4 ; matrix size , 256 256 ; fov , 4 cm 4 cm ; and slice
thickness , 1 mm .
for
the longitudinal study , [ f]fdg pet was performed 1 , 3 ,
10 , and 16 days after mcao ( n = 36 ) , while
static [ f]dpa-714 imaging was performed on days 0 , 1 ,
3 , 7 , 10 , and 16 with another group of mcao mice ( n = 46 ) .
another eight mice were used as sham controls for
[ f]fdg and [ f]dpa-714 imaging . for the acquisition
of static images , a single dose of 3.7 mbq ( 100 ci ) of [ f]fdg or [ f]dpa-714 was injected into animals
intravenously under isoflurane anesthesia .
one hour after the tracer
had been injected , a 15 min static pet scan was performed with a heating
pad to keep anesthetized animals warm .
for the [ f]dpa-714
displacement study , a 1 h dynamic acquisition was performed on mice
6 days after mcao with ( n = 3 ) or without ( n = 3 ) 5 mg of unlabeled pk11195/kg administered via an
intravenous catheter 30 min after tracer injection . for the treatment
study ,
static [ f]dpa-714 imaging with the same parameters
that were used in the longitudinal study was performed on 3 and 7
days after mcao surgery in both the amd3100-treated group ( n = 10 ) and the saline - treated control group ( n = 10 ) . all pet images were acquired with an inveon small animal
pet scanner ( siemens preclinical solutions ) .
all the images were reconstructed
using a two - dimensional ordered - subset expectation maximum algorithm
( 2d osem ) .
three - dimensional ellipsoidal regions of interest ( rois )
were manually defined on the ischemic region in the ipsilateral hemisphere
under the guidance of t2-weighted mr images of each mouse
obtained at the same time points of pet imaging .
another roi with
the same shape was drawn on the corresponding region in the contralateral
brain sphere as normal background signals .
the mean pixel value of
each roi was measured with inveon research workshop software ( siemens
preclinical solution ) .
the value was then converted to the concentration
of radioactivity in units of megabecquerels per milliliter .
the image - derived
tissue uptake , presented as percent injected dose per gram ( % id / g ) ,
was obtained with tissue radioactivity divided by injected dose assuming
a tissue density of 1 g / ml .
brain cryosections with
a thickness of 8 m were obtained using an ultrapro 5000 cryostat
( vibratome ) . both brain sections and cells
were fixed with z - fix solution
for 15 min and then blocked with phosphate - buffered saline ( pbs ) containing
1% bovine serum albumin ( bsa ) for 0.5 h. slices were incubated with
primary antibodies at 4 c overnight and with secondary antibodies
in a dark area for 60 min at room temperature . after each step ,
slices
were washed gently three times with pbs containing 0.05% tween 20
( pbst ) for 5 min . for different staining targets , the primary antibodies
and concentrations are as follows : rat anti - mouse tspo antibody ( 1:100 ;
abcam ) , rabbit anti - mouse cd11b antibody ( 1:100 ; abcam ) , goat anti - mouse
gfap antibody ( 1:100 ; abcam ) , rabbit anti - mouse macrophage antibody
( 1:100 ; abcam ) , rat anti - mouse myeloperoxidase ( mpo ) antibody ( 1:100 ;
abcam ) , and rabbit anti - mouse cxcr4 antibody ( 1:100 ; abcam ) .
secondary
antibodies are cy3-conjugated donkey anti - rabbit secondary antibody
( 1:200 ; jackson immunoresearch laboratories ) , dylight 488-conjugated
donkey anti - rabbit secondary antibody ( 1:200 ; jackson immunoresearch
laboratories ) , and cy3-conjugated donkey anti - goat secondary antibody
( 1:200 ; jackson immunoresearch laboratories ) .
all brain tissue slices
were mounted with medium containing 4,6-diamidino-2-phenylindole
( dapi ) and then observed with an epifluorescence microscope ( x81 ;
olympus ) .
brain tissues were sonicated in t - per
buffer ( thermo scientific ) containing a protease inhibitor cocktail
( roche ) .
after that , the protein suspension ( 40 g ) was separated
on a 4 to 12% bis - tris gel using sodium dodecyl sulfate
the membrane was blocked with blocking buffer ( thermo scientific )
for 1 h at room temperature and incubated with specific primary antibody
for tspo ( 1:5000 ; abcam ) or sdf-1 ( 1:1000 ; abcam ) overnight at 4 c .
after being washed three times with tris - buffered saline containing
0.05% tween 20 , the membrane was subjected to a horseradish peroxidase - conjugated
secondary antibody and detected by a supersignal west pico chemiluminescence
kit detection system ( pierce ) .
all data were expressed as means
the standard deviation ( means sd ) .
statistical analysis
was performed with spss software ( version 18.0 , spss , inc . ,
one - way analysis of variance and a student s t test were used for comparison of data between multiple groups and
two groups , respectively .
the successful
establishment of the mcao model was confirmed by t2-weighted
mri ( figure s1 of the supporting
information ) .
the high signal intensity that was observed in
brain parenchyma was recognized as ischemic area and used as a reference
to draw roi on the corresponding pet images . a glucose uptake defect
on the infarcted site by [ f]fdg pet
can be seen on both
days 1 and 3 , followed by an obviously increased level of signal uptake
on day 10 . on day 16 , the magnitude of the signal in the ischemic
area dropped to a level similar to that in the sham ( figure s2 of
the supporting information ) . to evaluate
tspo overexpression in mcao mice without any therapeutic intervention ,
the longitudinal pet using [
f]dpa-714 was performed on
0 , 1 , 3 , 7 , 10 , and 16 days after surgery . as shown in figure 1a , starting from day 3 , an increased level of local
uptake of [ f]dpa-714 in the ischemic area was observed ,
which peaked around day 10 , and then the signal intensity dropped
slowly until day 16 . to further confirm the location of the increased
signal intensity on pet and facilitate the quantification of data ,
we performed t2-weighted mri before pet imaging .
as shown
in figure 1b , the fused images clearly showed
a colocalization of the region of enhanced [ f]dpa-714
accumulation from pet and the ischemic area identified by mri .
obviously ,
a high signal intensity can be observed on both the cortex and the
basal area .
the lesion volume decreased gradually along with the pathologic
progress , indicating the self - recovery and scar formation at late
time points .
( a ) representative
coronal pet images of [ f]dpa-714 at the lesion area after
surgery .
the white dashed line shows the brain area and the white
arrow the damaged region .
( b ) representative coronal t2-weighted mri , [ f]dpa-714 pet , and coregistered images
at different time points after mcao surgery .
the uptake
ratio increased significantly from day 1 to day 16 compared with the
baseline level before surgery ( * p < 0.05 ; * * p < 0.01 ) .
quantitative results showed a significantly higher lesion - to - normal
ratio in mcao mice 1 day after surgery ( 1.37 0.09 ) compared
with the baseline level before surgery ( 1.09 0.19 ) ( p < 0.05 ) .
the ratio increased to 1.57 0.17 , 1.67
0.29 , and 2.04 0.38 on days 3 , 7 , and 10 , respectively .
after that , the lesion - to - normal uptake ratio value dropped slightly
to 1.80 0.30 on day 16 , which was still significantly higher
than that in the control mice ( figure 1c ) . to confirm the increased signal intensity in the ischemic area
is a result of specific binding of [ f]dpa-714 to tspo ,
a displacement study with unlabeled pk11195 , a competitive tspo ligand ,
was performed on three mcao mice 6 days after surgery . as shown in
figure 2a , within a few minutes of injection
of the tracer , the time
activity curve over the lesion area
reached a plateau and showed a very slow downslope until 60 min . upon
injection of pk11195 at 30 min during the dynamic acquisition ,
rois over the contralateral
brain tissue generated a tac with a much lower amplitude , and pk11195
injection showed a much less significant effect ( figure 2a ) .
the signal intensity changes were also identified on pet
images acquired before and after pk11195 displacement ( figure 2b ) .
the amount of tracer uptake was significantly
decreased after displacement at the lesion area [ 3.89 0.63%id / g
vs 1.95 0.77%id / g ( p < 0.05 ) ] but not in
contralateral brain tissue [ 1.90 0.30%id / g vs 1.62 0.80%id / g
( p > 0.05 ) ] ( figure 2c ) .
( a ) [ f]dpa-714 displacement study using 5 mg of pk11195/kg
6 days after stroke .
time activity curves of roi placed on
both the lesion and contralateral mirror area with or without pk11195
displacement , expressed as % id / g of tissue .
the arrow indicates the
time point of pk11195 injection during the 1 h dynamic pet acquisition .
( b ) representative coronal [ f]dpa-714 pet images of the
brain acquired before and after pk11195 displacement .
( c ) [ f]dpa-714 uptake ( % id / g ) of the lesion and healthy brain tissue before
and after pk11195 injection ( * p < 0.05 ) .
because
amd3100 treatment interferes with the inflammatory process after ischemic
brain injury , we performed in vivo pet imaging on
both treated and control mice to follow the change in the level of
tspo .
three consecutive doses of treatment using amd3100 did not fully
block tspo upregulation in the ischemic area .
however , an apparently
reduced [ f]dpa-714 signal intensity and signal volume
can be seen in the amd3100-treated group 3 days after surgery ( figure 3a ) , with a lesion - to - normal ratio of 1.47
0.14 versus a value of 1.73 0.17 ( p < 0.05 ,
compared with the saline only group ) . on day 7 , the local level of
tracer uptake is still somewhat lower in amd3100-treated mice , but
there was no significant difference between the two groups [ 1.64
0.17 vs 1.75 0.36 ( p > 0.05 ) ] ( figure 3b ) . [
( a ) representative coronal pet images of the amd3100-treated
group and normal saline - treated control group .
( b ) quantification
of the lesion - to - normal ratio showing a decreased signal intensity
after amd3100 treatment on days 3 and 7 ( * p <
0.05 ) .
( c ) western blot assay showing the reduced tspo protein level
and sdf-1 expression in the amd3100-treated group at targeted time
points . to confirm the pet findings , we
collected the brain tissue and
performed a western blot study using tspo specific antibodies . in
normal brain tissues
three days after surgery , the tspo level increased dramatically and
amd3100 treatment reduced the tspo protein level .
seven days after
surgery , the tspo level was still high and a decreased tspo protein
level in the amd3100-treated group can be observed , but to a lesser
extent ( figure 3c ) . to evaluate the sdf-1 expression
after amd3100 treatment
, we performed a western blot study in brain
tissue with and without amd3100 treatment on days 3 and 7 .
there was
a low level of sdf-1 expression in healthy brain tissue and a significantly
increased level of sdf-1 expression 3 and 7 days after stroke , and
the sdf-1 level was decreased after amd3100 treatment . during
neuroinflammation ,
tspo upregulation was identified on several types
of cells , including activated microglia , infiltrated neutrophils and
macrophages , and astrocytes .
accordingly , we evaluated tspo expression
on brain slices harvested at different time points after mcao , and
cd11b and gfap were chosen as markers for microglia and astrocytes ,
respectively .
we found that the intensity of tspo positive cells increased
along with time with a peak appearing on day 10 , which showed a time
course similar to that for in vivo pet results ( figure 4 ) .
costaining with cd11b and gfap revealed that
microglia were the main cell type of tspo positive cells at early
time points .
however , local astrocytes can also overexpress tspo at
late stages of cerebral ischemia . ( a )
immunofluorescence staining of brain
tissues harvested at different
time points after mcao surgery with anti - tspo ( red ) and anti - cd11b
( green ) antibodies .
( b ) immunofluorescence staining of brain slices
at different time points after stroke with anti - tspo ( red ) and anti - gfap
( green ) antibodies .
nuclei are counterstained with dapi ( blue ) . with amd3100 treatment , the decreased
intensity of tspo positive
cells in ischemic mouse brain
was observed on day 3 . on day 7 , both
treated and untreated groups presented a higher level of tspo positive
cells , but there was no significant difference between the two ( figure 5a ) . to further differentiate the source of tspo
protein , we performed immunofluorescence staining of macrophages and
leukocytes .
as shown in figure 5b , amd3100
treatment partially inhibited the accumulation of both macrophages
and leukocytes in infarcted brain parenchyma on day 3 , and the difference
was diminished by day 7 .
the colocalized staining of macrophages and
tspo was performed on brain slices harvested on day 7 .
tspo is positive
for infiltrated macrophages after stroke but not limited to macrophages
( figure s4 of the supporting information ) .
( a ) immunofluorescence staining of brain tissues from both the
amd3100-treated group and the control group with anti - tspo ( red ) and
anti - cd11b ( green ) antibodies .
( b ) immunofluorescence staining of
brain slices from both the amd3100-treated group and the control group
with anti - macrophage and anti - mpo antibodies .
tspo
can be overexpressed on activated microglia and other cell
types when the homogeneity in cns is broken by internal or external
factors .
hence , it has been intensively investigated as an imaging
target for monitoring the inflammatory responses in different types
of brain disease , including cerebral ischemia .
in particular , inhibition of
the sdf-1/cxcr4 pathway during the acute stage after stroke promotes
long - term recovery , mainly by depressing the extent of invasion of
the ischemic territory by peripheral immune cells . therefore , for the first time , to the best of our knowledge ,
we applied [ f]dpa-714 pet in monitoring the change in
neuroinflammation induced by amd3100 treatment in a mouse brain ischemia / reperfusion
model .
consistent with previous studies , the
time frame of the overexpression of tspo can be dynamically monitored
via longitudinal pet imaging using [ f]dpa-714 as the
imaging tracer .
the specificity of [ f]dpa-714 to tspo was confirmed by a displacement study with
a mass amount of unlabeled pk11195 . the local level of accumulation
of [ f]dpa-714 increased significantly in ipsilateral
ischemic side starting from day 3 after surgery and reached a peak
on day 10 .
our
study ended on day 16 after surgery instead of day 30 as in the previous
report .
it is partially because mice
are less tolerant to brain ischemia injury than rats .
the high mortality
rate of the model makes it difficult for long - term observation .
after ischemic injury in brain , the infarcted center and surrounding
edema area have an increased amount of intracellular and extracellular
water , leading to an obvious signal change on t2-weighted
mri .
coregistration of [ f]dpa-714 pet images with t2-weighted mri could provide
anatomical reference to locate the pet signal . in most cases
, we saw
a relatively good overlay of the increased magnitude of the pet signal
within the lesion site indicated by t2-weighted mri .
however ,
at a late time point ( day 16 ) , the lesion area presented on mri images
was much smaller than the signal increased area on pet images .
this
may be due to the alleviation of edema at the late stage of the injury ,
and t2-weighted mri is not sufficiently sensitive to detect
the lesion site .
the small molecule
sdf-1/cxcr4 inhibitor , amd3100 , has been shown
to have a promising effect in attenuating ischemic injury in different
animal models such as myocardial infarction , hindlimb ischemia , and
stroke .
the therapeutic
effect of amd3100 results from a reduction of the extent of infiltration
of ischemic brain parenchyma byperipheral inflammatory cells . with
[ f]dpa-714 pet
, we observed a significant decrease in
the magnitude of the signal after amd3100 treatment on day 3 , indicating
that amd3100 treatment downregulated the local tspo protein level .
this result
however , the magnitude
of the decrease in the signal intensity on pet images did not match
perfectly with that from western blots .
in addition , the tspo level
decreased on day 7 with amd3100 treatment on western blot but was
not detectable with pet .
we speculate that this is partially due to
the small size of the mouse brain and partial volume effect of pet
imaging .
it may also be related to the
small dose of amd3100 and the short drug delivery period .
a decreased sdf-1 level was also observed after
amd3100 treatment , as shown by the western blot study .
overexpression
of tspo in the ischemic brain area has been correlated
with pathophysiological heterogeneity , such as microglia and astrocyte
activation .
infiltrated peripheral immune
cells , such as monocytes , leukocytes , and lymphocytes , also contribute
to the overexpression of tspo .
the signal
of pet reflects the overall level of tspo , which has no ability to
differentiate the origination of the protein .
because amd3100 can
inhibit the sdf-1/cxcr4 pathway , resulting in decreased levels of
migration of macrophages and/or leukocytes , it is highly possible
that the decrease in the magnitude of the signal on pet images is
caused by a decreased number of infiltrated peripheral lymphocytes .
however , because of the nonspecificity of the cell maker of these
cells , we can not exclude the contribution
of activated microglia and astrocytes to the pet signal change .
because of the complexity of the neuroinflammatory response
after
brain injury and intertwining of various cytokines and inflammatory
cells , the dose and timing of therapeutic intervention would be very
critical to the exertion of a beneficial effect and the prevention
of any detrimental consequence in both acute and chronic phases .
although
amd3100 is promising in the treatment of experimental stroke , further
investigation to optimize the therapy regimen is still needed for
further clinical translation .
we believe the accurate evaluation of
neuroinflammation with noninvasive imaging strategy would accelerate
this process .
tspo - targeted pet imaging
using [ f]dpa-714 can be
used to monitor the inflammatory response after stroke dynamically
and noninvasively .
herein , [ f]dpa-714 can be a valuable
probe for understanding the pathological process of neuroinflammation
after stroke and provide a useful method for evaluating the therapeutic
efficiency of the anti - inflammation treatment . | chemokine
receptor 4 and stromal - cell - derived factor 1 have been
found to be related to the initiation of neuroinflammation in ischemic
brain . herein , we aimed to monitor the changes of neuorinflammation
after amd3100 treatment using a translocator protein ( tspo ) specific
pet tracer in a mouse model of stroke .
the transient mcao model was
established with balb / c mice .
the success of the model was confirmed
by magnetic resonance imaging and fdg pet .
the treatment started the
same day after surgery via daily intraperitoneal injection of 1 mg
of amd3100/kg for three consecutive days .
[ 18f]dpa-714
was used as the tspo imaging tracer . in vivo pet
was performed at different time points after surgery in both control
and treated mice .
ex vivo histological and immunofluorescence
staining of brain slices was performed to confirm the lesion site
and inflammatory cell activation .
the tspo level was also evaluated
using western blotting .
longitudinal pet scans revealed that the level
of [ 18f]dpa-714 uptake was significantly increased in the
ischemic brain area with a peak accumulation at around day 10 after
surgery , and the level of uptake remained high until day 16 .
the in vivo pet data were consistent with those from ex vivo immunofluorescence staining .
after amd3100 treatment ,
the signal intensity was significantly decreased compared with that
of normal saline - treated control group . in conclusion , tspo - targeted
pet imaging using [ 18f]dpa-714 can be used to monitor inflammatory
response after stroke and provide a useful method for evaluating the
efficacy of anti - inflammation treatment . | Introduction
Materials and Methods
Results
Discussion
Conclusions | although the pathologic mechanisms
of the life - threatening disease are still not clear , it has been well
accepted that neuroinflammation is a key component of the pathogenic
cascade after ischemic damage in the brain . indeed , the inflammatory response during the
brain remodeling period after injury is considered to be a negative
factor for the regenerative process . the long - lasting neuroinflammation
induced by wallerian degeneration in local infarction , the remote
area , and the connecting fibers could further lead to the loss of
brain function . chemokine receptor 4 ( cxcr4 ) and stromal - cell - derived factor
1
( sdf-1 ) are constitutively expressed by microglia and astrocytes in
both developing and mature central neural systems ( cnss ) . after experimental stroke , the level of expression
of sdf-1 is elevated for several days and closely associated with
the infiltration of cxcr4-expressing cells . when ischemic injury happens , the level of expression of sdf-1 is
elevated in the peri - infarct and infarct region . this phenomenon appears to be related to the initiation
of a cascade of events leading to neuroinflammation , such as local
microglia activation and peripheral monocyte and leukocyte migration
to the central neural system ( cns ) against a sdf-1 concentration gradient . in view of this
, the sdf-1 pathway has been proposed to be an attractive
pharmacological target for modulating the recruitment of immune cells
into the ischemic territory and improving functional recovery after
stroke . recently , pet imaging of translocator protein ( tspo ) has been investigated
as a noninvasive strategy for evaluating neuroinflammation . in cerebral ischemia , the level of expression
of tspo is increased in activated microglia as well as in macrophages
and neutrophils entering the brain from the peripheral blood within
the first days after the insult . studies of
[ f]dpa-714 in healthy volunteers further confirm its
potential use in the diagnosis of cns diseases and perhaps therapeutic
efficacy monitoring . in this study
,
we developed a mouse mcao model and treated the
mice that had suffered strokes with amd3100 to alleviate injury - induced
neuroinflammation . we expected to identify
the causal relationship between amd3100 treatment and the change in
tspo expression level after stroke and investigate its potential anti - inflammation
property . the long - term goal of this study is to apply tspo pet imaging
for optimizing the anti - inflammatory intervention of ischemic brain
injuries . transient mcao was induced in male balb / c
mice weighing 2530 g as previously reported with modifications . a midline skin incision was made at the neck region followed
by careful exposure of the external , internal , and common carotid
arteries ( eca , ica , and cca , respectively ) . after that , the suture was moved out for reperfusion of the
ischemic brain area . for treatment , amd3100 ( santa cruz
biotechnology ) was injected
intraperitoneally for three consecutive days with the first injection
performed immediately after the surgery with a dose of 1 mg / kg / day . the mice in the control group were injected with
the same volume of saline . all mri was performed
on a high - field micro - mr scanner ( 7.0 t , bruker , pharmascan ) with
a body coil . for
the longitudinal study , [ f]fdg pet was performed 1 , 3 ,
10 , and 16 days after mcao ( n = 36 ) , while
static [ f]dpa-714 imaging was performed on days 0 , 1 ,
3 , 7 , 10 , and 16 with another group of mcao mice ( n = 46 ) . for the treatment
study ,
static [ f]dpa-714 imaging with the same parameters
that were used in the longitudinal study was performed on 3 and 7
days after mcao surgery in both the amd3100-treated group ( n = 10 ) and the saline - treated control group ( n = 10 ) . three - dimensional ellipsoidal regions of interest ( rois )
were manually defined on the ischemic region in the ipsilateral hemisphere
under the guidance of t2-weighted mr images of each mouse
obtained at the same time points of pet imaging . another roi with
the same shape was drawn on the corresponding region in the contralateral
brain sphere as normal background signals . the image - derived
tissue uptake , presented as percent injected dose per gram ( % id / g ) ,
was obtained with tissue radioactivity divided by injected dose assuming
a tissue density of 1 g / ml . for different staining targets , the primary antibodies
and concentrations are as follows : rat anti - mouse tspo antibody ( 1:100 ;
abcam ) , rabbit anti - mouse cd11b antibody ( 1:100 ; abcam ) , goat anti - mouse
gfap antibody ( 1:100 ; abcam ) , rabbit anti - mouse macrophage antibody
( 1:100 ; abcam ) , rat anti - mouse myeloperoxidase ( mpo ) antibody ( 1:100 ;
abcam ) , and rabbit anti - mouse cxcr4 antibody ( 1:100 ; abcam ) . the successful
establishment of the mcao model was confirmed by t2-weighted
mri ( figure s1 of the supporting
information ) . a glucose uptake defect
on the infarcted site by [ f]fdg pet
can be seen on both
days 1 and 3 , followed by an obviously increased level of signal uptake
on day 10 . on day 16 , the magnitude of the signal in the ischemic
area dropped to a level similar to that in the sham ( figure s2 of
the supporting information ) . to evaluate
tspo overexpression in mcao mice without any therapeutic intervention ,
the longitudinal pet using [
f]dpa-714 was performed on
0 , 1 , 3 , 7 , 10 , and 16 days after surgery . as shown in figure 1a , starting from day 3 , an increased level of local
uptake of [ f]dpa-714 in the ischemic area was observed ,
which peaked around day 10 , and then the signal intensity dropped
slowly until day 16 . to further confirm the location of the increased
signal intensity on pet and facilitate the quantification of data ,
we performed t2-weighted mri before pet imaging . as shown
in figure 1b , the fused images clearly showed
a colocalization of the region of enhanced [ f]dpa-714
accumulation from pet and the ischemic area identified by mri . obviously ,
a high signal intensity can be observed on both the cortex and the
basal area . the lesion volume decreased gradually along with the pathologic
progress , indicating the self - recovery and scar formation at late
time points . ( a ) representative
coronal pet images of [ f]dpa-714 at the lesion area after
surgery . ( b ) representative coronal t2-weighted mri , [ f]dpa-714 pet , and coregistered images
at different time points after mcao surgery . the uptake
ratio increased significantly from day 1 to day 16 compared with the
baseline level before surgery ( * p < 0.05 ; * * p < 0.01 ) . quantitative results showed a significantly higher lesion - to - normal
ratio in mcao mice 1 day after surgery ( 1.37 0.09 ) compared
with the baseline level before surgery ( 1.09 0.19 ) ( p < 0.05 ) . after that , the lesion - to - normal uptake ratio value dropped slightly
to 1.80 0.30 on day 16 , which was still significantly higher
than that in the control mice ( figure 1c ) . to confirm the increased signal intensity in the ischemic area
is a result of specific binding of [ f]dpa-714 to tspo ,
a displacement study with unlabeled pk11195 , a competitive tspo ligand ,
was performed on three mcao mice 6 days after surgery . as shown in
figure 2a , within a few minutes of injection
of the tracer , the time
activity curve over the lesion area
reached a plateau and showed a very slow downslope until 60 min . upon
injection of pk11195 at 30 min during the dynamic acquisition ,
rois over the contralateral
brain tissue generated a tac with a much lower amplitude , and pk11195
injection showed a much less significant effect ( figure 2a ) . the signal intensity changes were also identified on pet
images acquired before and after pk11195 displacement ( figure 2b ) . the amount of tracer uptake was significantly
decreased after displacement at the lesion area [ 3.89 0.63%id / g
vs 1.95 0.77%id / g ( p < 0.05 ) ] but not in
contralateral brain tissue [ 1.90 0.30%id / g vs 1.62 0.80%id / g
( p > 0.05 ) ] ( figure 2c ) . ( a ) [ f]dpa-714 displacement study using 5 mg of pk11195/kg
6 days after stroke . time activity curves of roi placed on
both the lesion and contralateral mirror area with or without pk11195
displacement , expressed as % id / g of tissue . because
amd3100 treatment interferes with the inflammatory process after ischemic
brain injury , we performed in vivo pet imaging on
both treated and control mice to follow the change in the level of
tspo . three consecutive doses of treatment using amd3100 did not fully
block tspo upregulation in the ischemic area . however , an apparently
reduced [ f]dpa-714 signal intensity and signal volume
can be seen in the amd3100-treated group 3 days after surgery ( figure 3a ) , with a lesion - to - normal ratio of 1.47
0.14 versus a value of 1.73 0.17 ( p < 0.05 ,
compared with the saline only group ) . [
( a ) representative coronal pet images of the amd3100-treated
group and normal saline - treated control group . ( b ) quantification
of the lesion - to - normal ratio showing a decreased signal intensity
after amd3100 treatment on days 3 and 7 ( * p <
0.05 ) . ( c ) western blot assay showing the reduced tspo protein level
and sdf-1 expression in the amd3100-treated group at targeted time
points . to confirm the pet findings , we
collected the brain tissue and
performed a western blot study using tspo specific antibodies . in
normal brain tissues
three days after surgery , the tspo level increased dramatically and
amd3100 treatment reduced the tspo protein level . seven days after
surgery , the tspo level was still high and a decreased tspo protein
level in the amd3100-treated group can be observed , but to a lesser
extent ( figure 3c ) . to evaluate the sdf-1 expression
after amd3100 treatment
, we performed a western blot study in brain
tissue with and without amd3100 treatment on days 3 and 7 . there was
a low level of sdf-1 expression in healthy brain tissue and a significantly
increased level of sdf-1 expression 3 and 7 days after stroke , and
the sdf-1 level was decreased after amd3100 treatment . accordingly , we evaluated tspo expression
on brain slices harvested at different time points after mcao , and
cd11b and gfap were chosen as markers for microglia and astrocytes ,
respectively . we found that the intensity of tspo positive cells increased
along with time with a peak appearing on day 10 , which showed a time
course similar to that for in vivo pet results ( figure 4 ) . costaining with cd11b and gfap revealed that
microglia were the main cell type of tspo positive cells at early
time points . ( a )
immunofluorescence staining of brain
tissues harvested at different
time points after mcao surgery with anti - tspo ( red ) and anti - cd11b
( green ) antibodies . ( b ) immunofluorescence staining of brain slices
at different time points after stroke with anti - tspo ( red ) and anti - gfap
( green ) antibodies . with amd3100 treatment , the decreased
intensity of tspo positive
cells in ischemic mouse brain
was observed on day 3 . to further differentiate the source of tspo
protein , we performed immunofluorescence staining of macrophages and
leukocytes . as shown in figure 5b , amd3100
treatment partially inhibited the accumulation of both macrophages
and leukocytes in infarcted brain parenchyma on day 3 , and the difference
was diminished by day 7 . the colocalized staining of macrophages and
tspo was performed on brain slices harvested on day 7 . ( a ) immunofluorescence staining of brain tissues from both the
amd3100-treated group and the control group with anti - tspo ( red ) and
anti - cd11b ( green ) antibodies . ( b ) immunofluorescence staining of
brain slices from both the amd3100-treated group and the control group
with anti - macrophage and anti - mpo antibodies . in particular , inhibition of
the sdf-1/cxcr4 pathway during the acute stage after stroke promotes
long - term recovery , mainly by depressing the extent of invasion of
the ischemic territory by peripheral immune cells . therefore , for the first time , to the best of our knowledge ,
we applied [ f]dpa-714 pet in monitoring the change in
neuroinflammation induced by amd3100 treatment in a mouse brain ischemia / reperfusion
model . consistent with previous studies , the
time frame of the overexpression of tspo can be dynamically monitored
via longitudinal pet imaging using [ f]dpa-714 as the
imaging tracer . the specificity of [ f]dpa-714 to tspo was confirmed by a displacement study with
a mass amount of unlabeled pk11195 . the local level of accumulation
of [ f]dpa-714 increased significantly in ipsilateral
ischemic side starting from day 3 after surgery and reached a peak
on day 10 . our
study ended on day 16 after surgery instead of day 30 as in the previous
report . the high mortality
rate of the model makes it difficult for long - term observation . in most cases
, we saw
a relatively good overlay of the increased magnitude of the pet signal
within the lesion site indicated by t2-weighted mri . however ,
at a late time point ( day 16 ) , the lesion area presented on mri images
was much smaller than the signal increased area on pet images . this
may be due to the alleviation of edema at the late stage of the injury ,
and t2-weighted mri is not sufficiently sensitive to detect
the lesion site . the therapeutic
effect of amd3100 results from a reduction of the extent of infiltration
of ischemic brain parenchyma byperipheral inflammatory cells . with
[ f]dpa-714 pet
, we observed a significant decrease in
the magnitude of the signal after amd3100 treatment on day 3 , indicating
that amd3100 treatment downregulated the local tspo protein level . this result
however , the magnitude
of the decrease in the signal intensity on pet images did not match
perfectly with that from western blots . in addition , the tspo level
decreased on day 7 with amd3100 treatment on western blot but was
not detectable with pet . we speculate that this is partially due to
the small size of the mouse brain and partial volume effect of pet
imaging . it may also be related to the
small dose of amd3100 and the short drug delivery period . a decreased sdf-1 level was also observed after
amd3100 treatment , as shown by the western blot study . overexpression
of tspo in the ischemic brain area has been correlated
with pathophysiological heterogeneity , such as microglia and astrocyte
activation . infiltrated peripheral immune
cells , such as monocytes , leukocytes , and lymphocytes , also contribute
to the overexpression of tspo . the signal
of pet reflects the overall level of tspo , which has no ability to
differentiate the origination of the protein . because amd3100 can
inhibit the sdf-1/cxcr4 pathway , resulting in decreased levels of
migration of macrophages and/or leukocytes , it is highly possible
that the decrease in the magnitude of the signal on pet images is
caused by a decreased number of infiltrated peripheral lymphocytes . however , because of the nonspecificity of the cell maker of these
cells , we can not exclude the contribution
of activated microglia and astrocytes to the pet signal change . because of the complexity of the neuroinflammatory response
after
brain injury and intertwining of various cytokines and inflammatory
cells , the dose and timing of therapeutic intervention would be very
critical to the exertion of a beneficial effect and the prevention
of any detrimental consequence in both acute and chronic phases . although
amd3100 is promising in the treatment of experimental stroke , further
investigation to optimize the therapy regimen is still needed for
further clinical translation . tspo - targeted pet imaging
using [ f]dpa-714 can be
used to monitor the inflammatory response after stroke dynamically
and noninvasively . herein , [ f]dpa-714 can be a valuable
probe for understanding the pathological process of neuroinflammation
after stroke and provide a useful method for evaluating the therapeutic
efficiency of the anti - inflammation treatment . | [
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] |
bone - tendon junction injuries are common problem in athletes . due to a change in lifestyle , there has been an increase in the occurrence of bone - tendon junction ( btj ) injuries like patellar tendinitis , lateral epicondylitis , rotator cuff injury and the rupture of the achilles tendon in recent years.123 as a result , there has been a rising interest in the research of btj - related injuries .
the btj generally consists of four groups of tissue , namely fibrous tissue , uncalcified fibrocartilage ( uf ) , calcified fibrocartilage ( cf ) , and bone .
based on the properties of the bone - tendon tissues , it can also be categorized into fibro - tissues and bone , with a spacing known as the fibrocartilage zone.4 the short transition distance between the soft and hard tissues results in a slower rate of recovery .
the fibrocartilage zone connecting the bone to the tendon at the btj plays an important role in the repair and reconstruction of the btj .
the successful reconstruction of the fibrocartilage zone will allow for an improvement in the histological and mechanical recovery of the btj .
however , related studies have shown that the reconstruction of the fibrocartilage zone is extremely slow and difficult.56 there are several traditional treatments for btj injuries .
in recent years , non - invasive techniques or therapies like low intensity pulsed ultrasound ( lipus ) , extracorporeal shock - wave therapy , and electrical stimulation have gained recognition as well.2 the sox9 gene , which belongs to a family of genes known as sox and can be found in the chromosome region 17q24ii-25,7 is crucial to the embryonic development in the early stages.8 sox9 is a transcription factor that has c - terminal high - mobility group ( hmg ) domain and n - terminal transactivation domain .
it participates in various embryonic developmental processes such as chondrogenesis.9 translocations upstream of the coding sequence suggested that the altered expression of sox9 is capable of severely impacting chondrogenesis and thus lead to abnormal skeletal development . as a transcriptional activator , the sox9 gene is able to co - regulate the proliferation and differentiation of the chondrocytes together with other signal pathways through direct or indirect means.1011 this can be achieved through the combination with the chondrocytes - specific enhancer type ii collagen gene ( col2al ) and l - sox5 , sox6 .
currently , main treatments for bone - tendon healing include surgical treatment and conservative therapy .
however , there are obvious limitations to these treatments.12 gene therapy involves the use of gene transfer techniques to replace a mutated gene with an exogenous gene that encodes a therapeutic protein in order to treat certain diseases13 previous studies have confirmed that the target genes of sox9 promote the healing of bone and cartilage damage.1314 therefore , the synthesis of various matrix components can be regulated by the insertion or transformation of the therapeutic gene sox9 .
the gene therapy should ideally provide a precise transference of the target gene to target cells to achieve an efficient , specific , and controllable expression.15 however , at present , the transfers of such genes have poor tissue specificity , which leads to problems like low transfer efficiency and difficulties in gene expression . there is an extensive use of adenovirus in current gene therapies due to advantages such as susceptibility to infection , effective expression , high stability , high genome capacity , propensity to proliferate and purification and high viral titer.16 in this study , we injected sox9 into an established bone - tendon healing model and aimed to observe its effect on the healing of the btj by determining the biomechanical properties of the junction .
48 adult new zealand white rabbits ( 18 weeks old ) , weighing between 2.0 and 2.9 kg , were randomly divided into four equal groups : a , b , c , and d. the research protocol was approved in accordance with the institutional guidelines of the animal care and use committee at wuhan university .
the animals received an intravenous injection of 2.5% pentobarbital sodium ( 0.5 ml ) and 10% ketamine hydrochloride ( 0.5 ml / kg ) into the marginal ear vein before the surgery .
establishment of animal model was reported in previous report.17 during our study , we found that simply injecting sox9 gene resulted in low transcript and using adenovirus vector could improve this situation to a certain level . in our study , we constructed recombinant adenovirus vector sox9 , adsox9 , and it was used to treat btj .
sox9 was injected into the btj of rabbits in group a and adsox9 was transfected into the btj of rabbits in group c. rabbits in group b served as control and did not receive any injection during surgery .
rabbits in group d were transfected with adenovirus and served as a control for group c. for the rabbits in group a , the seam between the bone and tendon was injected with a concentration of 200 ng / ml sox9 that was dissolved in 0.9% sodium chloride ( sigma , mdl number : mfcd03097288 , product number : s4444 ) .
the btjs of the rabbits in group c were injected with adsox9 that had been previously diluted to a titer of 3.0 106 pfu / ml .
the epidermis of the right lower limb of the rabbits in all groups was sutured after the incision and they served as false - positive groups .
the operated limbs were fixed with plaster casts and the rabbits received 400,000 u / kg / d of penicillin via intramuscular injection 3 days after surgery . the surgical incision and blood supply in the limbs were observed .
the plaster casts were removed 4 weeks later and the rabbits were allowed to roam freely within the cage .
four groups of rabbits were sacrificed 4 weeks , 8 weeks , and 12 weeks after surgery .
after the rabbits in group a were sacrificed , two were selected for biomechanical testing and two for histological examinations .
the btj of the rabbits in group c were divided into two parts : one part was subjected to biomechanical testing ( two samples ) and the other part was subjected to histological examinations ( two samples ) . after the biomechanical testing was conducted , the samples were collected and used for mrna detection .
the experimental limb was extracted whole from the quadriceps femoris to the tibia while muscles and other tissues from the attachment points were detached .
next , the distal patellar tendon was removed from the tibial tubercle and the patella and patellar ligament were kept intact .
the structure , comprising of the patellar ligament and the patella with some quadriceps femoris attached , was sliced along the middle in a vertical axis .
the structure was later fixed with 10% formaldehyde for 24 hours and decalcified with 10% formic acid for 4 weeks .
it was subsequently dehydrated with progressively concentrated amounts of ethanol , treated with clearing and infiltration agents and then embedded in paraffin .
the specimen was stained using the hematoxylin and eosin stain ( h and e stain ) and the morphology of the btj osteocytes , chondrocytes , and tendon tissue ground substance were observed under a light microscope . the connective tissues and sutures from the surgery surrounding the knee of the specimen were first removed . using a vernier caliper , triplicate measurements of the thickness , width , and cross section of the healing surface from the end of the quadriceps femoris to the proximal tibial osteotomy
the end of the distal quadriceps femoris and the proximal tibial osteotomy were secured on a fixture and a 10 anteversion angle between the tibia and the load bearing axis was created .
the structure was then pulled at a velocity of 20 mm / min using a dynamometer with the force of 2 kn and the breaking load was recorded . the rupture surface created during experiment
total rna was extracted from 100 mg of btj tissues and the rt - pcr was used to identify the sox9 gene expression .
statistical analysis was performed with 4 2 factorial analysis of variance between the treatment and control groups using the spss 13.0 statistical software .
the animals received an intravenous injection of 2.5% pentobarbital sodium ( 0.5 ml ) and 10% ketamine hydrochloride ( 0.5 ml / kg ) into the marginal ear vein before the surgery .
establishment of animal model was reported in previous report.17 during our study , we found that simply injecting sox9 gene resulted in low transcript and using adenovirus vector could improve this situation to a certain level . in our study , we constructed recombinant adenovirus vector sox9 , adsox9 , and it was used to treat btj .
sox9 was injected into the btj of rabbits in group a and adsox9 was transfected into the btj of rabbits in group c. rabbits in group b served as control and did not receive any injection during surgery .
rabbits in group d were transfected with adenovirus and served as a control for group c. for the rabbits in group a , the seam between the bone and tendon was injected with a concentration of 200 ng / ml sox9 that was dissolved in 0.9% sodium chloride ( sigma , mdl number : mfcd03097288 , product number : s4444 ) .
the btjs of the rabbits in group c were injected with adsox9 that had been previously diluted to a titer of 3.0 106 pfu / ml .
the epidermis of the right lower limb of the rabbits in all groups was sutured after the incision and they served as false - positive groups .
the operated limbs were fixed with plaster casts and the rabbits received 400,000 u / kg / d of penicillin via intramuscular injection 3 days after surgery . the surgical incision and blood supply in the limbs were observed .
the plaster casts were removed 4 weeks later and the rabbits were allowed to roam freely within the cage .
four groups of rabbits were sacrificed 4 weeks , 8 weeks , and 12 weeks after surgery .
after the rabbits in group a were sacrificed , two were selected for biomechanical testing and two for histological examinations .
the btj of the rabbits in group c were divided into two parts : one part was subjected to biomechanical testing ( two samples ) and the other part was subjected to histological examinations ( two samples ) .
after the biomechanical testing was conducted , the samples were collected and used for mrna detection .
the experimental samples were collected from different groups of animals . the experimental limb was extracted whole from the quadriceps femoris to the tibia while muscles and other tissues from the attachment points were detached .
next , the distal patellar tendon was removed from the tibial tubercle and the patella and patellar ligament were kept intact .
the structure , comprising of the patellar ligament and the patella with some quadriceps femoris attached , was sliced along the middle in a vertical axis .
the structure was later fixed with 10% formaldehyde for 24 hours and decalcified with 10% formic acid for 4 weeks .
it was subsequently dehydrated with progressively concentrated amounts of ethanol , treated with clearing and infiltration agents and then embedded in paraffin .
the specimen was stained using the hematoxylin and eosin stain ( h and e stain ) and the morphology of the btj osteocytes , chondrocytes , and tendon tissue ground substance were observed under a light microscope .
the connective tissues and sutures from the surgery surrounding the knee of the specimen were first removed . using a vernier caliper , triplicate measurements of the thickness , width , and cross section of the healing surface from the end of the quadriceps femoris to the proximal tibial osteotomy
the end of the distal quadriceps femoris and the proximal tibial osteotomy were secured on a fixture and a 10 anteversion angle between the tibia and the load bearing axis was created .
the structure was then pulled at a velocity of 20 mm / min using a dynamometer with the force of 2 kn and the breaking load was recorded .
total rna was extracted from 100 mg of btj tissues and the rt - pcr was used to identify the sox9 gene expression .
statistical analysis was performed with 4 2 factorial analysis of variance between the treatment and control groups using the spss 13.0 statistical software .
the healing effect of sox9 on btj was observed through the histological changes and detection of the biomechanical properties of the junction .
after the surgery , varying degrees of swelling to the surgical incisions were observed in the animals .
they were given symptomatic treatments like antibiotics and none of them succumbed to infection . according to observations made on the samples collected from group a 4 weeks after surgery , fibrous tissue hyperplasia could be seen on the dorsal surface of the patella with no obvious adhesions on the skin .
in addition , the cut surface of the surgery was unclear and the surface of the bone - tendon showed trends of fusion .
the dorsal surface of the patella of group b , which was treated with 0.9% sodium chloride , had smaller amounts of fibrous tissue hyperplasia than that of group a. a certain degree of adhesion could be seen between the incision tissues and the skin .
the cut surface in group b was clearer in comparison to that of group a. there was a notable increase of fibrous tissue hyperplasia in group a animals that were sacrificed 8 weeks after the surgery , compared to those sacrificed after 4 weeks .
there was an increase in the fusion in the btj region and the cut surface was quite unclear . for the corresponding group b , fibrous tissue hyperplasia could be seen and there were fewer adhesions between the incision tissues and the skin . however , the cut surface was clear and animals in group a still healed faster than those in group b. in the case of the specimens from group a that were collected 12 weeks after the surgery , most of the btj had fused , the granulation tissues were clearer and neatly arranged , and the cut surface could not be distinguished .
in contrast , the patella and the dorsal surface of the patellar tendon of the corresponding control group had a little fibrous tissue hyperplasia , the granulation tissues were unclear and arranged irregularly , and the cut surface was a little unclear .
the morphological changes of the osteocytes , chondrocytes , and tendon tissue ground substance of the btj at different times were observed under the light microscope after h and e staining [ figure 1 ] .
large amounts of fibroblasts and chondrocyte - like cells could be found in the sox9 group 4 weeks after the surgery .
there was an obvious increase in fibroblasts and part of the cells and collagen were aligned along the vertical axis of the bone - tendon .
there was a disorder in the arrangement of a large portion of the cells and newly formed cancellous bones that grew from the cut surface toward the distal patellar tendon .
some chondrocyte - like cells that displayed a focal distribution could be seen in the bone - tendon contact area .
a large amount of fibrous connective tissue cover was seen on the dorsal surface of the patella and patellar tendon [ figure 1a ] . in the corresponding group b
the cut surface of the patellar and patellar tendon was fairly clear and new bone growth could still be found in the btj [ figure 1b ] .
histological observations of bone tendon junction healing in group a and b treated with sox9 ; a and b ( 4 weeks ) ; c and d ( 8 weeks ) ; e and f ( 12 weeks ) .
the arrow in a shows the fibrous connective tissue , while the arrow in figure 1b shows the chondrocyte - like cells , ( h and e , 10 ) .
fibroblast proliferation could also be seen in samples collected from group a 8 weeks after the surgery .
the formation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was not obvious .
new cancellous bone could be seen growing towards the inside of the patellar tendon at the distal end of the btj .
part of the patellar tendon and cancellous bone showed a fusion trend and the cut surface was unclear [ figure 1c ] .
fibroblast proliferation could be seen and the formation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was not obvious in the corresponding control group .
the chondrocyte - like cells showed a regular arrangement and new cancellous bone in the residual distal patella was seen growing toward the inside of the patellar tendon .
part of the patellar tendon and cancellous bone showed a fusion trend and the cut surface was unclear [ figure 1d ] .
the cut surface of samples in group a collected 12 weeks after the surgery was found to be almost completely fused and a large amount of new and actively proliferating cancellous bone could be seen .
there was a notable increase in collagen fibers , which had an orderly arrangement along the vertical axis [ figure 1e ] .
part of the cut surface had fused for samples in group b that were collected 12 weeks after the surgery .
there were fewer chondrocyte - like cells in a bead - like arrangement and collagen fibers had a more regular pattern compared to that of samples from 4 weeks after surgery .
the tidemark line was yet to form on the cells and collagen fibers that were arranged along the vertical axis [ figure 1f ] .
the results of comparison of the cross - sectional area , breaking load , and ultimate tensile strength of the btj of group a and b are shown in table 1 and figure 2 .
the cross - sectional areas of group a at 4 , 8 , and 12 weeks after the surgery are 24.38 4.53 , 18.65 3.49 , and 14.82 1.57 mm2 , respectively .
the cross - sectional areas for control group are 33.42 7.25 , 22.12 4.52 , and 20.81 4.14 mm2 , respectively . the results of the comparison of the cross - sectional areas between group a and b for 4 and 12 weeks after the surgery had a statistical significance ( p < 0.05 ) .
the breaking load of group a at 4 , 8 , and 12 weeks after the surgery were found to be 112.23 21.29 , 165.31 18.32 , and 359.25 28.31
in contrast , the breaking load of group b at 4 , 8 , and 12 weeks after the surgery were much smaller than in group a , at 64.76 12.84 , 114.92 18.46 , and 268.32 23.29
results showed that the breaking load of the three samples in group a are significantly higher than those of samples from group b ( p < 0.05 ) .
the results showed that there were significant differences ( p < 0.05 ) between group a and b at 4 , 8 , and 12 weeks after the surgery .
the ultimate tensile strength values ( mpa ) of group b at 4 , 8 , and 12 weeks after the surgery were shown to be 2.72 2.43 , 4.53 3.21 , and 14.54 4.65 mpa , respectively .
these values are much lower than the corresponding values of group a at 9.79 4.66 , 13.89 5.78 , and 26.72 12.54 mpa , respectively .
results of biomechanical test of group a and group b at different time after the surgery a comparison of the cross - sectional area ( a ) breaking load ( b ) and ultimate tensile strength ( c ) of the samples in group a ( sox9 ) and group b ( control ) at 4 , 8 , and 12 weeks after the surgery .
* indicates a significant difference at p < 0.05 in comparison to the adenovirus group d , observations of the samples in group c at 4 weeks after the surgery showed fibrous tissue hyperplasia on the dorsal surface of the patella , with no obvious adhesions on the skin .
there was a notable increase in the fibrous tissue in group c samples collected 8 weeks after the surgery compared to those collected after 4 weeks .
in addition , there was an increase in the fusion in the btj region and the cut surface was quite unclear . in the case of the samples of group c collected 12 weeks after the surgery , most of the btj had fused , the granulation tissues were clearer and neatly arranged , and the cut surface could not be distinguished .
large amounts of fibroblasts and chondrocyte - like cells could be found in the adtrack - sox9 group 4 weeks after surgery .
part of the cells and collagen were aligned along the vertical axis of the bone - tendon and most of the cells had a disordered arrangement while a portion was arranged in a regular pattern .
newly formed cancellous bone was seen and some chondrocyte - like cells that displayed a focal distribution were also seen in the bone - tendon contact area . there was a notable increase in the fibrous tissue hyperplasia in group c specimens collected 8 weeks after the surgery .
the generation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was visible .
there was also a tremendous growth of chondrocyte - like cells with a relatively regular arrangement .
new cancellous bone could be seen growing toward the inside of the patellar tendon at the distal end of the btj .
in the case of samples collected 12 weeks after surgery , the cut surface had fused and a large amount of new cancellous bone had formed .
the nucleus pulposus of the animals which was injected with adtrack - sox9 after 1 week was used for pcr analysis .
the pcr results showed that the product size of the transcription of the sox9 gene was between 400 and 500 bp ( base - pairs ) , which is in accordance with the length of the design .
the relative level of the mrna expression of the sox9 gene was measured through a quantitative analysis of the pcr product 4 weeks after the surgery . after injecting the sox9 vector
that was transfected with the recombinant adenovirus 8 weeks after the surgery for group c , there was an obvious improvement of 532% and 205% in the expression level for sox9 and type ii collagen mrna , respectively ( p < 0.05 ) .
the corresponding values for group d were lower in comparison to that of group c , at 76% for sox9 and 69% for type ii collagen mrna [ table 2 and figures 3 and 4 ] .
the gene expression in the nucleus pulposus tissues after the btj was injected with sox9 that was transduced with the recombinant adenovirus vector after surgery a comparison of the cross - sectional area , breaking load and ultimate tensile strength of group a and b at 4 , 8 , and 12 weeks after the surgery .
0.05 the level of mrna expression of the sox9 gene that was transduced with recombinant adenovirus vector in the nucleus pulposus is shown .
lines 1 , 3 , and 5 represent the mrna expression of the sox9 gene that was transduced with adenovirus in group d at 1 , 4 , and 8 weeks , respectively .
pcr results showed that there was no mrna expression during the treatment period ; lines 2 , 4 , and 6 represent the mrna expression of the sox9 gene that was transduced with adsox9 .
pcr results showed that there was a gradual increase in the expression of sox9 in group c with time the cross - sectional area , breaking load , and ultimate tensile strength of the samples in groups c and d were determined [ table 3 ] .
the results showed a significant difference ( p < 0.05 ) in the cross - sectional area of group c compared to that of group d. the cross - sectional areas of samples in group c collected at 4 , 8 , and 12 weeks after the surgery were found to be 20.62 4.17 , 15.43 2.87 , and 13.56 1.92 mm2 , respectively .
results of biomechanical test of group c at different time after the surgery the breaking load values of the samples in group c that were treated with adtrack - sox9 at 4 , 8 , and 12 weeks after the surgery were 123.14 25.32 , 206.57 32.87 , and 341.52 34.65 n , respectively .
there was a statistical significance ( p < 0.05 ) in relation to the values collected from the control group .
the ultimate tensile strengths of the samples in group c that were treated with adtrack - sox9 at 4 , 8 , and 12 weeks after the surgery were found to be 7.56 3.43 , 14.82 6.32 , and 28.62 8.32 mpa , respectively .
there was also a statistical significance ( p < 0.05 ) in relation to the values from the control group [ figure 5 ] .
a comparison of the cross - sectional area ( a ) , breaking load ( b ) , and ultimate tensile strength ( c ) of the samples in group c and group d at 4 , 8 , and 12 weeks after the surgery ( ) , n=4 .
according to observations made on the samples collected from group a 4 weeks after surgery , fibrous tissue hyperplasia could be seen on the dorsal surface of the patella with no obvious adhesions on the skin .
in addition , the cut surface of the surgery was unclear and the surface of the bone - tendon showed trends of fusion .
the dorsal surface of the patella of group b , which was treated with 0.9% sodium chloride , had smaller amounts of fibrous tissue hyperplasia than that of group a. a certain degree of adhesion could be seen between the incision tissues and the skin .
the cut surface in group b was clearer in comparison to that of group a. there was a notable increase of fibrous tissue hyperplasia in group a animals that were sacrificed 8 weeks after the surgery , compared to those sacrificed after 4 weeks .
there was an increase in the fusion in the btj region and the cut surface was quite unclear . for the corresponding group b , fibrous tissue hyperplasia could be seen and there were fewer adhesions between the incision tissues and the skin . however , the cut surface was clear and animals in group a still healed faster than those in group b. in the case of the specimens from group a that were collected 12 weeks after the surgery , most of the btj had fused , the granulation tissues were clearer and neatly arranged , and the cut surface could not be distinguished .
in contrast , the patella and the dorsal surface of the patellar tendon of the corresponding control group had a little fibrous tissue hyperplasia , the granulation tissues were unclear and arranged irregularly , and the cut surface was a little unclear .
the morphological changes of the osteocytes , chondrocytes , and tendon tissue ground substance of the btj at different times were observed under the light microscope after h and e staining [ figure 1 ] .
large amounts of fibroblasts and chondrocyte - like cells could be found in the sox9 group 4 weeks after the surgery .
there was an obvious increase in fibroblasts and part of the cells and collagen were aligned along the vertical axis of the bone - tendon .
there was a disorder in the arrangement of a large portion of the cells and newly formed cancellous bones that grew from the cut surface toward the distal patellar tendon .
some chondrocyte - like cells that displayed a focal distribution could be seen in the bone - tendon contact area .
a large amount of fibrous connective tissue cover was seen on the dorsal surface of the patella and patellar tendon [ figure 1a ] . in the corresponding group b
the cut surface of the patellar and patellar tendon was fairly clear and new bone growth could still be found in the btj [ figure 1b ] .
histological observations of bone tendon junction healing in group a and b treated with sox9 ; a and b ( 4 weeks ) ; c and d ( 8 weeks ) ; e and f ( 12 weeks ) .
the arrow in a shows the fibrous connective tissue , while the arrow in figure 1b shows the chondrocyte - like cells , ( h and e , 10 ) .
fibroblast proliferation could also be seen in samples collected from group a 8 weeks after the surgery .
the formation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was not obvious .
new cancellous bone could be seen growing towards the inside of the patellar tendon at the distal end of the btj .
part of the patellar tendon and cancellous bone showed a fusion trend and the cut surface was unclear [ figure 1c ] .
fibroblast proliferation could be seen and the formation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was not obvious in the corresponding control group .
the chondrocyte - like cells showed a regular arrangement and new cancellous bone in the residual distal patella was seen growing toward the inside of the patellar tendon .
part of the patellar tendon and cancellous bone showed a fusion trend and the cut surface was unclear [ figure 1d ] .
the cut surface of samples in group a collected 12 weeks after the surgery was found to be almost completely fused and a large amount of new and actively proliferating cancellous bone could be seen .
there was a notable increase in collagen fibers , which had an orderly arrangement along the vertical axis [ figure 1e ] .
part of the cut surface had fused for samples in group b that were collected 12 weeks after the surgery .
there were fewer chondrocyte - like cells in a bead - like arrangement and collagen fibers had a more regular pattern compared to that of samples from 4 weeks after surgery .
the tidemark line was yet to form on the cells and collagen fibers that were arranged along the vertical axis [ figure 1f ] .
the results of comparison of the cross - sectional area , breaking load , and ultimate tensile strength of the btj of group a and b are shown in table 1 and figure 2 .
the cross - sectional areas of group a at 4 , 8 , and 12 weeks after the surgery are 24.38 4.53 , 18.65 3.49 , and 14.82 1.57 mm2 , respectively .
the cross - sectional areas for control group are 33.42 7.25 , 22.12 4.52 , and 20.81 4.14 mm2 , respectively . the results of the comparison of the cross - sectional areas between group a and b for 4 and 12 weeks after the surgery had a statistical significance ( p < 0.05 ) . the breaking load of group a at 4 , 8 , and 12 weeks after the surgery were found to be 112.23 21.29 , 165.31 18.32 , and 359.25 28.31 n , respectively .
in contrast , the breaking load of group b at 4 , 8 , and 12 weeks after the surgery were much smaller than in group a , at 64.76 12.84 , 114.92 18.46 , and 268.32 23.29
results showed that the breaking load of the three samples in group a are significantly higher than those of samples from group b ( p < 0.05 ) .
the results showed that there were significant differences ( p < 0.05 ) between group a and b at 4 , 8 , and 12 weeks after the surgery .
the ultimate tensile strength values ( mpa ) of group b at 4 , 8 , and 12 weeks after the surgery were shown to be 2.72 2.43 , 4.53 3.21 , and 14.54 4.65 mpa , respectively .
these values are much lower than the corresponding values of group a at 9.79 4.66 , 13.89 5.78 , and 26.72 12.54 mpa , respectively .
results of biomechanical test of group a and group b at different time after the surgery a comparison of the cross - sectional area ( a ) breaking load ( b ) and ultimate tensile strength ( c ) of the samples in group a ( sox9 ) and group b ( control ) at 4 , 8 , and 12 weeks after the surgery .
in comparison to the adenovirus group d , observations of the samples in group c at 4 weeks after the surgery showed fibrous tissue hyperplasia on the dorsal surface of the patella , with no obvious adhesions on the skin .
there was a notable increase in the fibrous tissue in group c samples collected 8 weeks after the surgery compared to those collected after 4 weeks .
in addition , there was an increase in the fusion in the btj region and the cut surface was quite unclear . in the case of the samples of group c collected 12 weeks
after the surgery , most of the btj had fused , the granulation tissues were clearer and neatly arranged , and the cut surface could not be distinguished .
large amounts of fibroblasts and chondrocyte - like cells could be found in the adtrack - sox9 group 4 weeks after surgery .
part of the cells and collagen were aligned along the vertical axis of the bone - tendon and most of the cells had a disordered arrangement while a portion was arranged in a regular pattern .
newly formed cancellous bone was seen and some chondrocyte - like cells that displayed a focal distribution were also seen in the bone - tendon contact area . there was a notable increase in the fibrous tissue hyperplasia in group c specimens collected 8 weeks after the surgery .
the generation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was visible .
there was also a tremendous growth of chondrocyte - like cells with a relatively regular arrangement .
new cancellous bone could be seen growing toward the inside of the patellar tendon at the distal end of the btj .
part of the patellar tendon and cancellous bone showed a fusion trend . in the case of samples collected 12 weeks after surgery
, the cut surface had fused and a large amount of new cancellous bone had formed .
the nucleus pulposus of the animals which was injected with adtrack - sox9 after 1 week was used for pcr analysis .
the pcr results showed that the product size of the transcription of the sox9 gene was between 400 and 500 bp ( base - pairs ) , which is in accordance with the length of the design .
the relative level of the mrna expression of the sox9 gene was measured through a quantitative analysis of the pcr product 4 weeks after the surgery . after injecting the sox9 vector
that was transfected with the recombinant adenovirus 8 weeks after the surgery for group c , there was an obvious improvement of 532% and 205% in the expression level for sox9 and type ii collagen mrna , respectively ( p < 0.05 ) .
the corresponding values for group d were lower in comparison to that of group c , at 76% for sox9 and 69% for type ii collagen mrna [ table 2 and figures 3 and 4 ] .
the gene expression in the nucleus pulposus tissues after the btj was injected with sox9 that was transduced with the recombinant adenovirus vector after surgery a comparison of the cross - sectional area , breaking load and ultimate tensile strength of group a and b at 4 , 8 , and 12 weeks after the surgery .
* indicates a significant difference at p < 0.05 the level of mrna expression of the sox9 gene that was transduced with recombinant adenovirus vector in the nucleus pulposus is shown .
lines 1 , 3 , and 5 represent the mrna expression of the sox9 gene that was transduced with adenovirus in group d at 1 , 4 , and 8 weeks , respectively .
pcr results showed that there was no mrna expression during the treatment period ; lines 2 , 4 , and 6 represent the mrna expression of the sox9 gene that was transduced with adsox9 .
pcr results showed that there was a gradual increase in the expression of sox9 in group c with time
the cross - sectional area , breaking load , and ultimate tensile strength of the samples in groups c and d were determined [ table 3 ] .
the results showed a significant difference ( p < 0.05 ) in the cross - sectional area of group c compared to that of group d. the cross - sectional areas of samples in group c collected at 4 , 8 , and 12 weeks after the surgery were found to be 20.62 4.17 , 15.43 2.87 , and 13.56 1.92 mm2 , respectively .
results of biomechanical test of group c at different time after the surgery the breaking load values of the samples in group c that were treated with adtrack - sox9 at 4 , 8 , and 12 weeks after the surgery were 123.14 25.32 , 206.57 32.87 , and 341.52 34.65
there was a statistical significance ( p < 0.05 ) in relation to the values collected from the control group .
the ultimate tensile strengths of the samples in group c that were treated with adtrack - sox9 at 4 , 8 , and 12 weeks after the surgery were found to be 7.56 3.43 , 14.82 6.32 , and 28.62 8.32 mpa , respectively .
there was also a statistical significance ( p < 0.05 ) in relation to the values from the control group [ figure 5 ] .
a comparison of the cross - sectional area ( a ) , breaking load ( b ) , and ultimate tensile strength ( c ) of the samples in group c and group d at 4 , 8 , and 12 weeks after the surgery ( ) , n=4 .
although ongoing researches on the improvement to these techniques are underway , most of them are focussed on the development of the tissue structure .
the joint has to be immobilized after surgery as the btj is situated near the joint .
long term immobilization gives rise to a range of problems like joint stiffness , which negatively impacts patient 's daily work and life.1819 therefore , it is important to have early treatment programs that can provide a quicker healing of btj injuries .
previous research has reported that the injection of osteoprotegerin into the bone - tendon interface in a btj damage repair model of rabbits can suppress the formation , differentiation , activation , and resorption of osteoclasts , which ultimately improves the healing of btj injuries.20 researchers used liposomal clodronate on the damage repair model of sprague - dawley rats to diminish macrophage - induced tgf- production , which can promote the rejuvenation of the btj.21 although such literature is relatively abundant , most of it is focused on the histology of bone - tendon healing .
the main causes of tendon adhesion formation are thought to be the extrinsic healing mechanism , which is dependent on the blood supply and repair cells , which originate from the peritenon tissue adhesions .
soft tissues such as tendons and ligaments in btj can turn into hard tissues ( like bone ) through a range of tissue types in a short distance , the stress requirements for the btj are relatively large.2223 a simple histology - based treatment of the btj can not satisfy the body 's needs as the choice of treatment program not only depends on the histology but also on the patient 's tolerance to stress .
results have shown that the animals in sox9-treated groups had a higher cross - sectional area , breaking load , and ultimate tensile strength than those in the control groups .
taking into consideration that the fourth week after surgery is the inflammatory phase , a large amount of granulation tissue had filled the btj , resulting in an increase in the cross - sectional area for all groups .
however , as the healing of the btj in the control group is slower than that of the sox9 group , the cross - sectional area of the sox9 group is still larger . as a result of the new bone formation stage that accompanies the structural formation of the fibrocartilage transition zone , the regeneration of the woven bone , maturation of the fibrocartilage node as well as the bone remodeling phase,2425 the cross - sectional area of the btj of the sox9 group gradually became smaller
the breaking load and ultimate tensile strength for all sox9 group samples collected over the three periods were higher than that of the control group .
these indicate that the sox9 can not only improve the healing of the btj , but also increase the biomechanical strength , which in turn results in the acceleration of the healing process .
the rt - pcr results in the present study demonstrated that sox9 can be expressed at a high level in btj that had been injected with adsox9 , thus enhancing the healing process in the btj .
the subsequent biomechanical tests showed that the cross - sectional area , breaking load , and ultimate tensile strength for the sox9-treated group samples collected 4 , 8 , and 12 weeks after the surgery were significantly higher ( p < 0.05 ) than that of the control group , proving that adsox9 can improve the healing of the btj . in this study , we used recombinant adenovirus sox9 ( adsox9 ) in this research , the transgenic technology involved insertion of the sox9 coding gene into specific target cells and transfection of the animal model .
and then the healing effect of sox9 on btj was detected . in summary , based on our findings we considered that gene therapy may be applicable in the healing process of the bone - tendon junction . | background : sox9 is an operon that positively regulates the transcription of type ii collagen . the generation of type ii collagen plays a critical role in the healing process of the bone - tendon junction ( btj).materials and methods : sox9 was injected into an established bone - tendon healing model in order to observe its effect on the healing by determining the biomechanical properties of the btj .
in addition , the recombinant adenovirus sox9 was used to transduce the animal model samples and in vivo observations of the effect of the adenovirus - mediated sox9 transduction as well as its promotion of the healing properties were made.results:sox9 was not only able to promote the healing , but also increased the biomechanical strength .
the recombinant sox9 delivered by adenoviral vector can be expressed at a high level in the damaged tissues of the bone - tendon junction , which can stimulate the production of type ii collagen and improve the healing of the btj.conclusions:based on the results of this study , we considered that gene therapy may be applicable in the healing process of the bone - tendon junction . | I
M
Establishment of animal model and grouping
Histological examination
Biomechanical testing
RT-PCR analysis of mRNA expression
Statistical analysis
R
General examination
Results of histological observation
Results of biomechanical test
Results of general observations in groups treated with AdTrack-Sox9 at different times
Results of H and E, staining of specimen in groups treated with AdTrack-Sox9 at different times
Results of mRNA expression of specimen in groups treated with AdTrack-Sox9 at different times
Results of the biomechanics of specimen in groups treated with AdTrack-Sox9 at different times
D | bone - tendon junction injuries are common problem in athletes . due to a change in lifestyle , there has been an increase in the occurrence of bone - tendon junction ( btj ) injuries like patellar tendinitis , lateral epicondylitis , rotator cuff injury and the rupture of the achilles tendon in recent years.123 as a result , there has been a rising interest in the research of btj - related injuries . based on the properties of the bone - tendon tissues , it can also be categorized into fibro - tissues and bone , with a spacing known as the fibrocartilage zone.4 the short transition distance between the soft and hard tissues results in a slower rate of recovery . the fibrocartilage zone connecting the bone to the tendon at the btj plays an important role in the repair and reconstruction of the btj . the successful reconstruction of the fibrocartilage zone will allow for an improvement in the histological and mechanical recovery of the btj . in recent years , non - invasive techniques or therapies like low intensity pulsed ultrasound ( lipus ) , extracorporeal shock - wave therapy , and electrical stimulation have gained recognition as well.2 the sox9 gene , which belongs to a family of genes known as sox and can be found in the chromosome region 17q24ii-25,7 is crucial to the embryonic development in the early stages.8 sox9 is a transcription factor that has c - terminal high - mobility group ( hmg ) domain and n - terminal transactivation domain . it participates in various embryonic developmental processes such as chondrogenesis.9 translocations upstream of the coding sequence suggested that the altered expression of sox9 is capable of severely impacting chondrogenesis and thus lead to abnormal skeletal development . as a transcriptional activator , the sox9 gene is able to co - regulate the proliferation and differentiation of the chondrocytes together with other signal pathways through direct or indirect means.1011 this can be achieved through the combination with the chondrocytes - specific enhancer type ii collagen gene ( col2al ) and l - sox5 , sox6 . currently , main treatments for bone - tendon healing include surgical treatment and conservative therapy . however , there are obvious limitations to these treatments.12 gene therapy involves the use of gene transfer techniques to replace a mutated gene with an exogenous gene that encodes a therapeutic protein in order to treat certain diseases13 previous studies have confirmed that the target genes of sox9 promote the healing of bone and cartilage damage.1314 therefore , the synthesis of various matrix components can be regulated by the insertion or transformation of the therapeutic gene sox9 . the gene therapy should ideally provide a precise transference of the target gene to target cells to achieve an efficient , specific , and controllable expression.15 however , at present , the transfers of such genes have poor tissue specificity , which leads to problems like low transfer efficiency and difficulties in gene expression . there is an extensive use of adenovirus in current gene therapies due to advantages such as susceptibility to infection , effective expression , high stability , high genome capacity , propensity to proliferate and purification and high viral titer.16 in this study , we injected sox9 into an established bone - tendon healing model and aimed to observe its effect on the healing of the btj by determining the biomechanical properties of the junction . establishment of animal model was reported in previous report.17 during our study , we found that simply injecting sox9 gene resulted in low transcript and using adenovirus vector could improve this situation to a certain level . in our study , we constructed recombinant adenovirus vector sox9 , adsox9 , and it was used to treat btj . sox9 was injected into the btj of rabbits in group a and adsox9 was transfected into the btj of rabbits in group c. rabbits in group b served as control and did not receive any injection during surgery . rabbits in group d were transfected with adenovirus and served as a control for group c. for the rabbits in group a , the seam between the bone and tendon was injected with a concentration of 200 ng / ml sox9 that was dissolved in 0.9% sodium chloride ( sigma , mdl number : mfcd03097288 , product number : s4444 ) . using a vernier caliper , triplicate measurements of the thickness , width , and cross section of the healing surface from the end of the quadriceps femoris to the proximal tibial osteotomy
the end of the distal quadriceps femoris and the proximal tibial osteotomy were secured on a fixture and a 10 anteversion angle between the tibia and the load bearing axis was created . the rupture surface created during experiment
total rna was extracted from 100 mg of btj tissues and the rt - pcr was used to identify the sox9 gene expression . establishment of animal model was reported in previous report.17 during our study , we found that simply injecting sox9 gene resulted in low transcript and using adenovirus vector could improve this situation to a certain level . in our study , we constructed recombinant adenovirus vector sox9 , adsox9 , and it was used to treat btj . sox9 was injected into the btj of rabbits in group a and adsox9 was transfected into the btj of rabbits in group c. rabbits in group b served as control and did not receive any injection during surgery . rabbits in group d were transfected with adenovirus and served as a control for group c. for the rabbits in group a , the seam between the bone and tendon was injected with a concentration of 200 ng / ml sox9 that was dissolved in 0.9% sodium chloride ( sigma , mdl number : mfcd03097288 , product number : s4444 ) . using a vernier caliper , triplicate measurements of the thickness , width , and cross section of the healing surface from the end of the quadriceps femoris to the proximal tibial osteotomy
the end of the distal quadriceps femoris and the proximal tibial osteotomy were secured on a fixture and a 10 anteversion angle between the tibia and the load bearing axis was created . total rna was extracted from 100 mg of btj tissues and the rt - pcr was used to identify the sox9 gene expression . the healing effect of sox9 on btj was observed through the histological changes and detection of the biomechanical properties of the junction . in addition , the cut surface of the surgery was unclear and the surface of the bone - tendon showed trends of fusion . the dorsal surface of the patella of group b , which was treated with 0.9% sodium chloride , had smaller amounts of fibrous tissue hyperplasia than that of group a. a certain degree of adhesion could be seen between the incision tissues and the skin . there was an increase in the fusion in the btj region and the cut surface was quite unclear . however , the cut surface was clear and animals in group a still healed faster than those in group b. in the case of the specimens from group a that were collected 12 weeks after the surgery , most of the btj had fused , the granulation tissues were clearer and neatly arranged , and the cut surface could not be distinguished . there was an obvious increase in fibroblasts and part of the cells and collagen were aligned along the vertical axis of the bone - tendon . there was a disorder in the arrangement of a large portion of the cells and newly formed cancellous bones that grew from the cut surface toward the distal patellar tendon . some chondrocyte - like cells that displayed a focal distribution could be seen in the bone - tendon contact area . in the corresponding group b
the cut surface of the patellar and patellar tendon was fairly clear and new bone growth could still be found in the btj [ figure 1b ] . the formation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was not obvious . fibroblast proliferation could be seen and the formation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was not obvious in the corresponding control group . the chondrocyte - like cells showed a regular arrangement and new cancellous bone in the residual distal patella was seen growing toward the inside of the patellar tendon . the results of comparison of the cross - sectional area , breaking load , and ultimate tensile strength of the btj of group a and b are shown in table 1 and figure 2 . the results of the comparison of the cross - sectional areas between group a and b for 4 and 12 weeks after the surgery had a statistical significance ( p < 0.05 ) . results of biomechanical test of group a and group b at different time after the surgery a comparison of the cross - sectional area ( a ) breaking load ( b ) and ultimate tensile strength ( c ) of the samples in group a ( sox9 ) and group b ( control ) at 4 , 8 , and 12 weeks after the surgery . * indicates a significant difference at p < 0.05 in comparison to the adenovirus group d , observations of the samples in group c at 4 weeks after the surgery showed fibrous tissue hyperplasia on the dorsal surface of the patella , with no obvious adhesions on the skin . in addition , there was an increase in the fusion in the btj region and the cut surface was quite unclear . in the case of the samples of group c collected 12 weeks after the surgery , most of the btj had fused , the granulation tissues were clearer and neatly arranged , and the cut surface could not be distinguished . part of the cells and collagen were aligned along the vertical axis of the bone - tendon and most of the cells had a disordered arrangement while a portion was arranged in a regular pattern . newly formed cancellous bone was seen and some chondrocyte - like cells that displayed a focal distribution were also seen in the bone - tendon contact area . the generation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was visible . the nucleus pulposus of the animals which was injected with adtrack - sox9 after 1 week was used for pcr analysis . the pcr results showed that the product size of the transcription of the sox9 gene was between 400 and 500 bp ( base - pairs ) , which is in accordance with the length of the design . after injecting the sox9 vector
that was transfected with the recombinant adenovirus 8 weeks after the surgery for group c , there was an obvious improvement of 532% and 205% in the expression level for sox9 and type ii collagen mrna , respectively ( p < 0.05 ) . the corresponding values for group d were lower in comparison to that of group c , at 76% for sox9 and 69% for type ii collagen mrna [ table 2 and figures 3 and 4 ] . the gene expression in the nucleus pulposus tissues after the btj was injected with sox9 that was transduced with the recombinant adenovirus vector after surgery a comparison of the cross - sectional area , breaking load and ultimate tensile strength of group a and b at 4 , 8 , and 12 weeks after the surgery . 0.05 the level of mrna expression of the sox9 gene that was transduced with recombinant adenovirus vector in the nucleus pulposus is shown . in addition , the cut surface of the surgery was unclear and the surface of the bone - tendon showed trends of fusion . the dorsal surface of the patella of group b , which was treated with 0.9% sodium chloride , had smaller amounts of fibrous tissue hyperplasia than that of group a. a certain degree of adhesion could be seen between the incision tissues and the skin . there was an increase in the fusion in the btj region and the cut surface was quite unclear . however , the cut surface was clear and animals in group a still healed faster than those in group b. in the case of the specimens from group a that were collected 12 weeks after the surgery , most of the btj had fused , the granulation tissues were clearer and neatly arranged , and the cut surface could not be distinguished . the morphological changes of the osteocytes , chondrocytes , and tendon tissue ground substance of the btj at different times were observed under the light microscope after h and e staining [ figure 1 ] . there was an obvious increase in fibroblasts and part of the cells and collagen were aligned along the vertical axis of the bone - tendon . some chondrocyte - like cells that displayed a focal distribution could be seen in the bone - tendon contact area . a large amount of fibrous connective tissue cover was seen on the dorsal surface of the patella and patellar tendon [ figure 1a ] . in the corresponding group b
the cut surface of the patellar and patellar tendon was fairly clear and new bone growth could still be found in the btj [ figure 1b ] . the formation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was not obvious . fibroblast proliferation could be seen and the formation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was not obvious in the corresponding control group . the results of comparison of the cross - sectional area , breaking load , and ultimate tensile strength of the btj of group a and b are shown in table 1 and figure 2 . the results of the comparison of the cross - sectional areas between group a and b for 4 and 12 weeks after the surgery had a statistical significance ( p < 0.05 ) . in comparison to the adenovirus group d , observations of the samples in group c at 4 weeks after the surgery showed fibrous tissue hyperplasia on the dorsal surface of the patella , with no obvious adhesions on the skin . in addition , there was an increase in the fusion in the btj region and the cut surface was quite unclear . in the case of the samples of group c collected 12 weeks
after the surgery , most of the btj had fused , the granulation tissues were clearer and neatly arranged , and the cut surface could not be distinguished . part of the cells and collagen were aligned along the vertical axis of the bone - tendon and most of the cells had a disordered arrangement while a portion was arranged in a regular pattern . newly formed cancellous bone was seen and some chondrocyte - like cells that displayed a focal distribution were also seen in the bone - tendon contact area . the generation of the transition zone of the chondrocytes on the contact surfaces of the patellar tendon and cancellous bone was visible . the nucleus pulposus of the animals which was injected with adtrack - sox9 after 1 week was used for pcr analysis . the pcr results showed that the product size of the transcription of the sox9 gene was between 400 and 500 bp ( base - pairs ) , which is in accordance with the length of the design . after injecting the sox9 vector
that was transfected with the recombinant adenovirus 8 weeks after the surgery for group c , there was an obvious improvement of 532% and 205% in the expression level for sox9 and type ii collagen mrna , respectively ( p < 0.05 ) . the corresponding values for group d were lower in comparison to that of group c , at 76% for sox9 and 69% for type ii collagen mrna [ table 2 and figures 3 and 4 ] . the gene expression in the nucleus pulposus tissues after the btj was injected with sox9 that was transduced with the recombinant adenovirus vector after surgery a comparison of the cross - sectional area , breaking load and ultimate tensile strength of group a and b at 4 , 8 , and 12 weeks after the surgery . * indicates a significant difference at p < 0.05 the level of mrna expression of the sox9 gene that was transduced with recombinant adenovirus vector in the nucleus pulposus is shown . previous research has reported that the injection of osteoprotegerin into the bone - tendon interface in a btj damage repair model of rabbits can suppress the formation , differentiation , activation , and resorption of osteoclasts , which ultimately improves the healing of btj injuries.20 researchers used liposomal clodronate on the damage repair model of sprague - dawley rats to diminish macrophage - induced tgf- production , which can promote the rejuvenation of the btj.21 although such literature is relatively abundant , most of it is focused on the histology of bone - tendon healing . soft tissues such as tendons and ligaments in btj can turn into hard tissues ( like bone ) through a range of tissue types in a short distance , the stress requirements for the btj are relatively large.2223 a simple histology - based treatment of the btj can not satisfy the body 's needs as the choice of treatment program not only depends on the histology but also on the patient 's tolerance to stress . however , as the healing of the btj in the control group is slower than that of the sox9 group , the cross - sectional area of the sox9 group is still larger . as a result of the new bone formation stage that accompanies the structural formation of the fibrocartilage transition zone , the regeneration of the woven bone , maturation of the fibrocartilage node as well as the bone remodeling phase,2425 the cross - sectional area of the btj of the sox9 group gradually became smaller
the breaking load and ultimate tensile strength for all sox9 group samples collected over the three periods were higher than that of the control group . these indicate that the sox9 can not only improve the healing of the btj , but also increase the biomechanical strength , which in turn results in the acceleration of the healing process . the rt - pcr results in the present study demonstrated that sox9 can be expressed at a high level in btj that had been injected with adsox9 , thus enhancing the healing process in the btj . the subsequent biomechanical tests showed that the cross - sectional area , breaking load , and ultimate tensile strength for the sox9-treated group samples collected 4 , 8 , and 12 weeks after the surgery were significantly higher ( p < 0.05 ) than that of the control group , proving that adsox9 can improve the healing of the btj . in this study , we used recombinant adenovirus sox9 ( adsox9 ) in this research , the transgenic technology involved insertion of the sox9 coding gene into specific target cells and transfection of the animal model . and then the healing effect of sox9 on btj was detected . in summary , based on our findings we considered that gene therapy may be applicable in the healing process of the bone - tendon junction . | [
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] |
the last half - century has seen considerable improvements for dental health . unlike previous generations ,
more and more older adults are maintaining their natural teeth into old age [ 13 ] .
this is a welcome trend but results in new patterns of disease that become especially significant for those who are frail and who must depend on others for their personal care and hygiene [ 46 ] .
mouth care is an integral part of personal care yet it is inadequate [ 4 , 6 , 7 ] and given low priority for residents in long - term care [ 8 , 9 ] .
poor oral hygiene resulting from inadequate mouth care causes considerable morbidity such as mucosal inflammation , caries ( tooth decay ) , and periodontal disease ( bone loss around teeth ) [ 7 , 10 ] . evidence demonstrating links between dental disease and systemic conditions such as respiratory infections [ 11 , 12 ] , diabetes [ 1316 ] , and cardiovascular disease also continues to emerge .
tooth loss , pain , and poorly functioning dentures result in problems chewing [ 18 , 19 ] which is linked to poor nutrition , low body mass index , and involuntary weight loss .
dental diseases and dysfunction impact quality of life are known to diminish the pleasures of eating , speaking and social interactions [ 12 , 22 ] .
overall , the oral health status of residents in long - term care is poor [ 7 , 10 , 2325 ] and those with dementia experience even higher rates of oral disease .
oral care for dependent older adults in long - term care is becoming a challenge that is expected to grow in importance as our population ages . over the past several decades ,
research focusing on attitudes and behaviors of care providers ; education and oral hygiene intervention programs ; and more recently , environmental , organizational , or social influences on the delivery of care is shedding new light on the complexity of factors influencing the ability to provide adequate oral care .
practical barriers to oral care include a perceived lack of time [ 2729 ] , inadequate staffing levels [ 27 , 28 ] , lack of readily available oral care equipment [ 30 , 31 ] , resistant behaviors by patients / residents [ 27 , 28 , 30 , 31 ] , and high care staff turnover rates that undermine oral care education programs [ 32 , 33 ] .
social barriers include embarrassment and repulsion , lack of care staff confidence in their knowledge or ability to provide care [ 28 , 34 ] , a perception of oral care as invasive to the dignity and privacy of residents [ 35 , 36 ] or unwanted by the resident , and the perception by nurses that oral care is professionally unrewarding .
factors identified to facilitate care staff 's ability and/or willingness to provide oral care include availability of oral health equipment , influence of and examples set by people seen as influential leaders , education and/or demonstration in oral health care procedures [ 27 , 29 , 35 , 37 ] , adequate time to provide care , and believing that oral health and oral care are important [ 28 , 29 ] .
research involving staff education - based interventions directed toward improving oral health status of long - term care residents demonstrates conflicting results [ 3840 ] .
some studies have demonstrated a decrease in disease indicators [ 32 , 39 ] while others did not . a similarly designed comprehensive personal mouth care program introduced in multiple facilities found different effects on oral hygiene practices and health status of residents of the different facilities .
in fact , structural variables expected to influence quality of care ( e.g. , on - site services , routines , and resources ) explained very little of the difference between effective and ineffective programs [ 41 , 42 ] . rather , effectiveness appeared contingent upon the organizational context within facilities , comprised of both programmatic strategies and the organizational culture that supports or inhibits them .
creating effective strategies to address the issue of oral health in long - term care is further complicated because oral health care has traditionally been peripheral to mainstream health considerations .
the lack of interaction between dentistry and other domains of health care has fostered isolation in approaches for managing oral health .
this has been recognized as a shortcoming and has resulted in a call for an interdisciplinary and collaborative approach to both research and practice [ 8 , 9 ] .
the purpose of this paper is to describe the experience of developing a meaningful interdisciplinary collaboration and to highlight the processes used to design a comprehensive set of actions to integrate oral care into organizational policy and practice within three long - term care facilities in rural nova scotia on the east coast of canada .
a collaborative approach is advantageous because the synergy created by the blending of perspectives , resources , and skills of various participants enables the group to create something that is not attainable by single agents . in this study , an interdisciplinary network of researchers and stakeholders
were brought together with administrators and front - line care staff to think about the work in creative and practical ways ; develop realistic goals ; plan and carry out comprehensive interventions that connect multiple programs , services , and sectors ; understand and document the impact of its actions ; incorporate the perspectives and priorities of stakeholders including the target population ; and communicate how actions will address problems .
a common interest was established between the principal investigator ( m. e. mcnally ) and a senior administrator of three long - term care facilities during a provincial oral health policy workshop . over the course of a year
, multiple face - to - face meetings , telephone , and email consultations were undertaken with administrative staff to : discuss the current level of oral care and associated challenges ; develop realistic and practical research objectives ; establish a formal commitment to the research ; clarify roles and expectations ; and establish mutual benefits . through this process
, a formal collaboration was established consisting of an interdisciplinary research team in partnership with health service managers and nurse managers of the three facilities ( i.e. , the site team ) .
all members of the collaboration were involved in establishing goals and strategies for the project .
the nurse managers were further involved as direct liaisons to each of the sites and assisted with recruitment and data collection . to help focus the research goals and ongoing knowledge exchange , the site team identified training and resources as desired outcomes .
they are rurally situated within 1 hour of each other and are within 1 - 2-hour drive of a metropolitan area . the number of residents per site ranged from 25 to 40 , which is typical of the majority of long - term care facilities found in rural settings in the region .
the three long - term care facilities are administered under the same health district where there is sharing of resources , budgets , and policy .
examining multiple sites within the same organizational structure was undertaken to provide a deeper understanding of how subcultures influence delivery of care at a micro - organizational level .
a case study approach was used to explore the individual , organizational , and system factors associated with the integration of oral care in three long - term care settings .
the case study method was selected in order to gain a holistic understanding of how the development and implementation of actions may be influenced by the cultural systems of action that exist within each setting .
this research is consistent with the elements of a program planning cycle that include assessment , planning , implementation , and evaluation of a set of actions to integrate oral care into both organizational policy and daily personal care practice .
this paper describes details associated with the first two phases , assessment and action planning .
assessment involved an exploration of the internal and external factors that influence the provision of oral care and oral disease prevention and was undertaken through a document review , focus groups , and one - on - one interviews .
the action - planning phase brought care providers and stakeholders ( government representatives , educators , and dental professionals ) together for a workshop to design a set of actions to integrate oral care into organizational policy and practice in each of the settings .
the action plan is being implemented in each of the settings over a 12-month period and experiences of the health care team ( front - line care staff and administrators ) explored . the process and outcomes associated with the implementation of actions are being evaluated and are providing the basis of recommendations to revise organizational policy and oral care practice .
the latter two phases of the project will be described in a subsequent manuscript at their completion . a systematic analysis of multiple forms of evidence was used to enhance understanding of the context and the people within it .
sources of triangulation of data include multiple perspectives among investigators ( i.e. , dentistry , nursing , medicine , administration , policy decision - makers ) , multiple methods ( i.e. , document analysis , focus groups , interviews , journaling ) , multiple data sources ( i.e. , personal care providers , administrators , residents , and families ) , and multiple settings .
it was also recognized that the term oral care connotes the broad spectrum of oral health care needs associated with both professional and personal care .
this research is primarily concerned with the latter , daily mouth care , for frail older adults .
however , it was recognized that the scope of this work might also overlap with clinical and policy considerations of professional dentistry and public health .
the term oral care is therefore used to encompass the broader range of considerations in the research .
documents were selected on the basis of their topic relevance and included those expected to inform the provision of oral care for long - term care in the region ( e.g. , job descriptions , clinical guidelines , health assessment tools , accreditation guidelines ) .
documents were collected directly from the site team , from government , academia , the health services and community college education sectors , and through internet searches of relevant government and education websites .
they were individually read , coded , and organized according to four criteria : ( 1 ) general health terminology ( that may or may not include oral care ) , ( 2 ) oral care ( including terms oral , mouth , and dental ) , ( 3 ) foot care , and ( 4 ) wound care .
references to foot care and wound care were included to provide a basis for comparing oral care to other aspects of care that may be similarly addressed through relevant documents .
members of the collaboration suggested that it would be useful to examine oral care for consistency with existing and familiar clinical domains that may ultimately provide a useful framework for oral care .
this approach is constructivist and interpretivist [ 52 , 53 ] seeking to distil from personal accounts the experiences and meaning behind oral health care for dependent older adults .
qualitative methods are particularly well suited to finding answers to what questions ( what are people doing , what does it mean ) and how questions ( how are things done , how is meaning produced ) . ethics approval was obtained from the nova scotia capital district health authority ( cdha - rs/2009 - 033 ) .
members of the site team identified potential participants who were invited by letter and a follow - up telephone call .
one - on - one semistructured interviews were held with administrators and health professionals who provide a variety of health services to residents and clients associated with the three facilities .
two focus groups were held in each of the three facilities with ( 1 ) personal care providers ( i.e. , those who provide personal care within their job scope including personal care workers , continuing care assistants , and licensed practical nurses ) and ( 2 ) residents and family members . residents who had capacity to provide consent ( as determined by the nurse manager ) were invited to participate and family members were included to represent experiences of those not able to speak for themselves .
semistructured questions for both focus groups and individual interviews were designed to guide and generate discussion to elicit participants ' description of practices associated with the provision of oral care , perceptions of barriers and facilitators to oral care in the care settings , attitudes toward oral health and oral health care , and relevant knowledge of formal oral health policies .
this approach allowed for structure but was flexible enough for participants to raise issues not anticipated .
each verbatim interview underwent open coding to identify thematically group - related phrases and patterns arising from the data [ 54 , 55 ] using hyperresearch qualitative analysis tool ( version 2.8.2 ) .
a one - day interdisciplinary oral care action - planning workshop was held at a location central to the research sites .
members of the collaboration established workshop goals and identified invitees to ensure a broad range of relevant expertise and experience .
the purpose of the workshop was to design a set of actions that would integrate oral care into organizational policy and personal care practices in each of the three long - term care settings .
the two key goals were to : ( 1 ) identify and prioritize education , training , tools , and program strategies and ( 2 ) establish a detailed plan for implementing , tracking , and evaluating both professional and daily personal mouth care delivery .
forty - six invitees were contacted individually by m. e. mcnally or k. p. mcneil .
the workshop was attended by 34 participants : clinical researchers from dentistry , dental hygiene , nursing , and medicine ( n = 7 ) ; researchers from health promotion and organizational management ( n = 4 ) ; administrators ( n = 5 ) ; nurse managers ( n = 2 ) ; regulated and unregulated front line care staff ( n = 5 ) ; policy makers in the health and continuing care sectors ( n = 3 ) ; representatives of organized dentistry and dental hygiene ( n = 3 ) ; community college educators ( n = 2 ) ; speech language pathologist ( n = 1 ) ; dietician ( n = 1 ) ; as well as a representative from a seniors ' government advisory organization .
findings from the document review and experiential data were presented to provide participants with an understanding of context . a review of current best practices and a model of oral health care in long - term care provided an evidence base to inform workshop discussion .
relevant topics identified through the document review , focus groups , and interviews were prioritized by the collaboration for discussion in one - hour breakout sessions and a discussion guide was developed for each topic ( table 1 ) .
each session included 68 participants organized to ensure input from a variety of perspectives and disciplines .
responses to the discussion questions were recorded by individual groups and reported back in a plenary session where further input was gathered from the larger group . at the plenary ,
the group was asked to consider the following : how to best synthesize the information to create a comprehensive oral care action plan , how to best communicate the action plan to end users , and how to determine the biggest indicators of success over the next 18 months .
findings from the workshop were collated and synthesized into a draft oral care action plan for integration at each of the three long - term care sites .
strategies for implementing and evaluating actions were finalized as part of this process . following the workshop , details of the action plan were prepared into a report and relevant materials circulated to the site team for final approval .
the principal investigator and research coordinator met with both managers and front line care staff at each of the sites to ensure that the proposed action plan accommodated circumstances unique to each setting .
forty - two internal ( n = 28 ) and external ( n = 14 ) documents were collected and reviewed .
internal documents included information that was directly applicable to the sites ( e.g. , mission and values , job descriptions , accreditation standards ) and external documents provided information on potentially influential outside factors ( e.g. , continuing care provincial policy , professional standards of practice ) .
overall the document scan revealed a general lack of specific reference to oral health and oral care .
consistent with other findings , external documents clearly acknowledged a need for or commitment to whole body health and optimal well - being .
however , internal documents reflected more direct activities of practice and referred to general terms such as personal care
, assessing all body systems , or am / pm care without the specific mention of oral care . where oral care was included ( n = 10 ) , terms such as mouth / denture care or
overall , the scan revealed negligible references to oral or mouth care as an explicit domain of personal care .
there are no government standards with specific reference to mouth or oral care in long - term care .
similar references and level of detail were provided for foot care ( n = 13 ) .
conversely , the five documents mentioning wound care included details regarding scopes of practice , an interdisciplinary clinic manual , a health services operational report , and a comprehensive manual developed by the provincial government .
details provided for wound care were not surprising given its recognition as specialized care with established best practices for managing wound pathology .
this is informative for oral care where the consequences of unchecked oral disease have similar negative implications for health and quality of life .
thirteen one - on - one semistructured interviews ranging from 30 to 60 minutes were undertaken with administrators and health professionals who service the three facilities .
participants represented two distinct groups , 5 internal professionals who were involved with the day - to - day care of residents living in long - term care facilities ( i.e. , long - term care coordinators , nurse managers ) , and 8 external professionals who provided care to residents but who are not present on a daily basis ( i.e. , physician , dietitian , physiotherapist , occupational therapist , social worker , nurse practitioner , acute care coordinators ) .
the former group was more involved in addressing and recognizing the needs of residents on a daily basis and their perspectives were very much aligned with those of front - line care staff described hereinafter .
external professionals recognized the importance of oral care but generally felt removed from oral care and its implications .
neither group was aware of existing formal policies or supports related to oral care in long - term care .
we certainly have concerns about the people pocketing food , going to bed after lunch and that they could choke on that food .
lack of consideration for oral care in routine health assessments undertaken by the various professions was also identified as problematic :
in a routine screening , i probably would n't ask about teeth unless i noticed something and that is probably not a good thing . it is probably something we should be asking about .
both groups acknowledged the importance of daily oral care : [ oral health is important ] for overall general health , for nutrition status , for comfort , for self esteem .
it 's really important for basic health and advocated for more educational opportunities for care staff : i think that front line people need more education on oral health
i am not sure it is focused enough in the program they take there does n't seem to be a lot of emphasis on oral health and why it 's important in what i see in the people who come here to work .
all interviewees were generally aware of difficulties residents have in accessing professional dental services : for a lot of our people , they find they ca n't do anything .
if they do have a problem with their dental or oral health , they ca n't really afford to do anything about it so they tend to leave it .
focus groups held at each of the three sites included 17 residents and family members ( n = 8 , 3,6 ) and 14 front - line personal care providers ( n = 5 , 3,6 ) .
residents and family members expressed feedback about availability of mouth care products and good communication between care staff and residents as being important features of mouth care .
responses to direct questions about daily brushing and denture care met with positive responses by residents : they are a great bunch of girls . even with probing questions about daily hygiene care , residents and family members associated mouth care with professional dental care
current residents who did have complaints about their teeth indicated that they would make do or put up with the discomfort : i 'll put up with my teeth
; i ca n't bite with [ my dentures ] like i used to with some things but i think they 'll do me .
access to professional services was limited by residents ' mobility and funding issues associated with transportation costs to move residents off - site for professional care , costs that must be borne by residents themselves :
it 's really expensive to go to a dentist and get a cap or a filling , or even just to have your dentures fixed because some of the elderly , their dentures are loose and they ca n't afford a new set of dentures .
it 's practically a whole day by the time you get to the dentist 's and back again very draining .
i get there but by the time i get home , i 'm dead . ( resident ) . not surprisingly , the most significant input came from front line care staff most involved with the day - to - day care of residents summarized in table 2 .
however , they identified numerous factors that either hindered or helped with carrying out these tasks that are consistent with other reported findings .
for instance , although oral care is included in their personal care training , many felt a repulsion and lack of comfort ( fear ) when providing mouth care [ 28 , 34 ] .
this was intensified when residents exhibit resistant behavior as a result of dementia [ 28 , 34 ] disability or indifference to the value of mouth care .
participants acknowledged that the proportion of residents and clients with advanced frailty and dementia - related disease is increasing , placing greater demands for providing care .
the number of residents with natural teeth is also increasing and many enter long - term care with very poor oral health [ 1 , 34 ] .
in fact , the oral health status of residents was also seen to be an important factor influencing the quality of care they received especially if poor oral health is accompanied by sensitivity or pain
. constraints of resources and time for completing personal care tasks often leave mouth care low on care staff 's list of priorities [ 31 , 34 ] .
although one of the facilities did have a formal oral care protocol , it was acknowledged that there was little guidance for oral assessments , care planning , and accountability . along with barriers ,
key facilitating influences were also identified and are consistent with the earlier findings [ 28 , 34 ] .
the level of residents ' functional abilities and a good relationship with their care provider were seen as beneficial .
having a good routine , availability of mouth care products , and sufficient time were also identified as important for facilitating care . with respect to perspectives about education
, there was a strong indication that standardized and in - depth oral health education during personal care and nursing training programs would be key to achieving improved and consistent daily oral care .
care staff were generally receptive to in house education and training opportunities as well .
reminders and visuals ( such as those commonly posted for hand - washing ) would be useful tools for raising awareness .
they were unanimous in expressing a resistance to being monitored daily through check lists saying : tick sheets are definitely not the answer .
positive reinforcements , available resources , visual reminders , and education would be more readily accepted by care staff for enhancing mouth care .
overall these findings provided a unique window into the continuing care environment and direct responsibilities of a range of front - line care staff , managers , and administrators working within the three facilities .
this feedback , coupled with findings of the document analysis and input from the collaboration , provided the basis for establishing priorities for the action planning workshop , evaluation of prospective activities arising from the workshop , and planning next steps for introducing an oral care action plan .
as described previously , this one - day interdisciplinary workshop was held to develop an oral care action plan to be integrated into organizational policy and practice . following formal presentations , significant contributions of the workshop
there was some overlap between topics but feedback was collated into the following summaries .
education / training required to strengthen the delivery of daily oral careeducation for residents may be important to heighten awareness regarding oral health .
these posters would be used as a visual reminder and would include information on the importance of proper oral care , steps outlining proper care , and so on
train the trainers approach would be appropriate to enhance sustainability of the action plan .
this would involve training nursing staff or a designate who would take a leadership role in providing ongoing oral health care support for personal care providers and other relevant staff within the facilities .
these posters would be used as a visual reminder and would include information on the importance of proper oral care , steps outlining proper care , and so on .
train the trainers approach would be appropriate to enhance sustainability of the action plan .
this would involve training nursing staff or a designate who would take a leadership role in providing ongoing oral health care support for personal care providers and other relevant staff within the facilities .
planning and tracking daily oral care activities ( daily and professional)there is a need to change the built environment to provide appropriate space for oral health ( i.e. , designated space for oral care ) . to adequately plan and track oral care activities within long - term care , specific tools and resources were suggested .
( i ) oral health kits should be created for each resident including the necessary tools to complete oral care such as toothbrushes , denture brushes , toothpaste , mouth rinses , and a towel to protect dentures in sink .
( ii ) care cards should be developed and color coded according to tooth and denture status ( i.e. , natural teeth , dentures , partial dentures , no teeth ) and would facilitate an individualized oral care plan for each resident .
cards could be used by care providers and , if residents go home for visits , by family members .
( iii ) a tool to enable personal care providers to conduct oral assessments as a part of providing oral care should be developed .
the tool would provide guidance for a visual exam of the mouth and a record of any problems .
care providers would need to be educated on what to look for and recognized as being the eyes of oral care within the facility .
they need to be involved in decision making about what they will be asked / required to do .
( iv ) a strategy for including oral care in dysphagia assessment that is performed by the dysphagia team should be developed .
by documenting issues related to oral care , it will increase the likelihood that something will be done about it .
there is a need to change the built environment to provide appropriate space for oral health ( i.e. , designated space for oral care ) . to adequately plan and track oral care activities within long - term care ,
( i ) oral health kits should be created for each resident including the necessary tools to complete oral care such as toothbrushes , denture brushes , toothpaste , mouth rinses , and a towel to protect dentures in sink .
( ii ) care cards should be developed and color coded according to tooth and denture status ( i.e. , natural teeth , dentures , partial dentures , no teeth ) and would facilitate an individualized oral care plan for each resident .
cards could be used by care providers and , if residents go home for visits , by family members .
( iii ) a tool to enable personal care providers to conduct oral assessments as a part of providing oral care should be developed .
the tool would provide guidance for a visual exam of the mouth and a record of any problems .
care providers would need to be educated on what to look for and recognized as being the eyes of oral care within the facility .
they need to be involved in decision making about what they will be asked / required to do .
( iv ) a strategy for including oral care in dysphagia assessment that is performed by the dysphagia team should be developed .
this would allow for a more formalized system of information sharing . by documenting issues related to oral care
special support needed to manage resistant residents / residents with dementiaa multidisciplinary approach to care planning is necessary .
there is a need to raise the profile of oral care for these residents and look at preexisting tools , daily report sheets , and white boards to improve communication regarding oral care .
role - playing may help to put care providers in the residents ' shoes .
there is a need to raise the profile of oral care for these residents and look at preexisting tools , daily report sheets , and white boards to improve communication regarding oral care .
role - playing may help to put care providers in the residents ' shoes .
access to professional dental servicesthis is an issue that needs to involve everyone from frontline care staff , senior administration , to government .
taking residents to a dentist requires funding for transportation in addition to the cost of service .
bringing a dentist to the residents requires funding for space , equipment , and costs of professional service .
oral care could be set up similar to foot care where a mobile unit makes site visits .
good communication across silos of continuing care and professional dental services is required to improve resident care and potentially save money in the health care system .
the value of professional dental care depends on the personal values of residents and their families .
participants recognized that access to professional dental services was an issue beyond the scope of the workshop and of the three facilities involved in the research .
this is an issue that needs to involve everyone from frontline care staff , senior administration , to government .
taking residents to a dentist requires funding for transportation in addition to the cost of service .
bringing a dentist to the residents requires funding for space , equipment , and costs of professional service .
oral care could be set up similar to foot care where a mobile unit makes site visits .
good communication across silos of continuing care and professional dental services is required to improve resident care and potentially save money in the health care system .
the value of professional dental care depends on the personal values of residents and their families .
participants recognized that access to professional dental services was an issue beyond the scope of the workshop and of the three facilities involved in the research .
the strategic action plan evolving from the workshop included each of the activities identified for action in the workshop .
the plan emphasized targeted education and training for administrators , nursing staff , and daily personal care providers .
the manuals would include education materials , pamphlets , and prepared forms to guide oral assessment , care planning , and intervention / referral documents , detailed work - plans , required oral hygiene products such as toothbrushes , toothpaste , denture care products , as well as individualized oral health toolkits for residents .
the site team liaison ( or an appropriate designate ) assumed responsibility for coordinating activities associated with the action plan .
the research coordinator visited each of the three research sites at 6-week intervals to review progress , to provide support , and to gather data .
proposed hands - on education workshops were provided by qualified research team members at the outset of action plan intervention and during regularly scheduled visits .
the site team liaison assumed responsibility for overseeing the care and reinforcing the skills with individual care providers as needed .
relevant research team members and the research coordinator provided ongoing support . to ensure that proposed methods of collecting data to evaluate the action plan activities were relevant and acceptable , the draft evaluation framework was reviewed and refined by the research team based on feedback at the action - planning workshop ( table 3 ) . according to thorne et al .
, success in oral health programs in long - term care is contingent upon effective programmatic strategies ( e.g. , routine oral hygiene , oral assessment , availability of professional dental services ) as well as the organizational culture influencing them ( e.g. , administrative capacity to support and control a caring environment , the presence of champions , organizational values ) . recalling that the unit of analysis for this case study was institutional ,
the evaluation plan was designed to examine the institutional context and to consider both organizational culture and the programmatic strategies arising from the oral care action plan .
this paper highlights a variety of important considerations for developing meaningful collaborative and applied intervention research . a defining feature of the brushing up on mouth care project has been an enduring and positive collaboration with end - users .
it has also necessitated that frequent project updates and face - to - face meetings are balanced with ensuring that end - users do not become burned out .
prior to the launch of this project , the collaboration ( members of both the research and project site teams ) invested time in getting to know each other and in coming to a common understanding of where the research should go .
these early communications created a level of comfort and familiarity enabling all voices to be heard and respected . at the outset , this led to the development of realistic research goals about what could be achieved .
the document scan , focus groups , and interviews all contributed to our understanding of the institutional context and organizational culture influencing the delivery of mouth care .
the document scan revealed significant gaps in policy , education , and clinical standards available to guide oral care in long - term care .
our understanding of influences on the delivery of oral care was further informed through the workshop group discussions involving a broader stakeholder group . here ,
mechanisms for addressing gaps were also identified and have become integrated into considerations for our ongoing work .
one specific example has been the uptake of the brushing up on mouth care
action plan by local community colleges responsible for training relevant entry - level care staff .
engagement with government policy makers , directors of care , educators , health administrators , and a broad spectrum of health professionals has also been fruitful in creating awareness about the need for relevant policy as well as guidelines that consider the interdisciplinary nature of this realm of care .
finally , the creation of an oral care action plan that is suitable and applicable to end - users is benefiting both care staff and those who depend on them for care . | research focusing on the introduction of daily mouth care programs for dependent older adults in long - term care has met with limited success .
there is a need for greater awareness about the importance of oral health , more education for those providing oral care , and organizational structures that provide policy and administrative support for daily mouth care .
the purpose of this paper is to describe the establishment of an oral care action plan for long - term care using an interdisciplinary collaborative approach
. methods .
elements of a program planning cycle that includes assessment , planning , implementation , and evaluation guided this work and are described in this paper .
findings associated with assessment and planning are detailed .
assessment involved exploration of internal and external factors influencing oral care in long - term care and included document review , focus groups and one - on - one interviews with end - users .
the planning phase brought care providers , stakeholders , and researchers together to design a set of actions to integrate oral care into the organizational policy and practice of the research settings .
findings .
the establishment of a meaningful and productive collaboration was beneficial for developing realistic goals , understanding context and institutional culture , creating actions suitable and applicable for end - users , and laying a foundation for broader networking with relevant stakeholders and health policy makers . | 1. Introduction
2. Methods
3. Results and Discussion
4. Conclusions | unlike previous generations ,
more and more older adults are maintaining their natural teeth into old age [ 13 ] . this is a welcome trend but results in new patterns of disease that become especially significant for those who are frail and who must depend on others for their personal care and hygiene [ 46 ] . mouth care is an integral part of personal care yet it is inadequate [ 4 , 6 , 7 ] and given low priority for residents in long - term care [ 8 , 9 ] . overall , the oral health status of residents in long - term care is poor [ 7 , 10 , 2325 ] and those with dementia experience even higher rates of oral disease . oral care for dependent older adults in long - term care is becoming a challenge that is expected to grow in importance as our population ages . over the past several decades ,
research focusing on attitudes and behaviors of care providers ; education and oral hygiene intervention programs ; and more recently , environmental , organizational , or social influences on the delivery of care is shedding new light on the complexity of factors influencing the ability to provide adequate oral care . social barriers include embarrassment and repulsion , lack of care staff confidence in their knowledge or ability to provide care [ 28 , 34 ] , a perception of oral care as invasive to the dignity and privacy of residents [ 35 , 36 ] or unwanted by the resident , and the perception by nurses that oral care is professionally unrewarding . factors identified to facilitate care staff 's ability and/or willingness to provide oral care include availability of oral health equipment , influence of and examples set by people seen as influential leaders , education and/or demonstration in oral health care procedures [ 27 , 29 , 35 , 37 ] , adequate time to provide care , and believing that oral health and oral care are important [ 28 , 29 ] . research involving staff education - based interventions directed toward improving oral health status of long - term care residents demonstrates conflicting results [ 3840 ] . a similarly designed comprehensive personal mouth care program introduced in multiple facilities found different effects on oral hygiene practices and health status of residents of the different facilities . , on - site services , routines , and resources ) explained very little of the difference between effective and ineffective programs [ 41 , 42 ] . creating effective strategies to address the issue of oral health in long - term care is further complicated because oral health care has traditionally been peripheral to mainstream health considerations . this has been recognized as a shortcoming and has resulted in a call for an interdisciplinary and collaborative approach to both research and practice [ 8 , 9 ] . the purpose of this paper is to describe the experience of developing a meaningful interdisciplinary collaboration and to highlight the processes used to design a comprehensive set of actions to integrate oral care into organizational policy and practice within three long - term care facilities in rural nova scotia on the east coast of canada . a collaborative approach is advantageous because the synergy created by the blending of perspectives , resources , and skills of various participants enables the group to create something that is not attainable by single agents . in this study , an interdisciplinary network of researchers and stakeholders
were brought together with administrators and front - line care staff to think about the work in creative and practical ways ; develop realistic goals ; plan and carry out comprehensive interventions that connect multiple programs , services , and sectors ; understand and document the impact of its actions ; incorporate the perspectives and priorities of stakeholders including the target population ; and communicate how actions will address problems . a common interest was established between the principal investigator ( m. e. mcnally ) and a senior administrator of three long - term care facilities during a provincial oral health policy workshop . over the course of a year
, multiple face - to - face meetings , telephone , and email consultations were undertaken with administrative staff to : discuss the current level of oral care and associated challenges ; develop realistic and practical research objectives ; establish a formal commitment to the research ; clarify roles and expectations ; and establish mutual benefits . through this process
, a formal collaboration was established consisting of an interdisciplinary research team in partnership with health service managers and nurse managers of the three facilities ( i.e. they are rurally situated within 1 hour of each other and are within 1 - 2-hour drive of a metropolitan area . the number of residents per site ranged from 25 to 40 , which is typical of the majority of long - term care facilities found in rural settings in the region . the three long - term care facilities are administered under the same health district where there is sharing of resources , budgets , and policy . a case study approach was used to explore the individual , organizational , and system factors associated with the integration of oral care in three long - term care settings . this research is consistent with the elements of a program planning cycle that include assessment , planning , implementation , and evaluation of a set of actions to integrate oral care into both organizational policy and daily personal care practice . this paper describes details associated with the first two phases , assessment and action planning . assessment involved an exploration of the internal and external factors that influence the provision of oral care and oral disease prevention and was undertaken through a document review , focus groups , and one - on - one interviews . the action - planning phase brought care providers and stakeholders ( government representatives , educators , and dental professionals ) together for a workshop to design a set of actions to integrate oral care into organizational policy and practice in each of the settings . the process and outcomes associated with the implementation of actions are being evaluated and are providing the basis of recommendations to revise organizational policy and oral care practice . a systematic analysis of multiple forms of evidence was used to enhance understanding of the context and the people within it . , document analysis , focus groups , interviews , journaling ) , multiple data sources ( i.e. , personal care providers , administrators , residents , and families ) , and multiple settings . it was also recognized that the term oral care connotes the broad spectrum of oral health care needs associated with both professional and personal care . this research is primarily concerned with the latter , daily mouth care , for frail older adults . documents were selected on the basis of their topic relevance and included those expected to inform the provision of oral care for long - term care in the region ( e.g. they were individually read , coded , and organized according to four criteria : ( 1 ) general health terminology ( that may or may not include oral care ) , ( 2 ) oral care ( including terms oral , mouth , and dental ) , ( 3 ) foot care , and ( 4 ) wound care . members of the collaboration suggested that it would be useful to examine oral care for consistency with existing and familiar clinical domains that may ultimately provide a useful framework for oral care . this approach is constructivist and interpretivist [ 52 , 53 ] seeking to distil from personal accounts the experiences and meaning behind oral health care for dependent older adults . one - on - one semistructured interviews were held with administrators and health professionals who provide a variety of health services to residents and clients associated with the three facilities . two focus groups were held in each of the three facilities with ( 1 ) personal care providers ( i.e. semistructured questions for both focus groups and individual interviews were designed to guide and generate discussion to elicit participants ' description of practices associated with the provision of oral care , perceptions of barriers and facilitators to oral care in the care settings , attitudes toward oral health and oral health care , and relevant knowledge of formal oral health policies . a one - day interdisciplinary oral care action - planning workshop was held at a location central to the research sites . the purpose of the workshop was to design a set of actions that would integrate oral care into organizational policy and personal care practices in each of the three long - term care settings . the two key goals were to : ( 1 ) identify and prioritize education , training , tools , and program strategies and ( 2 ) establish a detailed plan for implementing , tracking , and evaluating both professional and daily personal mouth care delivery . the workshop was attended by 34 participants : clinical researchers from dentistry , dental hygiene , nursing , and medicine ( n = 7 ) ; researchers from health promotion and organizational management ( n = 4 ) ; administrators ( n = 5 ) ; nurse managers ( n = 2 ) ; regulated and unregulated front line care staff ( n = 5 ) ; policy makers in the health and continuing care sectors ( n = 3 ) ; representatives of organized dentistry and dental hygiene ( n = 3 ) ; community college educators ( n = 2 ) ; speech language pathologist ( n = 1 ) ; dietician ( n = 1 ) ; as well as a representative from a seniors ' government advisory organization . a review of current best practices and a model of oral health care in long - term care provided an evidence base to inform workshop discussion . relevant topics identified through the document review , focus groups , and interviews were prioritized by the collaboration for discussion in one - hour breakout sessions and a discussion guide was developed for each topic ( table 1 ) . at the plenary ,
the group was asked to consider the following : how to best synthesize the information to create a comprehensive oral care action plan , how to best communicate the action plan to end users , and how to determine the biggest indicators of success over the next 18 months . findings from the workshop were collated and synthesized into a draft oral care action plan for integration at each of the three long - term care sites . the principal investigator and research coordinator met with both managers and front line care staff at each of the sites to ensure that the proposed action plan accommodated circumstances unique to each setting . consistent with other findings , external documents clearly acknowledged a need for or commitment to whole body health and optimal well - being . however , internal documents reflected more direct activities of practice and referred to general terms such as personal care
, assessing all body systems , or am / pm care without the specific mention of oral care . where oral care was included ( n = 10 ) , terms such as mouth / denture care or
overall , the scan revealed negligible references to oral or mouth care as an explicit domain of personal care . there are no government standards with specific reference to mouth or oral care in long - term care . conversely , the five documents mentioning wound care included details regarding scopes of practice , an interdisciplinary clinic manual , a health services operational report , and a comprehensive manual developed by the provincial government . thirteen one - on - one semistructured interviews ranging from 30 to 60 minutes were undertaken with administrators and health professionals who service the three facilities . participants represented two distinct groups , 5 internal professionals who were involved with the day - to - day care of residents living in long - term care facilities ( i.e. , long - term care coordinators , nurse managers ) , and 8 external professionals who provided care to residents but who are not present on a daily basis ( i.e. external professionals recognized the importance of oral care but generally felt removed from oral care and its implications . neither group was aware of existing formal policies or supports related to oral care in long - term care . both groups acknowledged the importance of daily oral care : [ oral health is important ] for overall general health , for nutrition status , for comfort , for self esteem . it 's really important for basic health and advocated for more educational opportunities for care staff : i think that front line people need more education on oral health
i am not sure it is focused enough in the program they take there does n't seem to be a lot of emphasis on oral health and why it 's important in what i see in the people who come here to work . focus groups held at each of the three sites included 17 residents and family members ( n = 8 , 3,6 ) and 14 front - line personal care providers ( n = 5 , 3,6 ) . even with probing questions about daily hygiene care , residents and family members associated mouth care with professional dental care
current residents who did have complaints about their teeth indicated that they would make do or put up with the discomfort : i 'll put up with my teeth
; i ca n't bite with [ my dentures ] like i used to with some things but i think they 'll do me . access to professional services was limited by residents ' mobility and funding issues associated with transportation costs to move residents off - site for professional care , costs that must be borne by residents themselves :
it 's really expensive to go to a dentist and get a cap or a filling , or even just to have your dentures fixed because some of the elderly , their dentures are loose and they ca n't afford a new set of dentures . for instance , although oral care is included in their personal care training , many felt a repulsion and lack of comfort ( fear ) when providing mouth care [ 28 , 34 ] . the number of residents with natural teeth is also increasing and many enter long - term care with very poor oral health [ 1 , 34 ] . although one of the facilities did have a formal oral care protocol , it was acknowledged that there was little guidance for oral assessments , care planning , and accountability . having a good routine , availability of mouth care products , and sufficient time were also identified as important for facilitating care . with respect to perspectives about education
, there was a strong indication that standardized and in - depth oral health education during personal care and nursing training programs would be key to achieving improved and consistent daily oral care . overall these findings provided a unique window into the continuing care environment and direct responsibilities of a range of front - line care staff , managers , and administrators working within the three facilities . this feedback , coupled with findings of the document analysis and input from the collaboration , provided the basis for establishing priorities for the action planning workshop , evaluation of prospective activities arising from the workshop , and planning next steps for introducing an oral care action plan . as described previously , this one - day interdisciplinary workshop was held to develop an oral care action plan to be integrated into organizational policy and practice . following formal presentations , significant contributions of the workshop
there was some overlap between topics but feedback was collated into the following summaries . these posters would be used as a visual reminder and would include information on the importance of proper oral care , steps outlining proper care , and so on
train the trainers approach would be appropriate to enhance sustainability of the action plan . this would involve training nursing staff or a designate who would take a leadership role in providing ongoing oral health care support for personal care providers and other relevant staff within the facilities . these posters would be used as a visual reminder and would include information on the importance of proper oral care , steps outlining proper care , and so on . train the trainers approach would be appropriate to enhance sustainability of the action plan . this would involve training nursing staff or a designate who would take a leadership role in providing ongoing oral health care support for personal care providers and other relevant staff within the facilities . planning and tracking daily oral care activities ( daily and professional)there is a need to change the built environment to provide appropriate space for oral health ( i.e. to adequately plan and track oral care activities within long - term care , specific tools and resources were suggested . ( i ) oral health kits should be created for each resident including the necessary tools to complete oral care such as toothbrushes , denture brushes , toothpaste , mouth rinses , and a towel to protect dentures in sink . , natural teeth , dentures , partial dentures , no teeth ) and would facilitate an individualized oral care plan for each resident . ( iii ) a tool to enable personal care providers to conduct oral assessments as a part of providing oral care should be developed . care providers would need to be educated on what to look for and recognized as being the eyes of oral care within the facility . ( iv ) a strategy for including oral care in dysphagia assessment that is performed by the dysphagia team should be developed . by documenting issues related to oral care , it will increase the likelihood that something will be done about it . there is a need to change the built environment to provide appropriate space for oral health ( i.e. to adequately plan and track oral care activities within long - term care ,
( i ) oral health kits should be created for each resident including the necessary tools to complete oral care such as toothbrushes , denture brushes , toothpaste , mouth rinses , and a towel to protect dentures in sink . , natural teeth , dentures , partial dentures , no teeth ) and would facilitate an individualized oral care plan for each resident . ( iii ) a tool to enable personal care providers to conduct oral assessments as a part of providing oral care should be developed . care providers would need to be educated on what to look for and recognized as being the eyes of oral care within the facility . ( iv ) a strategy for including oral care in dysphagia assessment that is performed by the dysphagia team should be developed . there is a need to raise the profile of oral care for these residents and look at preexisting tools , daily report sheets , and white boards to improve communication regarding oral care . there is a need to raise the profile of oral care for these residents and look at preexisting tools , daily report sheets , and white boards to improve communication regarding oral care . participants recognized that access to professional dental services was an issue beyond the scope of the workshop and of the three facilities involved in the research . the strategic action plan evolving from the workshop included each of the activities identified for action in the workshop . the plan emphasized targeted education and training for administrators , nursing staff , and daily personal care providers . the manuals would include education materials , pamphlets , and prepared forms to guide oral assessment , care planning , and intervention / referral documents , detailed work - plans , required oral hygiene products such as toothbrushes , toothpaste , denture care products , as well as individualized oral health toolkits for residents . the site team liaison ( or an appropriate designate ) assumed responsibility for coordinating activities associated with the action plan . the research coordinator visited each of the three research sites at 6-week intervals to review progress , to provide support , and to gather data . proposed hands - on education workshops were provided by qualified research team members at the outset of action plan intervention and during regularly scheduled visits . , success in oral health programs in long - term care is contingent upon effective programmatic strategies ( e.g. , routine oral hygiene , oral assessment , availability of professional dental services ) as well as the organizational culture influencing them ( e.g. recalling that the unit of analysis for this case study was institutional ,
the evaluation plan was designed to examine the institutional context and to consider both organizational culture and the programmatic strategies arising from the oral care action plan . a defining feature of the brushing up on mouth care project has been an enduring and positive collaboration with end - users . it has also necessitated that frequent project updates and face - to - face meetings are balanced with ensuring that end - users do not become burned out . the document scan , focus groups , and interviews all contributed to our understanding of the institutional context and organizational culture influencing the delivery of mouth care . the document scan revealed significant gaps in policy , education , and clinical standards available to guide oral care in long - term care . our understanding of influences on the delivery of oral care was further informed through the workshop group discussions involving a broader stakeholder group . one specific example has been the uptake of the brushing up on mouth care
action plan by local community colleges responsible for training relevant entry - level care staff . engagement with government policy makers , directors of care , educators , health administrators , and a broad spectrum of health professionals has also been fruitful in creating awareness about the need for relevant policy as well as guidelines that consider the interdisciplinary nature of this realm of care . finally , the creation of an oral care action plan that is suitable and applicable to end - users is benefiting both care staff and those who depend on them for care . | [
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] |
chronic obstructive pulmonary disease ( copd ) is a leading cause of disability and death worldwide [ 13 ] .
although copd is more and more common , the molecular and cellular mechanisms that are responsible for the development of copd are not well understood .
early studies have shown that copd is marked by the accumulation of both cd4 and cd8 t cells in the airways and lung parenchyma , with cd8 t cells predominating .
recent findings suggest that copd is an autoimmune disease characterized by an association of antielastin antibody and th1 response [ 5 , 6 ] .
il-17 ( also known as il-17a ) was first cloned in 1993 and identified as cytotoxic t lymphocyte - associated antigen ( ctla)-8 .
evidence has indicated that il-17 is involved in inflammatory disorders of the lungs [ 9 , 10 ] .
il-17 may play an important role in the pathogenesis of copd because of its ability to induce neutrophilic airway inflammation by stimulation of neutrophil chemotaxis and mucin gene expression in bronchial epithelial cells [ 11 , 12 ] .
overexpression of il-17 in lungs of transgenic mice may induce lung inflammation with a copd - like phenotype .
th17 cells are the newly described subset of cd4 t cells and have significant role in the progression of several t cells driven by autoimmune diseases , such as rheumatoid arthritis and multiple sclerosis , which were previously thought to be exclusively mediated by th1 cells [ 14 , 15 ] .
more recently , th17 cells have been found in copd and emphysema [ 16 , 17 ] .
however , the role of th17 cells in regulating inflammatory response in emphysema remains to be demonstrated .
il-21 is a pleiotropic cytokine of the -chain family , which engages the common cytokine receptor -chain expressed on cells of both lymphoid and myeloid lineages .
this cytokine was originally thought to be restricted to cd4 t cells ( th1 and th2 cells ) and nkt cells , but it is now clear that il-21 is also produced by th17 cells [ 18 , 19 ] .
il-21 can serve to recruit th17 cells into the inflamed tissue , and also deliver intracellular signal through il-21r and influence t - cell activation and differentiation .
recently , zeng et al . demonstrated that in vitro exposure to il-21 can lead to the generation of cd8 t cell in increased numbers and with enhanced function .
these data suggest that th17 cells may play role in regulating cd8 cytotoxic t - cell function via il-21/il-21r . in the present study
, we hypothesized that th17 cells and il-21 are involved in the local and system inflammatory response using a murine model of emphysema induced by smoking .
we tested il-17 and il-21 protein levels in both peripheral blood and lung tissues of cigarette - smoke- ( cs- ) exposed mice and air - exposed mice , and analyzed the expression of th17 , il-21th17 , and cd8il-21r t cells in peripheral blood and lung tissues of mice , and their relationship with emphysematous lesions .
finally , we assessed the possible modulating effect of recombinant il-21 ( ril-21 ) on cd8 t cells in vitro .
specific pathogen - free inbred male balb / c mice ( 8 weeks of age , 2025 g body weight ) were purchased from the guangxi medical university laboratory animal center ( nanning , china ) .
all animal procedures were reviewed and approved by the laboratory animal ethics committee of guangxi medical university .
all mice were housed in sterilized cages and maintained on a 12 : 12 h light - dark cycle and received sterilized food and water ad libitum .
mice ( n = 10 ) were exposed whole - body to cs , as described previously .
briefly , groups of ten mice ( cs - exposed mice ) were exposed to five cigarettes ( nanning jiatianxia unfiltered cigarettes : 12 mg of tar and 0.9 mg of nicotine ) , 4 times a day with 30 min smoke - free intervals in a closed 0.75 m room , 5 days a week for 24 weeks .
mice tolerated cigarette smoke exposure without evidence of toxicity ( carboxyhemoglobin levels ~10% and no weight loss ) . an optimal smoke : air ratio of 1 : 6 was obtained . the control groups ( air - exposed mice )
the serum carboxyhemoglobin of cs - exposed mice was 8.4 1.2% versus 1.0 0.3% in air - exposed mice ( n = 10 ) , which is similar to carboxyhemoglobin blood concentrations of human smokers .
24 hours after the last air or smoke exposure , the mice were sacrificed with sodium pentobarbital anesthesia .
approximately 200 l of peripheral blood was obtained for flow cytometric analysis , and approximately 1500 l of peripheral blood was separated serum for enzyme - linked immunosorbent assay analysis ( elisa ) .
one part of the right lungs ( 30 to 50 mg ) was homogenized for elisa , and remanent parts were used for the preparation of single - cell suspensions .
the left lungs were inflated by instilling 10% formalin at a constant pressure of 25 cm h2o ( for 10 min ) and then ligated and removed .
inflated lungs were fixed for 24 h before embedding in paraffin . after paraffin embedding , 5 m sections were cut and stained with hematoxylin and eosin for histological analysis . for each animal , 10 fields at a magnification of 100x were captured randomly from the 4 different zones of the left lung .
we determined enlargement of alveolar spaces by quantifying the mean linear intercept ( lm ) and destruction of alveolar walls by measuring the destructive index ( di ) in cs- and air - exposed mice , as described previously [ 24 , 25 ] .
lung single - cell suspensions were prepared from part of the right lung , as detailed previously .
briefly , the lung was thoroughly minced , digested , passed through a 70 m cell strainer , washed and centrifuged twice with cold pbs at 1200 rpm for 10 min at 4c , and resuspended in pbs .
the mononuclear cells were isolated from the lung single - cell suspension by ficoll - hypaque gradient centrifugation ( pharmacia , uppsala , sweden ) , washed and centrifuged twice with cold pbs at 1200 rpm for 10 min at 4c , and kept on ice until labelling .
erythrocytes were lysed with rbc lysis buffer ( sigma - aldrich ) for 10 minutes at room temperature and the remaining cells were washed twice with cold pbs and centrifuged at 1200 rpm for 10 minutes .
fresh peripheral - blood mononuclear cells ( pbmcs ) were used for intracellular cytokine staining within 1 h. after mincing spleens , the cell suspensions were pipetted rapidly with a sterile pasteur pipette into 3 ml of rpmi 1640 ( gibco , usa ) , filtered through nylon mesh to eliminate debris , and centrifuged at 1000 rpm for 5 min .
the cell pellets of spleens were resuspended in pbs , and the lymphocyte fractions were isolated by ficoll - plaque ( solarbio science & technology , china ) gradient centrifugation .
lymphocytes were maintained in a 24-well flat - bottom tissue culture plate with rpmi 1640 supplemented with 10% fetal calf serum ( gibco , usa ) at 37c in a humidified atmosphere with 5% co2 .
the expression markers on t cells were determined by flow cytometry after surface staining or intracellular staining using phycoerythrin cyanine-5-conjugated anti - mouse cd4 ( pe - cy5-cd4 ) , fluorescein isothiocyanate - conjugated anti - mouse cd8 ( fitc - cd8 ) , phycoerythrin - conjugated anti - mouse il-17 ( pe - il-17 ) , phycoerythrin - conjugated anti - mouse il-21r ( pe - il-21r ) , and alexa fluor 647-conjugated anti - mouse il-21 ( alexa fluor 647-il-21 ) .
these mice abs were purchased from bd biosciences or ebioscience ( san diego , ca ) .
briefly , pbmcs were stimulated with phorbol myristate acetate ( pma , 25 ng / ml , sigma - aldrich , usa ) and ionomycin ( 10 g / ml , sigma - aldrich , usa ) in the presence of golgistop ( bd biosciences ) for 5 h. the cells were washed and then fixed / permeabilized in the ebioscience fixation / permeabilization and permeabilization buffers according to the manufacturer 's protocol , stained with fluorescent antibodies against cd4 , cd8 , il-21r , il-17 , and il-21 .
flow cytometry was performed on a bd facscalibur flowcytometer and analyzed by using fcs expressv4 software .
bulk cd8 t cells from spleen were positively selected using paramagnetic microbeads conjugated to anti - mouse cd8 ( ly-2 ) monoclonal antibody according to the manufacturer 's instructions ( macs , miltenyi biotec ) .
purified cd8 t cells were cultured at 1 10 cells / ml in rpmi 1640 medium containing 10% fbs , 100 u / ml penicillin , 100 g / ml streptomycin , 2 mm l - glutamine , and 50 m mercaptoethanol ( rpmi 1640 complete medium 2 ml ) in 96-well plates ( 200 l ) at 37c , 5% co2 , and 100% humidity with phytohemagglutinin ( pha , 10 ng / ml ) and treated with or without 50 ng / ml of mril-21 for 3 days .
a cytokine concentration of 50 ng / ml was chosen based on initial dose - response experiments with 10 to 100 ng / ml concentrations and published literature to achieve maximal effect on cd8 t - cell cytotoxic [ 21 , 28 ] .
the cells were washed once and restimulated with pma / ionomycin in the presence of golgistop ( bd biosciences ) for 5 h and then fixed / permeabilized with ebioscience fixation / permeabilization according to the manufacturer 's protocol and stained with antibodies specific for intracellular perforin ( pe - perforin ) and granzyme b ( apc - granzyme b ) .
the concentrations of il-17 and il-21 in the peripheral blood and the lungs , as well as perforin and granzyme b in culture supernatants , were measured by elisa kits according to the manufacturer 's protocols ( r&d systems , minneapolis , mn ) .
statistical analysis was performed by using spss statistical software version 16 ( spss inc . ,
emphysema is a structural disorder characterized by destruction of the alveolar walls and enlargement of the alveolar spaces .
histologically , the lungs sections from the air - exposed mice showed normal alveolar structure and exhibited normal size airspaces with thin septa ( figure 1(a ) ) .
in contrast , the lungs sections from the cs - exposed mice showed an increased air space enlargement and destruction .
some airspaces seemed irregular in size , and septa were thin ( figure 1(b ) ) .
the di was higher in cs - exposed mice ( 45.16 3.13 ) compared with air - exposed animals ( 28.86 2.07 , p < 0.001 ; figure 1(c ) ) ; exposure to cigarette smoke significantly induced airspace enlargement .
m ) compared with air - exposed animals ( 32.60 3.21 m , p < 0.001 ; figure 1(d ) ) . in the present study , using elisa we noted that the lung levels of il-17 and il-21 were significantly increased in cs - exposed mice compared to air - exposed mice ( p < 0.001 , figures 2(a ) and 2(b ) ) .
similarly , we also noted that the levels of il-17 and il-21 in the peripheral blood of cs - exposed mice were significantly higher than those of air - exposed mice ( p < 0.001 , figures 2(c ) and 2(d ) ) . to further confirm our results , we studied the relation between the levels of il-17 and il-21 and emphysematous lesions as measured by di and lm in cs - exposed mice .
the lung levels of il-17 and il-21 were positively correlated with di ( r = 0.87 , p = 0.001 and r = 0.707 , p = 0.022 , resp .
, figures 3(a ) and 3(b ) ) and with lm ( r = 0.747 , p = 0.013 and r = 0.821 , p = 0.004 , resp . , figures 3(c ) and 3(d ) ) .
similarly , the peripheral blood levels of il-17 and il-21 were positively correlated with di ( r = 0.757 , p = 0.011 and r = 0.738 , p = 0.015 , resp .
, figures 3(e ) and 3(f ) ) and with lm ( r = 0.817 , p = 0.004 and r = 0.736 , p = 0.015 , resp . , figures 3(g ) and 3(h ) ) .
we analyzed the frequencies of th17 , il-21th17 , and cd8il-21r t cells in the peripheral blood and the lungs by using flow cytometry .
the frequencies of th17 and il-21th17 cells in the peripheral blood of cs - exposed mice were significantly increased compared to air - exposed littermates ( p < 0.05 , figures 4(a ) and 4(b ) ) . also after chronic cs - exposure for 6 months , a significant increase in cd8il-21r t cells was observed in the peripheral blood from cs - exposed mice compared with those in air - exposed littermates ( p < 0.001 , figure 4(c ) ) .
in addition , the frequencies of th17 , il-21th17 , and cd8il-21r t cells in the lungs of cs - exposed mice were significantly higher than those of the controls ( p < 0.05 , figures 5(a ) , 5(b ) , and 5(c ) ) . the frequencies of peripheral blood th17 , il-21th17 , and cd8il-21r t cells were positively correlated with di ( r = 0.892 , p = 0.001 ; r = 0.777 , p = 0.008 and r = 0.697 , p = 0.025 , resp . )
( figures 6(a ) , 6(c ) , and 6(e ) ) and with lm ( r = 0.757 , p = 0.011 ; r = 0.789 , p = 0.007 and r = 0.716 , p = 0.020 , resp . ) ( figures 6(b ) , 6(d ) , and 6(f ) ) in cs - exposed mice .
in addition , il-21th17 cells were correlated positively with cd8il-21r t cells ( r = 0.648 , p = 0.005 ) ( figure 6(g ) ) .
the frequencies of lung th17 , il-21th17 , and cd8il-21r t cells were significantly and positively correlated with di ( r = 0.861 , p = 0.001 ; r = 0.700 , p = 0.024 and r = 0.818 , p = 0.004 , resp . )
( figures 7(a ) , 7(c ) , and 7(e ) ) and with lm ( r = 0.865 , p = 0.001 ; r = 0.785 , p = 0.007 and r = 0.885 , p = 0.001 , resp . ) ( figures 7(b ) , 7(d ) , and 7(f ) ) in cs - exposed mice .
in addition , il-21th17 cells were correlated positively with cd8il-21r t cells ( r = 0.73 , p = 0.017 ) ( figure 7(g ) ) .
our findings indicated that th17 cells might be able to regulate cd8 t cells via il-21/il-21r system . to evaluate the contribution of il-21 to the function of cd8 t cells in cs - exposed mice , we next cultured cd8 t cells in vitro with ril-21 to analyze cytokine production .
we isolated cd8 t cells by macs from cs - exposed mice and from air - exposed mice .
the purified cd8 t cells were cultured in the presence of pha ( 10 ng / ml ) and ril-21 ( 50 ng / ml ) or pha ( 10 ng / ml ) alone for 3 days .
results showed that administration of ril-21 and pha significantly upregulated the expression of perforin and granzyme b in cd8 t cells ; this effect was greater in cs - exposed mice than in air - exposed mice ( figure 8(a ) ) .
notably , there were more perforin+ cells ( 88.29 11.03% ) than granzyme b+ cells ( 69.47 5.31% , p < 0.01 ) in cd8 t cells of cs - exposed mice ( figure 8(a ) ) . in parallel , the protein levels of perforin and granzyme b in the culture supernatants significantly increased following 3 days of culture with il-21 and pha ; this effect was also greater in cs - exposed mice than in air - exposed mice ( figure 8(b ) ) .
the protein levels of perforin in the culture supernatants of cs - exposed mice ( 24.47 2.61
ng / l ) were significantly increased more than the levels of granzyme b ( 1.92 0.21 ng / l , p < 0.001 ) ( figure 8(b ) ) .
these data suggest that il-21rcd8 t cells have the capacity to secrete perforin and granzymes and further support the potential relevance of th17 cells in emphysema .
emphysema is considered a t - cell - mediated autoimmune disease , but its etiology and pathology have not been elucidated . in this study , we employed a murine model of cigarette smoke - induced lung emphysema to investigate the capacity of th17 cells to participate in emphysema pathogenesis .
we found that the numbers of th17 , il-21th17 , and cd8il-21r t cells and the levels of il-17 and il-21 in the peripheral blood and lungs of cs - exposed mice were much higher than those of air - exposed mice and correlated with emphysematous lesions . additionally , the number of il-21th17 cells positively correlated with the number of cd8il-21r t cells .
furthermore , il-21 significantly augmented the secretion of perforin and granzyme b in cd8 t cells from cs - exposed mice in vitro .
these data indirectly demonstrate that il-21 produced by th17 cells can act on cd8 t cells to promote cytotoxic function .
cd4 t cells are known as important key cells in immunoregulation , whereas cd8 t cells have cytotoxic function in copd .
classically , naive cd4 t cells have been thought to differentiate into two main lineages , th1 and th2 cells on the basis of their cytokines secretion and immune regulatory function .
regulatory t cells represent only a small subset of cd4 t cells in the peripheral circulation and are responsible for the balance of immune responses , which is essential for health .
early studies have suggested that infiltrating cd4 t cells in copd exhibit a th1 phenotype .
our previous study showed that decreased regulatory t cells were found in lungs of emphysema group .
more recently , we have reported that increased th17 cells could be found in lungs of smoke - exposed mice , and these th17 cells might be due to th17 differentiation stimulated by lung proinflammatory cytokines and to recruitment of th17 cells via ccr6/ccl20 . in the current study , we also demonstrated that increased th17 cells were present in peripheral blood and lungs of cs - exposed mice .
these findings are in agreement with the in vivo data of shan et al . , who demonstrated that th17 cells were present in lungs from patients with emphysema , and harrison et al .
thus , these studies indicated that th17 cells may have a relevant role in the local and system inflammatory process of copd .
th17 cells secrete not only il-17a but also il-17f , il-21 , and il-22 , these cytokines most likely induce tissue inflammation .
recent studies have been showed that il-17a and il-17f could stimulate chemokine production and promote neutrophil and macrophage recruitment to the lung [ 37 , 38 ] .
il-21 might act in a positive feedback loop , preserving and/or amplifying generation of th17 cells [ 39 , 40 ] , and serve to recruit th17 cells into the inflamed tissue .
moreover , leonard and spolski indicated that il-21 could significantly increase lymphocytes survival and cytolytic potential . given these findings , it is perhaps not surprising that exaggerated il-17 and il-21 responses are implicated in the pathogenesis of copd . in the present study , significantly elevated levels of il-17 and il-21
in addition , a positive correlation between levels of il-17 and il-21 and emphysematous lesions was found in cs - exposed mice .
although these findings suggest that the enhanced th17 cells and il-21 production in emphysema are biologically relevant , the mechanism for th17 cells in emphysema pathogenesis remains unidentified .
several studies have shown that the number of cd8 t cells found in the lungs of patients with copd correlates with disease severity , and lung cd8 t cells may directly cause cytotoxicity contributing to emphysema by inducing apoptosis through secretion of perforin , granzyme , and by fas / fas ligand ( fasl ) interactions .
further , it has been demonstrated that cd4 t cells are essential in the promotion of functional cd8 t - cell memory after an acute infection .
therefore , it is likely that th17 cells may participate in copd immunoregulation via generation of cd8 cytotoxic t cells .
it mediates its effects through a class i cytokine family receptor il-21r , which specifically binds il-21 . in the current study , we demonstrated that the number of il-21th17 and cd8il-21r t cells was significantly increased in the lungs and peripheral blood of cs - exposed mice .
more importantly , a significantly positive correlation between the number of il-21th17 and cd8il-21r t cells and emphysematous lesions was found in cs - exposed mice , which indicated the importance of il-21th17 and cd8il-21r t cells in the development of emphysema .
in addition , a significant correlation existed between il-21th17 and cd8il-21r t cells in the lungs and peripheral blood of cs - exposed mice .
this suggests that th17 cells could produce il-21 to mediate inflammatory response through il-21r , which is expressed on cd8 t cells .
thus , we presume that il-21 released from th17 cells might play a more important role in the immunopathology of emphysema through its actions on cd8 t cells . to further address this issue , we examined the capacity of ril-21 to promote cd8 t cells functions in vitro and found that coculture of il-21 and cd8 t cells results in cells producing increased amounts of perforin and granzyme - b upon chronic smoke expose .
interestingly , the cd8 perforin cells were proportionally higher than cd8 granzyme b cells , and the perforin levels were also much higher than the granzyme b levels .
perforin can form pores in the target cells ' membranes , while granzymes , as serine proteases , enter the cytoplasm of the target cells , altering their function and/or activating cell death .
these observations indirectly demonstrate that il-21 produced by th17 cells can promote cd8 t cells to induce apoptosis and tissue damage via the granzyme - b / perforin - mediated pathway .
however , the precise mechanism for the effect of th17 cells on the development of smoke - induced emphysema undoubtedly needed successive studies .
our data showed that the frequencies of th17 , il-21th17 , and cd8il-21r t cells and the levels of il-17 and il-21 in the peripheral blood and lungs of cs - exposed mice were significantly increased compared to controls and correlated with emphysematous lesions .
furthermore , expression of perforin and granzyme b by cd8 t cells was increased by in vitro stimulation with il-21 .
our findings support the concept that th17 cells and related cytokine il-21 were involved in the pathogenesis of copd .
however , to the best of our knowledge , although in the in vitro experiments il-21 promotes cd8 t cell cytotoxic responses , whether or not il-21 derived from the th17 cells contributes to the enhanced cd8 t cells function response remains unclear .
further research should be done by using adoptive transfer of th17 cells and il-21-null ( or il-21 deficient ) mice . | emphysema is a t - cell mediated autoimmune disease caused predominantly by cigarette smoking .
th17 cells and related cytokines may contribute to this disorder .
however , the possible implication of th17 cells in regulating inflammatory response in emphysema remains to be elucidated . in the current study , we tested the protein levels of il-17 and il-21 in peripheral blood and lung tissues from cigarette - smoke- ( cs- ) exposed mice and air - exposed mice , analyzed the frequencies of cd4+il-17+(th17 ) cells , il-21+th17 cells , and cd8+il-21r+ t cells in peripheral blood and lung tissues of mice , and their relationship with emphysematous lesions , and explored the impact of il-21 on cytotoxic cd8 + t cells function in vitro .
it was found that the frequencies of th17 , il-21+th17 , and cd8+il-21r+ t cells and the levels of il-17 and il-21 of cs - exposed mice were much higher than those of the air - exposed mice and correlated with emphysematous lesions . additionally , the number of il-21+th17 cells positively correlated with the number of cd8+il-21r+ t cells .
the in vitro experiments showed that il-21 significantly augmented the secretion of perforin and granzyme b in cd8 + t cells from cs - exposed mice .
these data indirectly provide evidence that th17 cells could be involved in the control of the local and system inflammatory response in emphysema by regulating cd8 + cytotoxic t - cell function . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Summary | early studies have shown that copd is marked by the accumulation of both cd4 and cd8 t cells in the airways and lung parenchyma , with cd8 t cells predominating . evidence has indicated that il-17 is involved in inflammatory disorders of the lungs [ 9 , 10 ] . th17 cells are the newly described subset of cd4 t cells and have significant role in the progression of several t cells driven by autoimmune diseases , such as rheumatoid arthritis and multiple sclerosis , which were previously thought to be exclusively mediated by th1 cells [ 14 , 15 ] . however , the role of th17 cells in regulating inflammatory response in emphysema remains to be demonstrated . il-21 is a pleiotropic cytokine of the -chain family , which engages the common cytokine receptor -chain expressed on cells of both lymphoid and myeloid lineages . this cytokine was originally thought to be restricted to cd4 t cells ( th1 and th2 cells ) and nkt cells , but it is now clear that il-21 is also produced by th17 cells [ 18 , 19 ] . il-21 can serve to recruit th17 cells into the inflamed tissue , and also deliver intracellular signal through il-21r and influence t - cell activation and differentiation . these data suggest that th17 cells may play role in regulating cd8 cytotoxic t - cell function via il-21/il-21r . in the present study
, we hypothesized that th17 cells and il-21 are involved in the local and system inflammatory response using a murine model of emphysema induced by smoking . we tested il-17 and il-21 protein levels in both peripheral blood and lung tissues of cigarette - smoke- ( cs- ) exposed mice and air - exposed mice , and analyzed the expression of th17 , il-21th17 , and cd8il-21r t cells in peripheral blood and lung tissues of mice , and their relationship with emphysematous lesions . finally , we assessed the possible modulating effect of recombinant il-21 ( ril-21 ) on cd8 t cells in vitro . briefly , groups of ten mice ( cs - exposed mice ) were exposed to five cigarettes ( nanning jiatianxia unfiltered cigarettes : 12 mg of tar and 0.9 mg of nicotine ) , 4 times a day with 30 min smoke - free intervals in a closed 0.75 m room , 5 days a week for 24 weeks . the control groups ( air - exposed mice )
the serum carboxyhemoglobin of cs - exposed mice was 8.4 1.2% versus 1.0 0.3% in air - exposed mice ( n = 10 ) , which is similar to carboxyhemoglobin blood concentrations of human smokers . 24 hours after the last air or smoke exposure , the mice were sacrificed with sodium pentobarbital anesthesia . approximately 200 l of peripheral blood was obtained for flow cytometric analysis , and approximately 1500 l of peripheral blood was separated serum for enzyme - linked immunosorbent assay analysis ( elisa ) . one part of the right lungs ( 30 to 50 mg ) was homogenized for elisa , and remanent parts were used for the preparation of single - cell suspensions . we determined enlargement of alveolar spaces by quantifying the mean linear intercept ( lm ) and destruction of alveolar walls by measuring the destructive index ( di ) in cs- and air - exposed mice , as described previously [ 24 , 25 ] . lung single - cell suspensions were prepared from part of the right lung , as detailed previously . briefly , the lung was thoroughly minced , digested , passed through a 70 m cell strainer , washed and centrifuged twice with cold pbs at 1200 rpm for 10 min at 4c , and resuspended in pbs . the mononuclear cells were isolated from the lung single - cell suspension by ficoll - hypaque gradient centrifugation ( pharmacia , uppsala , sweden ) , washed and centrifuged twice with cold pbs at 1200 rpm for 10 min at 4c , and kept on ice until labelling . fresh peripheral - blood mononuclear cells ( pbmcs ) were used for intracellular cytokine staining within 1 h. after mincing spleens , the cell suspensions were pipetted rapidly with a sterile pasteur pipette into 3 ml of rpmi 1640 ( gibco , usa ) , filtered through nylon mesh to eliminate debris , and centrifuged at 1000 rpm for 5 min . the cell pellets of spleens were resuspended in pbs , and the lymphocyte fractions were isolated by ficoll - plaque ( solarbio science & technology , china ) gradient centrifugation . the expression markers on t cells were determined by flow cytometry after surface staining or intracellular staining using phycoerythrin cyanine-5-conjugated anti - mouse cd4 ( pe - cy5-cd4 ) , fluorescein isothiocyanate - conjugated anti - mouse cd8 ( fitc - cd8 ) , phycoerythrin - conjugated anti - mouse il-17 ( pe - il-17 ) , phycoerythrin - conjugated anti - mouse il-21r ( pe - il-21r ) , and alexa fluor 647-conjugated anti - mouse il-21 ( alexa fluor 647-il-21 ) . briefly , pbmcs were stimulated with phorbol myristate acetate ( pma , 25 ng / ml , sigma - aldrich , usa ) and ionomycin ( 10 g / ml , sigma - aldrich , usa ) in the presence of golgistop ( bd biosciences ) for 5 h. the cells were washed and then fixed / permeabilized in the ebioscience fixation / permeabilization and permeabilization buffers according to the manufacturer 's protocol , stained with fluorescent antibodies against cd4 , cd8 , il-21r , il-17 , and il-21 . bulk cd8 t cells from spleen were positively selected using paramagnetic microbeads conjugated to anti - mouse cd8 ( ly-2 ) monoclonal antibody according to the manufacturer 's instructions ( macs , miltenyi biotec ) . a cytokine concentration of 50 ng / ml was chosen based on initial dose - response experiments with 10 to 100 ng / ml concentrations and published literature to achieve maximal effect on cd8 t - cell cytotoxic [ 21 , 28 ] . the cells were washed once and restimulated with pma / ionomycin in the presence of golgistop ( bd biosciences ) for 5 h and then fixed / permeabilized with ebioscience fixation / permeabilization according to the manufacturer 's protocol and stained with antibodies specific for intracellular perforin ( pe - perforin ) and granzyme b ( apc - granzyme b ) . the concentrations of il-17 and il-21 in the peripheral blood and the lungs , as well as perforin and granzyme b in culture supernatants , were measured by elisa kits according to the manufacturer 's protocols ( r&d systems , minneapolis , mn ) . ,
emphysema is a structural disorder characterized by destruction of the alveolar walls and enlargement of the alveolar spaces . histologically , the lungs sections from the air - exposed mice showed normal alveolar structure and exhibited normal size airspaces with thin septa ( figure 1(a ) ) . in contrast , the lungs sections from the cs - exposed mice showed an increased air space enlargement and destruction . the di was higher in cs - exposed mice ( 45.16 3.13 ) compared with air - exposed animals ( 28.86 2.07 , p < 0.001 ; figure 1(c ) ) ; exposure to cigarette smoke significantly induced airspace enlargement . in the present study , using elisa we noted that the lung levels of il-17 and il-21 were significantly increased in cs - exposed mice compared to air - exposed mice ( p < 0.001 , figures 2(a ) and 2(b ) ) . similarly , we also noted that the levels of il-17 and il-21 in the peripheral blood of cs - exposed mice were significantly higher than those of air - exposed mice ( p < 0.001 , figures 2(c ) and 2(d ) ) . to further confirm our results , we studied the relation between the levels of il-17 and il-21 and emphysematous lesions as measured by di and lm in cs - exposed mice . the lung levels of il-17 and il-21 were positively correlated with di ( r = 0.87 , p = 0.001 and r = 0.707 , p = 0.022 , resp . similarly , the peripheral blood levels of il-17 and il-21 were positively correlated with di ( r = 0.757 , p = 0.011 and r = 0.738 , p = 0.015 , resp . we analyzed the frequencies of th17 , il-21th17 , and cd8il-21r t cells in the peripheral blood and the lungs by using flow cytometry . the frequencies of th17 and il-21th17 cells in the peripheral blood of cs - exposed mice were significantly increased compared to air - exposed littermates ( p < 0.05 , figures 4(a ) and 4(b ) ) . also after chronic cs - exposure for 6 months , a significant increase in cd8il-21r t cells was observed in the peripheral blood from cs - exposed mice compared with those in air - exposed littermates ( p < 0.001 , figure 4(c ) ) . in addition , the frequencies of th17 , il-21th17 , and cd8il-21r t cells in the lungs of cs - exposed mice were significantly higher than those of the controls ( p < 0.05 , figures 5(a ) , 5(b ) , and 5(c ) ) . the frequencies of peripheral blood th17 , il-21th17 , and cd8il-21r t cells were positively correlated with di ( r = 0.892 , p = 0.001 ; r = 0.777 , p = 0.008 and r = 0.697 , p = 0.025 , resp . ) ( figures 6(b ) , 6(d ) , and 6(f ) ) in cs - exposed mice . the frequencies of lung th17 , il-21th17 , and cd8il-21r t cells were significantly and positively correlated with di ( r = 0.861 , p = 0.001 ; r = 0.700 , p = 0.024 and r = 0.818 , p = 0.004 , resp . ) ( figures 7(b ) , 7(d ) , and 7(f ) ) in cs - exposed mice . our findings indicated that th17 cells might be able to regulate cd8 t cells via il-21/il-21r system . to evaluate the contribution of il-21 to the function of cd8 t cells in cs - exposed mice , we next cultured cd8 t cells in vitro with ril-21 to analyze cytokine production . we isolated cd8 t cells by macs from cs - exposed mice and from air - exposed mice . results showed that administration of ril-21 and pha significantly upregulated the expression of perforin and granzyme b in cd8 t cells ; this effect was greater in cs - exposed mice than in air - exposed mice ( figure 8(a ) ) . notably , there were more perforin+ cells ( 88.29 11.03% ) than granzyme b+ cells ( 69.47 5.31% , p < 0.01 ) in cd8 t cells of cs - exposed mice ( figure 8(a ) ) . in parallel , the protein levels of perforin and granzyme b in the culture supernatants significantly increased following 3 days of culture with il-21 and pha ; this effect was also greater in cs - exposed mice than in air - exposed mice ( figure 8(b ) ) . the protein levels of perforin in the culture supernatants of cs - exposed mice ( 24.47 2.61
ng / l ) were significantly increased more than the levels of granzyme b ( 1.92 0.21 ng / l , p < 0.001 ) ( figure 8(b ) ) . these data suggest that il-21rcd8 t cells have the capacity to secrete perforin and granzymes and further support the potential relevance of th17 cells in emphysema . emphysema is considered a t - cell - mediated autoimmune disease , but its etiology and pathology have not been elucidated . in this study , we employed a murine model of cigarette smoke - induced lung emphysema to investigate the capacity of th17 cells to participate in emphysema pathogenesis . we found that the numbers of th17 , il-21th17 , and cd8il-21r t cells and the levels of il-17 and il-21 in the peripheral blood and lungs of cs - exposed mice were much higher than those of air - exposed mice and correlated with emphysematous lesions . additionally , the number of il-21th17 cells positively correlated with the number of cd8il-21r t cells . furthermore , il-21 significantly augmented the secretion of perforin and granzyme b in cd8 t cells from cs - exposed mice in vitro . these data indirectly demonstrate that il-21 produced by th17 cells can act on cd8 t cells to promote cytotoxic function . cd4 t cells are known as important key cells in immunoregulation , whereas cd8 t cells have cytotoxic function in copd . regulatory t cells represent only a small subset of cd4 t cells in the peripheral circulation and are responsible for the balance of immune responses , which is essential for health . early studies have suggested that infiltrating cd4 t cells in copd exhibit a th1 phenotype . our previous study showed that decreased regulatory t cells were found in lungs of emphysema group . more recently , we have reported that increased th17 cells could be found in lungs of smoke - exposed mice , and these th17 cells might be due to th17 differentiation stimulated by lung proinflammatory cytokines and to recruitment of th17 cells via ccr6/ccl20 . in the current study , we also demonstrated that increased th17 cells were present in peripheral blood and lungs of cs - exposed mice . , who demonstrated that th17 cells were present in lungs from patients with emphysema , and harrison et al . thus , these studies indicated that th17 cells may have a relevant role in the local and system inflammatory process of copd . th17 cells secrete not only il-17a but also il-17f , il-21 , and il-22 , these cytokines most likely induce tissue inflammation . il-21 might act in a positive feedback loop , preserving and/or amplifying generation of th17 cells [ 39 , 40 ] , and serve to recruit th17 cells into the inflamed tissue . given these findings , it is perhaps not surprising that exaggerated il-17 and il-21 responses are implicated in the pathogenesis of copd . in the present study , significantly elevated levels of il-17 and il-21
in addition , a positive correlation between levels of il-17 and il-21 and emphysematous lesions was found in cs - exposed mice . although these findings suggest that the enhanced th17 cells and il-21 production in emphysema are biologically relevant , the mechanism for th17 cells in emphysema pathogenesis remains unidentified . several studies have shown that the number of cd8 t cells found in the lungs of patients with copd correlates with disease severity , and lung cd8 t cells may directly cause cytotoxicity contributing to emphysema by inducing apoptosis through secretion of perforin , granzyme , and by fas / fas ligand ( fasl ) interactions . further , it has been demonstrated that cd4 t cells are essential in the promotion of functional cd8 t - cell memory after an acute infection . therefore , it is likely that th17 cells may participate in copd immunoregulation via generation of cd8 cytotoxic t cells . in the current study , we demonstrated that the number of il-21th17 and cd8il-21r t cells was significantly increased in the lungs and peripheral blood of cs - exposed mice . more importantly , a significantly positive correlation between the number of il-21th17 and cd8il-21r t cells and emphysematous lesions was found in cs - exposed mice , which indicated the importance of il-21th17 and cd8il-21r t cells in the development of emphysema . in addition , a significant correlation existed between il-21th17 and cd8il-21r t cells in the lungs and peripheral blood of cs - exposed mice . this suggests that th17 cells could produce il-21 to mediate inflammatory response through il-21r , which is expressed on cd8 t cells . thus , we presume that il-21 released from th17 cells might play a more important role in the immunopathology of emphysema through its actions on cd8 t cells . to further address this issue , we examined the capacity of ril-21 to promote cd8 t cells functions in vitro and found that coculture of il-21 and cd8 t cells results in cells producing increased amounts of perforin and granzyme - b upon chronic smoke expose . interestingly , the cd8 perforin cells were proportionally higher than cd8 granzyme b cells , and the perforin levels were also much higher than the granzyme b levels . perforin can form pores in the target cells ' membranes , while granzymes , as serine proteases , enter the cytoplasm of the target cells , altering their function and/or activating cell death . these observations indirectly demonstrate that il-21 produced by th17 cells can promote cd8 t cells to induce apoptosis and tissue damage via the granzyme - b / perforin - mediated pathway . however , the precise mechanism for the effect of th17 cells on the development of smoke - induced emphysema undoubtedly needed successive studies . our data showed that the frequencies of th17 , il-21th17 , and cd8il-21r t cells and the levels of il-17 and il-21 in the peripheral blood and lungs of cs - exposed mice were significantly increased compared to controls and correlated with emphysematous lesions . furthermore , expression of perforin and granzyme b by cd8 t cells was increased by in vitro stimulation with il-21 . our findings support the concept that th17 cells and related cytokine il-21 were involved in the pathogenesis of copd . however , to the best of our knowledge , although in the in vitro experiments il-21 promotes cd8 t cell cytotoxic responses , whether or not il-21 derived from the th17 cells contributes to the enhanced cd8 t cells function response remains unclear . further research should be done by using adoptive transfer of th17 cells and il-21-null ( or il-21 deficient ) mice . | [
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central to neurovascular homeostasis is the function of the blood - brain barrier ( bbb ) .
the bbb is a highly regulated interface between the systemic circulation and brain parenchyma and is comprised of a monolayer of brain capillary endothelial cells on the blood side and perivascular cells on the brain side of microvessels .
the bbb functions to protect the parenchymal cells from fluctuations in plasma composition , such as during exercise and following meals , and against circulating neurotransmitters or xenobiotics capable of disrupting neural function . in this regard ,
the bbb acts as a mechanical barrier ; brain capillaries are ~50100 times tighter than peripheral microvessels , a property that is attributed to intercellular tight junctions between neighboring endothelial cells that restrict the paracellular diffusion of hydrophilic solutes
. only small molecules such as oxygen and co2 can freely diffuse across the lipid membranes of the endothelium . on the luminal and abluminal membranes , specific transport systems regulate the transcellular traffic of small hydrophilic molecules , such as glut-1 and l - system carrier 1 in the transport of glucose or leucine , respectively , thereby providing a selective transport barrier that facilitates nutrient entry .
the highly expressed p - glycoprotein transporter on endothelial luminal surface protects the brain from xenobiotics and the potentially toxic neurometabolite , glutamate .
examples include ectoenzymes such as peptidases and nucleotidases , which metabolize peptides and atp , respectively , and the intracellular enzymes monoamine oxidase and cytochrome p450 1a and 2b which inactivate blood - borne neuroactive compounds .
moreover , the cerebral endothelium exhibits specific systems for receptor - mediated and adsorptive endocytosis that allow for the transfer of specific peptides and lipoproteins to the brain .
such multiple functions of the bbb regulate the brain microenvironment and maintain parenchymal homeostasis . the glutathione / glutathione disulfide ( gsh / gssg ) couple is the most abundant thiol redox system that plays a key role in the maintenance of the redox environment in cells [ 3 , 4 ] . under physiological conditions ,
intracellular gsh homeostasis depends on de novo gsh synthesis , gsh redox cycling , and transmembrane gsh transport .
cellular gsh exists mainly in the reduced form with gssg constituting less than 10% of the total gsh pool .
the biological functions of gsh are attributed to its unique -glutamyl bond between the glutamate and cysteine residues and to the presence of a free thiol group .
reduced gsh is synthesized in the cytosol in two steps from its constituent amino acids ( glutamate , cysteine , glycine ) catalyzed by -glutamyl cysteine ligase ( gcl ) and gsh synthase .
gcl catalyzes the formation of -glutamylcysteine , the first and rate - limiting reaction in gsh synthesis , and enzyme function is controlled by gsh feedback inhibition or by transcriptional upregulation of enzyme subunits ( section 1.4 ) .
an important aspect of cellular gsh homeostasis is that increased gsh oxidation is generally followed by increases in the total pool size , notably through enhanced de novo gsh synthesis .
the versatility of gsh in contributing to a myriad of cellular functions is notable in its role in detoxication reactions ( e.g. , hydroperoxide and xenobiotic catabolism ) , regulation of amino acid transport into cells , maintenance of native three - dimensional protein structure in biosynthetic / metabolic processes ( e.g. , prostaglandins d2 and e2 synthesis ) , serving as a cofactor for enzyme systems ( e.g. , glyoxalase i ) , and redox signaling .
thiol - disulfide exchanges and protein s - glutathiolation are mechanisms by which gsh modulates the oxidative modification of redox active cysteines within proteins and thereby regulates the activity of a variety of enzyme functions , including those controlling proliferation , differentiation , or apoptosis [ 6 , 7 ] .
intracellular gsh is differentially distributed among the various subcellular compartments of cytosol , mitochondria , endoplasmic reticulum , and nucleus wherein distinct redox pools are formed [ 8 , 9 ] .
cytosolic gsh is highly reduced , and under physiological conditions cytosolic gsh concentrations are between 1 and 11 mm with the gsh to gssg ratio maintained in excess of 10 to 1 depending on cell types .
the redox state of a cell is generally represented by the ratio of gsh to gssg given the large gsh pool size .
quantitatively , the cytosolic pool accounts for > 70% of the total cellular gsh , while the nuclear and mitochondrial compartments comprise 10% to 30% of the total cellular gsh , respectively .
the uniqueness of the nuclear and mitochondrial gsh pools is evidenced by the differences in compartmental gsh turnover rate and sensitivity to chemical depletion .
specifically , the distinct characteristic of the nuclear gsh redox state is consistent with its physiological role in the nucleus , significantly during cell cycle ( section 3.2 below ) .
indeed , increased nuclear - to - cytosol gsh distribution is a crucial factor in cell proliferation wherein elevated nuclear gsh maintains the functional integrity of the nucleus in gene transcription .
while the biological importance of metabolically unique gsh compartments in redox regulation of various endothelial cell functions [ 14 , 15 ] is yet to be fully defined , it can be readily appreciated that such independent gsh pools would afford an elegant mechanism for specific control of redox - sensitive metabolic processes , the failure of which will have significant implications for endothelial pathobiology .
the reader is referred to previous excellent reviews for a full discussion of redox compartmentation and its integration in redox signaling [ 3 , 8 , 15 ] .
one of the undesired consequences for an organism living in an aerobic environment is an increased potential for oxidative damage by reactive oxygen species ( ros ) .
however , the ability to thrive within such an aerobic environment also implies an evolved capability to handle ros - mediated tissue damage .
the major intracellular sources of ros , namely , superoxide anion ( o2 ) , hydrogen peroxide ( h2o2 ) , or hydroxyl radical ( ho ) , are derived from mitochondrial respiration , arachidonic acid pathway , and activities of cellular oxidases , such as cytochrome p450 , glucose oxidase , amino acid oxidases , xanthine oxidase , nadh / nadph oxidases , or no synthases [ 17 , 18 ] .
elevated ros levels are damaging to cellular macromolecules like proteins , lipids , and dna and will induce a state of oxidative stress and redox imbalance .
central to maintaining intracellular redox balance is gsh - dependent ros elimination that includes gsh peroxidase - catalyzed hydroperoxide metabolism , gssg reductase - catalyzed , nadph - dependent gsh regeneration , or gsh s - transferase - catalyzed xenobiotic detoxication .
the recognition that ros can serve as important mediators of cell signaling and that signal transduction may be mediated by ros - induced gsh redox imbalance is major conceptual breakthrough in our understanding of gsh - dependent redox signaling [ 20 , 21 ] .
significantly , low ros levels participate in the signaling of proliferation , senescence , and apoptosis . for instance , h2o2-targeted proteins containing redox sensitive cysteine residues ( p - sh ) can result in the formation of reversible sulfenic ( p - soh ) as well as irreversible sulfinic ( p - so2h ) , and sulfonic ( p - so3h ) acid derivatives [ 22 , 23 ] . the protein sulfenic acid derivative
can further react with nitric oxide ( no ) to yield nitrosothiol ( p - sno ) or with another p - sh to form a disulfide bond ( p - ss - p ) [ 22 , 23 ] .
the latter posttranslational modification , termed s - glutathiolation ( also known as s - glutathiolation ) , refers to the formation of a mixed disulfide between the cysteine of gsh and a cysteine moiety of a protein .
reversible protein cysteine oxidation and protein mixed disulfide formation are catalyzed by the thioredoxin ( trx ) and glutaredoxin ( grx ) family of redox proteins .
this gsh - protein cysteine interaction protects against irreversible protein thiol oxidation and is an important redox mechanism in regulating protein function at low or modest levels of ros .
ros - dependent protein cysteine oxidation has been implicated in the redox regulation of a wide range of protein functions including enzyme activity , protein expression and abundance , subcellular protein localization , and interaction with other molecular partners in controlling new patterns of cell signaling and gene expression .
viewed simply , control of protein functions by reversible s - glutathiolation / deglutathiolation is akin to that of phosphorylation / dephosphorylation .
gsh exerts profound effects on vascular endothelial function , which include endothelial barrier permeability , cell apoptosis , chemotaxis , angiogenesis [ 26 , 27 ] , constitutive and agonist - induced adhesion molecule expression , leukocyte - endothelial adhesion response , and endothelial dependent vasodilation [ 28 , 30 ] .
the modulatory effects of gsh are accomplished through the scavenging of ros , an important second messenger in many endothelial functions .
for instance , gsh was shown to attenuate h2o2-induced decrease in transendothelial electrical resistance via negative regulation of the activation of p38 map kinase .
in other roles , reduced gsh acts as a substrate for the detoxication enzymes , gsh peroxidase , and gsh s - transferase .
our recent studies showed that gsh served as a cofactor in glyoxalase 1-catalyzed detoxication of methylglyoxal and prevented carbonyl stress - induced brain endothelial barrier dysfunction ( figure 1 ) .
a large body of evidence supports a role for s - glutathiolation in redox regulation of vascular function , ranging from cell signaling , apoptosis , protein folding , to cytoskeletal reorganization . in hypertensive vessels ,
the thiolation of endothelial nitric oxide synthase ( enos ) is pivotal in the redox control of vascular tone .
the bioactive nitric oxide ( no ) molecule plays a crucial role in normal endothelial function , including modulation of vascular dilator tone , inhibition of platelet activation , inhibition of leukocyte adhesion and migration , and inhibition of smooth muscle cell migration and proliferation .
therefore , altered no production , such as during oxidative stress , would compromise vascular homeostasis .
oxidative stress has been shown to mediate s - glutathiolation of enos that was associated with decreased nos activity , attenuated no production , increased o2 generation , and impaired endothelium - dependent vasodilation , dysregulated processes that were restored by thiol - specific reducing agents .
as for cell signaling , oxidants have been shown to trigger direct s - glutathiolation of p21ras at cys , which activated p21ras and mediated downstream phosphorylation of erk and akt in both endothelial and smooth muscle cells [ 34 , 35 ] .
similarly , oxidant - induced insulin resistance was mediated through s - glutathiolation of p21ras and erk - dependent inhibition of insulin signaling . during diamide - induced oxidative stress , activation of endothelial ca signaling
was associated with s - glutathiolation of the inositol-1,4,5-trisphosphate ( ip3 ) receptor ( ip3r ) and the plasmalemmal ca - atpase pump , which promoted ca release from ip3-sensitive internal ca stores and elevated basal [ ca]i in the absence of extracellular ca .
current evidence implicates the involvement of s - glutathiolation / deglutathiolation in apoptotic signaling . in tnf--mediated
apoptosis , grx - catalyzed deglutathiolation of procaspase-3 induced caspase-3 activation . in fas - mediated apoptosis , fas thiolation following caspase - dependent grx1 degradation resulted in the activation of caspases-8 and -3 .
molecular chaperones are an interesting class of proteins that are readily s - glutathiolated wherein thiolated proteins exhibited potentiation of chaperone activities , such as the correct folding of newly synthesized polypeptides .
the activities of several s - glutathiolated members of the glucose - related protein ( grp ) family of proteins including grp78 , heat shock protein 60 ( hsp60 ) , heat shock cognate 71-kda protein , and hsp90 were similarly increased by s - glutathiolation in diamide - treated endothelial cells .
remarkably , even endothelial cytoskeletal reorganization can be modulated by protein s - glutathiolation , notably that of actin and tubulin . under physiological conditions , s - glutathiolated actin ( at cys ) inhibited f - actin polymerization , which was reversed by egf via actin deglutathiolation , consistent with a dynamic role of actin assembly / disassembly in the biological process of cell division and cell growth .
notably , actin - glutathiolation also occurred under conditions of oxidative stress ; in this instance , intracellular actin disassembly or disrupted actin - junctional protein interactions would likely mediate the loss of endothelial barrier function . a role for s - glutathiolated annexin a2-actin interaction
similarly , while s - glutathiolation of endothelial -tubulin has been reported , the biological importance of this modification in endothelial barrier function remains to be defined .
gcl - catalyzed de novo synthesis is central to the preservation of tissue gsh balance , particularly during oxidative stress .
gcl is a heterodimeric protein composed of catalytic ( gclc ) and modifier ( gclm ) subunits .
the gclc subunit alone possesses all of the catalytic activities of the enzyme ; however , heterodimerization with the gclm subunit increases gcl activity ( vmax and kcat ) , substrate affinity ( km ) for glutamate and atp , and the ki for gsh feedback inhibition .
metabolic regulation of gcl is mediated by protein phosphorylation at serine and threonine moieties , which inhibits enzyme activity and transcriptional control of gcl function is through the expression of the catalytic and modulatory subunits .
the promoters of gclc and gclm subunits share common elements and coordinate transactivation results in overall increase in subunit expression .
key mediators of gcl expression are the redox sensitive transcription factors , nuclear factor kappa b ( nf-b ) , sp-1 , activator protein-1 and -2 ( ap-1 , ap-2 ) , and nuclear factor e2-related factor 2 ( nrf2 ) . the promoter of the human gclc gene contains consensus binding sites for ap-1 , nf-b , nrf2 , and for the antioxidant response ( are ) or electrophile responsive ( epre ) elements .
a proximal ap-1 element was crucial for the transcription of gclc induced by oxidative stress while nf-b was essential in tnf-mediated increase in gclc transcription either directly or indirectly via transactivation of ap-1 sites through induction of c - jun expression . among the four ares in the human gclc promoter ,
are4 was important in the constitutive expression of hepatic gclc induced by -naphthoflavone ( -nf ) or cytochrome p450 2e1 [ 46 , 47 ] .
in macrophages , elevated gclc expression caused by homocysteine was mediated by are4 and the merk - erk1/2 kinase pathway .
involvement of the pi3 kinase pathway was also described in adrenomedullin - induced transcriptional activation of the gclc promoter .
recent studies from our laboratory demonstrated a role for nrf2 in the constitutive and insulin - induced endothelial gclc expression .
significantly , the increase in gcl activity and gsh synthesis via insulin signaling and activation of the pi3k / akt / mtor / nrf2/gclc pathway prevented hyperglycemia - induced endothelial apoptosis .
interestingly , rat gclc promoter exhibited only three ares in the 5-flanking region , of which are3 was involved in nrf2-dependent expression of gclc , suggesting species differences in are requirements for gclc activation .
in contrast to insulin and hydrocortisone , which induced gclc gene expression , stress hormones such as glucagon and phenylephrine caused gclc phosphorylation through activating the protein kinases , pka , pkc , or ca - calmodulin kinase [ 54 , 55 ] .
notably , gclc phosphorylation decreased gclc activity . the transcriptional regulation of gclm is poorly understood .
current evidence shows that the human gclm promoter also contained an are site that mediated nrf2-dependent gclm upregulation induced by -nf and lipid peroxidation products [ 56 , 57 ] .
in rat liver , an are element similarly mediated the basal and tnf-induced of the gclm promoter activity . additionally , the rat gclm promoter has an ap-1 consensus site for constitutive and tert - butylhydroquinone - induced gclm expression . for reasons yet
unclear , nfb - dependent gclm expression appeared to be linked to ap-1 activation within the gclc promoter , suggesting possible cross - talk between the two promoters in subunit expression .
it is abundantly clear that oxidative stress induced by ros such as o2 , ho , or h2o2 can elicit endothelial barrier dysfunction .
moreover , oxidative stress also increased intracellular endothelial calcium concentration ( [ ca]i ) [ 59 , 60 ] ; in pulmonary artery endothelial cells , the blockade of ca entry abolished oxidative stress - induced solute permeability , indicating that oxidative stress was linked to elevated [ ca]i , an important modulator of endothelial permeability ( figure 1 ) .
in addition , oxidants like h2o2 were shown to increase the phosphorylation of myosin light chain kinase , suggesting that ros can alter endothelial contraction and contribute to endothelial barrier dysfunction ( figure 1 ) .
this means that oxidant modulation of the cytoskeletal architecture of the endothelial monolayer could be central to the loss of barrier integrity .
moreover , increased ros concentrations can decrease no bioavailability through chemical inactivation to form the powerful oxidizing agent , peroxynitrite .
tetrahydrobiopterin ( bh4 ) , a critical cofactor for enos function , is a crucial target for oxidation by peroxynitrite .
significantly , bh4 oxidation and depletion were shown to induce enos uncoupling , a process that was associated with increased o2 generation and decreased no production . in this
regard , uncoupled enos is akin to a dysfunctional o2 generating enzyme that could contribute to endothelial oxidative stress and vascular dysfunction .
the uncoupling of enos has been demonstrated in vitro and in hypertensive rat ( shr ) models of cardiovascular pathophysiology , such as angiotensin - ii - induced hypertension and diabetes .
control of paracellular permeability in the endothelium is a function of the intercellular endothelial adherens junctions ( aj ) and tight junctions ( tj ) , a complex structure comprised of specific junctional proteins .
the cadherins , -catenin , and -catenin proteins are components of the aj , while the transmembrane proteins , occludin , claudin , junction adhesion molecule , and the cytoplasmic accessory zonula occludin ( zo-1 , -2 , and -3 ) proteins comprised the tj .
h2o2-induced barrier disruption has been shown to occur through rearrangement of endothelial cadherin and -catenin and the disruption of -catenin / cytoskeletal association , but the signaling events are unresolved .
however , activation of erk1/erk2 signaling and occludin phosphorylation were shown to mediate the disorganization of occludin and the disruption of occludin - zo-1 interactions on endothelial cell surfaces .
ros activation of signaling pathways , such as pkc , may further regulate the phosphorylation state of other aj and tj proteins . in this regard
, a reversal of thrombin - induced loss of the cadherin junctional proteins , -catenin , -catenin , and p120 , by pkc inhibitor has been described .
carbonyl stress is the result of enhanced reactive carbonyl species ( rcs ) production and decreased carbonyl - scavenging capability , leading to tissue accumulation of reactive dicarbonyl species , such as methylglyoxal ( mg ) .
mg is produced from cellular glycolytic intermediates and can induce carbonyl stress through irreversible reaction with free arginine residues of proteins to form advanced mg - glycated end product ( age ) ( figure 1 ) . the generation of protein carbonyls or protein - glycated products could be a major problem in diabetic neurovascular pathology .
an mg - derived argpyrimidine adduct has been detected in human lens and kidney and in atherosclerotic lesions of diabetic patients [ 7173 ] , and argpyrimidine - modified heat shock protein 27 ( hsp 27 ) was shown to alter diabetic endothelial cell function .
moreover , diabetes - associated hyperglycemia and mg - induced modification of the corepressor msin3a gene were linked to elevated angiopoietin-2 transcription in microvascular endothelial cells .
other evidence revealed that the modification of 20s proteasome by mg decreased proteasomal chymotrypsin - like activity and impaired the chip and chaperone - dependent quality control of the protein , leading to the accumulation of toxic aggregates and endothelial cell death .
further , mg - induced glycation of vascular basement membrane type iv collagen yielded hotspots of arginine - derived hydroimidazolone residues at rgd and gfoger integrin - binding sites , causing endothelial cell detachment , anoikis , and inhibition of angiogenesis .
the crosslinking of mg and amino acids was shown to yield the o2 radical anion that can be quenched by o2 scavenger and membrane - permeable catalase .
it is noteworthy that mg - induced mitochondrial o2 generation stimulated enos activity , while mg - mediated enos phosphorylation ( at ser ) attenuated endothelial no production , suggesting that carbonyl stress modulation of endogenous endothelial no production is a complex process . in rat carotid arterial endothelium mg
was found to augment at1r - induced nadph oxidase - derived mediated o2 and h2o2 production , which increased ang ii - dependent vascular contraction .
similarly , mg - derived ros mediated the oxidative and hyperglycemic stress - induced impairment of endothelium - dependent vasorelaxation .
this oxidative stress response was attenuated by the overexpression of glyoxalase i which promoted mg degradation . in recent studies
, we found that mg - occludin glycation induced barrier dysfunction of human brain microvascular endothelial cells ; surprisingly , mg - dependent endogenous ros generation did not contribute majorly to barrier dysfunction .
our study further revealed that the endothelial gsh status is a determinant of barrier integrity by facilitating glyoxalase i - catalyzed mg metabolism and thereby decreasing the availability of free mg ( li and aw , unpublished data , figure 1 ) .
moreover , gsh depletion significantly promoted mg - induced endothelial oxidative stress and cell apoptosis [ 25 , 83 ] .
altered cell morphology , aberrant cytoskeletal rearrangement , and zo-1 loss were notable biological consequences of glyoxal , another sugar - derived aldehyde product .
additionally , glyoxal also elicited mitochondrial dysfunction , inhibition of dna and cell replication , and cell cytotoxicity through protein carbonyl formation .
collectively , these findings underscore the wide - ranging cellular effects of carbonyl stress on vascular endothelial function .
the mammalian cell cycle is characterized by a quiescent g0 phase of nondividing cells followed by cell entry into the cell cycle at g1 and progression through the s , g2 , and m phases in response to environmental or cellular cues that overcome the biological constraint of a mitotic block .
dna replication takes place during the s phase , and accurate replication commits cell progression into the m phase while aberrant dna replication induces transient g2 arrest that allows for dna repair .
cell progression through the cell division cycle is governed by regulatory checkpoints controlled by specific serine / threonine cyclin - dependent kinases ( cdks ) and their respective cyclin subunits .
specifically , the checkpoints for cell transitions from g0/g1 to s , late g1 to early s , s to g2 , and g2 to m are , respectively , regulated by d - type cyclin d1 , d2 and associated with cdk4 - 6 , cyclin e1/cdk2 complex , cyclin a / cdk2 kinase complex , and cyclin b1/cdk1 kinase complex in association with cdc25 phosphatase ( figure 2 ) .
the progression of cells through the cell cycle has been linked to dynamic changes in the intracellular redox environment particularly that of the gsh / gssg redox couple from a more oxidized state prior to cell cycle initiation to a more reduced state throughout cell cycle until cell cycle exit after prometaphase and cytokinesis ( figure 2 ) .
specifically , studies have documented that cell exit from the quiescent stage at g0/early g1 and entry into cell cycle was characterized by a relatively more oxidizing milieu [ 87 , 88 ] than that during progression from g1 through s to g2/m [ 87 , 89 ] .
the redox status of cysteine residues of cell cycle regulatory proteins and their functions were highly sensitive to the intracellular redox environment , which is impacted by cellular production and/or removal of ros .
for example , in actively dividing cells , redox - dependent activation of specific cyclin / cdks complexes by locally produced ros allowed for checkpoint bypass at the g1 restriction point or at late g1 to s transition [ 7 , 14 , 90 ] . similarly , growth - factor - mediated ros production and redox regulation of p16 , p27 , and cyclin d1 , which drove terminally differentiated cells into cell cycle [ 91 , 92 ] , governed the reentry of quiescent cells into the cell cycle [ 91 , 92 ] .
e2f , prb , map kinase , cdc25 phosphatase , and cyclin are other important cell cycle proteins shown to undergo redox changes and/or modifications during cell cycle progression [ 90 , 9395 ] .
a role for ros in mitogenic signaling is underscored by the finding that treatment of serum - starved cells with the thiol antioxidant , n - acetylcysteine ( nac ) , elicited cell cycle arrest at g1 , a delay of g0 to g1 progression that correlated with defective redox control .
interestingly , during exponential growth of cultured mouse embryonic fibroblasts , nac treatment arrested cells at the g1 to s transition but allowed cell transit through the s , g2 , and m phases , indicating that redox control at the early event at g1 governed cell progression from g1 to s. an increase in mnsod activity was implicated in nac - induced inhibition of g1 to s entry .
collectively , these studies illustrate the importance of ros in mitogenic signaling during cell cycle , a redox process that appears to be coordinated through defined cellular mechanisms for ros generation and elimination . a reduced intracellular redox environment protected genomic dna from oxidative damage upon breakdown of the nuclear envelope and was therefore essential to enhance dna synthesis during cell transition from g1 to g2/m .
early accumulation of soluble thiols at the mitotic spindle was observed during mitosis in sea urchin eggs .
similarly a graduation of low to high gsh content was associated with the transition of chinese hamster ovary fibroblasts through g1 to s to g2/m , consistent with a well - defined dynamics of redox changes in the intracellular environment during cell cycle .
intracellular redox homeostasis is maintained by the thiol / disulfide redox systems of gsh / gssg , thioredoxin ( trx / trss ) , and cysteine ( cys / cyss ) .
the product of reducing potential and reducing capacity of the redox couples determined the cellular redox environment , which in most cells are largely governed by that of the gsh / gssg couple .
indeed , the cellular gsh / gssg redox status provides a good quantitative indicator of the intracellular redox state , often expressed as the redox potential , eh . under physiological conditions , eh for gsh
/ gssg , as calculated by the nernst equation , is between 260 mv and 200 mv .
notably , a change in gsh / gssg eh from a reduced value of ~260 mv to an oxidized value of 170 mv was associated with phenotypic cell transition from proliferation to growth arrest and apoptosis . as discussed in section 1.2.1 , specificity of redox signaling and independent redox regulation of the functions of single proteins or protein sets are in part attributed to the existence of distinct compartments of gsh within the subcellular organelles .
recent evidence suggests that the dynamic cytosol - to - nuclear gsh distribution was a crucial factor in cell cycle progression in that nuclear gsh accumulation provided an intranuclear redox environment that enabled proper regulation of redox signaling events during the various stages of the cell cycle .
a novel concept of a nuclear gsh cycle that operated during cell cycle has been proposed as illustrated in figure 2 . according to this hypothesis ,
gsh was recruited and sequestrated into nucleus in early g1 phase , likely through a bcl-2-dependent import mechanism .
increased cytosolic - to - nuclear gsh translocation transiently caused gsh imbalance within the cytosol that initiated de novo gsh synthesis , resulting in progressive increases in the total cytosolic gsh pool .
cell transition through g2/m and the dissolution of the nuclear envelope during mitosis enabled the reequilibration of the cytosolic and nuclear gsh pools , and this return to a pre - cell cycle nuclear - to - cytosolic gsh ratio of 1 to 1 was maintained in non - proliferating cells at g0/g1 .
it was further proposed that it was the transient decrease in cytosolic gsh that promoted early g1 signaling . moreover , the increased gsh presence in the nucleus during the s phase coincided with the activation of dna replication as evidenced by elevated s - glutathiolation of histones , telomerase , and polyadp ribose polymerase [ 13 , 100 ] .
additionally , dna synthesis and replication could be further facilitated by gsh - dependent reorganization of the nuclear matrix and chromatin structure .
the details of gsh control of cell cycle checkpoints during endothelial cell proliferation are sketchy and are the subjects of current investigation in our laboratory . as an organ
that is highly dependent on oxidative metabolism for its energy needs , the brain is susceptible to tissue gsh imbalance and oxidative damage mediated by increased formation of free radical species and lipid peroxidation [ 102 , 103 ] . given the location of the bbb at the interface between brain parenchyma and systemic blood , the endothelial monolayer is easily exposed to the oxidizing conditions of elevated ros or rcs associated with various pathological states ( section 4 below ) .
additionally , an often decreased tissue or systemic gsh level under these diseased states would enhance oxidative damage to the vascular endothelium and the consequent loss of vascular integrity will have important implications for cerebral homeostasis .
the enzyme -glutamyl transpeptidase ( -gt ) is regarded as a marker of bbb integrity in the mammalian brain .
it is noteworthy that -gt levels were lowest in the more primitive regions of the brain and highest in the more specialized regions of the brain , the reason of which is yet unknown .
importantly , within the brain , the microvesicular fractions exhibited significantly higher -gt activity than the neuronal or glial fractions , consistent with a microvesicular localization of the enzyme .
however , -gt activity in type i cells ( cobblestone phenotype ) increased 1012-fold after glial stimulation , indicating a role for type i cells in bbb function as well .
equally notable was the finding that membrane - associated -gt activity in the endothelium of capillaries was higher than that in larger vessels in the brain , implying that cerebral small vessel endothelial monolayers will likely be more sensitive to fluctuations in the plasma gsh levels in terms of both susceptibility to injury and efficiency of repair .
furthermore , given that -gt can catalyze the metabolism of not only gsh but also s - nitrosoglutathione ( gsno ) , cerebral microvascular -gt function could be pivotal in mediating the bioactivity of gsno and/or no ( section 4.2 ) .
the findings that gsh levels in endothelial cells in culture increased during the lag phase , were elevated during the initial exponential growth phase , and then fell as cells become confluent suggest that systemic gsh interruption would alter endothelial growth .
our recent studies in human microvascular endothelial cells showed that inhibition of gsh synthesis and gsh depletion elicited a delayed s - to - g2 transition reflected in a lengthening of the cell cycle s - phase resident time ( busu and aw , unpublished ) , in agreement with previous observations .
significantly , cellular gsh depletion was largely confined to the cytosolic pool while the nuclear gsh compartment remained relatively unchanged .
somewhat surprisingly , delayed s - to - g2 transition remained evident for 6 h despite the restoration of cytosolic gsh synthetic capacity and near normalization of basal cellular gsh levels ( busu and aw , unpublished ) , consistent with a significant time lag between restored cellular redox balance and recovery of normal cell cycle activity .
the reason for this temporal dissociation is unclear and is currently under investigation in our laboratory .
what is clear , however , is that through perturbing cell cycle events , a disruption in cellular gsh such as that occuring during oxidative or carbonyl stress could delay endothelial proliferation and tissue repair following oxidative damage to the endothelium , a deleterious scenario for brain function in cerebrovascular and neurodegenerative disorders .
increased bbb permeability has been demonstrated in patients with type ii diabetes and in the streptozotocin- ( stz- ) induced type i diabetic experimental rat model .
elevated activities of plasma metalloproteinases 2 and 9 were implicated in the loss of tight junctional proteins ( occludin , claudin-5 , zo-1 , and jam-1 ) and bbb failure .
interestingly , the receptor for age ( rage ) was upregulated during diabetes , suggesting that increased plasma - to - cellular mg uptake and enhanced gsh - dependent intracellular mg catabolism could provide a means to attenuate the elevated systemic mg levels associated with the diabetic state .
bbb disruption was notable during diabetic ketoacidosis wherein neurovascular inflammation , accompanying ccl-2 chemokine expression , nf-b activation , and nitrotyrosine formation were likely contributors to the attenuated bbb integrity and increased barrier permeability .
in stz - induced diabetic rats , bbb function was improved by the administration of growth hormone and insulin [ 113 , 114 ] .
our recent studies demonstrated that insulin - mediated protection of human microvascular endothelial cells against mg - induced apoptosis was the result of increased intracellular gsh through activation of the insulin - pi3k / akt / mtor / nrf2/gclc signaling pathway [ 50 , 52 ] .
it is well known that diabetes is associated with hyperglycemia , elevated oxidative and carbonyl stress , and low tissue and plasma levels of gsh [ 115120 ] , conditions that complicate the diabetic state , which would lead to further exacerbation of gsh loss .
thus , mechanisms that promote neurovascular gsh status or those that attenuate oxidative and/or carbonyl stress could preserve endothelial barrier function .
a viable approach could involve activation of insulin signaling to maintain cellular gsh balance and support gsh - dependent attenuation of oxidative or carbonyl stress mediated by ros or mg [ 25 , 51 , 52 , 121 ] .
furthermore , increasing gsh protection of redox sensitive thiols of membrane proteins , including those of the aj or tj , could preserve the functional integrity of the endothelium .
the question of whether acute or chronic gsh therapy would be effective in abrogating systemic hyperglycemia - linked oxidative and carbonyl stress and mitigate diabetes - associated bbb dysfunction remains an open question that warrants further investigation .
stroke is a cerebrovascular disorder wherein a blood clot or interrupted blood flow to a region of the brain leads to a rapid loss of brain function .
significantly , a lack or delayed flux of oxygen and glucose to the brain will result in neuronal death and brain damage .
clinical studies have shown that subjects at risk for stroke exhibited low tissue gsh levels and decreased gsh - to - gssg ratio and that the restoration of normal cerebral gsh balance could be as long as 72 h after the ischemic insult [ 122 , 123 ] .
importantly , acute ischemic stroke was associated with elevated oxidative stress , a major contributor to immediate and delayed ischemic brain injury and changes in the parenchymal gsh redox status [ 124 , 125 ] .
an increase in free radical production during acute cerebral ischemia can arise from multiple sources including stimulation of n - methyl - d - aspartate receptors , mitochondrial dysfunction , activation of neuronal no synthase ( nos ) [ 128 , 129 ] , autooxidation of catecholamines , and metabolism of free fatty acids .
the activation and migration of inflammatory cells , such as neutrophils , further contributed to o2 and h2o2 generation .
the restoration of endothelial integrity after thrombotic or hemorrhagic stroke is crucial to preserving bbb function and neurovascular homeostasis .
the proliferation of endothelial cells adjacent to the lesion or injury site is a pivotal step .
given the role of gsh in cell proliferation ( section 3 ) , maintaining cellular gsh balance is therefore essential for postdamage endothelial repair and wound healing .
s - nitrosoglutathione ( gsno ) is an important physiological metabolite produced by the reaction of no with gsh that is involved in no storage and release through the function of -gt .
the affinity of -gt for gsno ( km of 0.4 mm ) was comparable to other -glutamyl substrates , suggesting a physiological role for -gt - in gsno metabolism .
however , recent studies demonstrated that , at least in plasma , gsno levels are likely to be higher than previously reported due to the presence of exogenous -gt , further underscoring the significance of the enzyme in modulating gsno levels and bioactivity . reportedly , gsno functions in cellular signaling [ 134 , 135 ] and protection of the central nervous system ( cns ) against excitotoxicity , inflammation , and ros [ 136 , 137 ] .
notably , gsno protection against peroxynitrite - induced oxidative stress is severalfold more potent than gsh .
gsno - mediated cns protection against inflammation appeared to be through suppressing inos induction and promoting enos expression , and maintaining cerebral blood flow .
the anti - inflammatory activity of gsno in downregulating inos was mediated by inhibition of nf-b activation and decreased expression of icam-1 and ed-1 .
additionally , the expression of zo-1 and occludin at endothelial tight junctions was enhanced by gsno treatment . unlike conventional no donors that mediate quick no release
, gsno elicits slow no release that was implicated in neurovascular protection against ischemia reperfusion .
in addition to -gt , s - nitrosoglutathione reductase ( gsnor ) , which catalyzes the reduction of gsno , has been shown to be an important regulator of the endogenous gsno levels and no bioactivity .
the pathophysiological role of gsnor in sno signaling and no bioactivity in the regulation of vascular tone is incompletely understood ; recent evidence suggests that gsnor regulates airway sno levels in cell signaling and protects against nitrosative stress and cancer risk in human lung .
this notwithstanding , it remains unclear whether therapeutic strategies involving exogenous gsh and/or no supplementation during neurovascular inflammatory conditions , such as stroke , would be clinically efficacious in the short term in attenuating the oxidative burden and protecting the bbb , or in the long term in reducing brain edema and tissue damage .
ros- and/or rcs - induced endothelial dysregulation is an underlying concern in barrier failure , and , as such , much research has focused on the use of antioxidants as a strategy to attenuate oxidative or carbonyl stress and restore monolayer function .
the finding that gsh , a major cellular antioxidant , is able to afford cytoprotection supports the notion that antioxidant therapy is important in endothelial barrier preservation . in past years ,
more recent conceptual advances in redox cell biology have uncovered a fundamental role of gsh in signal transduction and redox signaling in cellular functions .
moreover , the finding that distinct pools of gsh exist in subcellular organelles that allow for independent redox regulation has revolutionized our thinking of gsh - dependent redox mechanisms in controlling metabolic processes .
one such biological process is that of cell proliferation . in the context of enhanced endothelial proliferation and self - repair surrounding lesion sites in response to systemic cues , for example
the dynamics of cytosol - to - nuclear gsh distribution appears to be pivotal in governing cell cycle responses .
the notion that cell proliferation and growth can be a relevant biological process for monolayer repair / restitution following endothelial injury in much the same way as epithelial cell restitution / proliferation restores postinjured epithelium suggests exciting new avenues for future research in endothelial biology .
importantly , an understanding of gsh control of endothelial cell proliferative potential under different oxidizing conditions and plasma gsh levels will expand our perspective for future development of therapeutic strategies .
targeting endothelial restoration after oxidative insult and tissue damage is likely to be clinically relevant to the neurovascular disorders of diabetes and stroke and additionally could have broader implications for neurodegenerative and neurological disorders as well . | the integrity of the vascular endothelium of the blood - brain barrier ( bbb ) is central to cerebrovascular homeostasis .
given the function of the bbb as a physical and metabolic barrier that buffers the systemic environment , oxidative damage to the endothelial monolayer will have significant deleterious impact on the metabolic , immunological , and neurological functions of the brain .
glutathione ( gsh ) is a ubiquitous major thiol within mammalian cells that plays important roles in antioxidant defense , oxidation - reduction reactions in metabolic pathways , and redox signaling .
the existence of distinct gsh pools within the subcellular organelles supports an elegant mode for independent redox regulation of metabolic processes , including those that control cell fate .
gsh - dependent homeostatic control of neurovascular function is relatively unexplored .
significantly , gsh regulation of two aspects of endothelial function is paramount to barrier preservation , namely , gsh protection against oxidative endothelial cell injury and gsh control of postdamage cell proliferation in endothelial repair and/or wound healing .
this paper highlights our current insights and hypotheses into the role of gsh in cerebral microvascular biology and pathobiology with special focus on endothelial gsh and vascular integrity , oxidative disruption of endothelial barrier function , gsh regulation of endothelial cell proliferation , and the pathological implications of gsh disruption in oxidative stress - associated neurovascular disorders , such as diabetes and stroke . | 1. Glutathione and Neurovascular Homeostasis
2. Oxidative Challenge and Endothelial Barrier Dysfunction
3. Endothelial Repair through Proliferation and Growth
4. Pathological Implications of Impaired Glutathione in Neurovascular Disease States
5. Summary and Perspective | central to neurovascular homeostasis is the function of the blood - brain barrier ( bbb ) . the bbb is a highly regulated interface between the systemic circulation and brain parenchyma and is comprised of a monolayer of brain capillary endothelial cells on the blood side and perivascular cells on the brain side of microvessels . the bbb functions to protect the parenchymal cells from fluctuations in plasma composition , such as during exercise and following meals , and against circulating neurotransmitters or xenobiotics capable of disrupting neural function . in this regard ,
the bbb acts as a mechanical barrier ; brain capillaries are ~50100 times tighter than peripheral microvessels , a property that is attributed to intercellular tight junctions between neighboring endothelial cells that restrict the paracellular diffusion of hydrophilic solutes
. on the luminal and abluminal membranes , specific transport systems regulate the transcellular traffic of small hydrophilic molecules , such as glut-1 and l - system carrier 1 in the transport of glucose or leucine , respectively , thereby providing a selective transport barrier that facilitates nutrient entry . the highly expressed p - glycoprotein transporter on endothelial luminal surface protects the brain from xenobiotics and the potentially toxic neurometabolite , glutamate . examples include ectoenzymes such as peptidases and nucleotidases , which metabolize peptides and atp , respectively , and the intracellular enzymes monoamine oxidase and cytochrome p450 1a and 2b which inactivate blood - borne neuroactive compounds . such multiple functions of the bbb regulate the brain microenvironment and maintain parenchymal homeostasis . the glutathione / glutathione disulfide ( gsh / gssg ) couple is the most abundant thiol redox system that plays a key role in the maintenance of the redox environment in cells [ 3 , 4 ] . the biological functions of gsh are attributed to its unique -glutamyl bond between the glutamate and cysteine residues and to the presence of a free thiol group . , glyoxalase i ) , and redox signaling . thiol - disulfide exchanges and protein s - glutathiolation are mechanisms by which gsh modulates the oxidative modification of redox active cysteines within proteins and thereby regulates the activity of a variety of enzyme functions , including those controlling proliferation , differentiation , or apoptosis [ 6 , 7 ] . indeed , increased nuclear - to - cytosol gsh distribution is a crucial factor in cell proliferation wherein elevated nuclear gsh maintains the functional integrity of the nucleus in gene transcription . while the biological importance of metabolically unique gsh compartments in redox regulation of various endothelial cell functions [ 14 , 15 ] is yet to be fully defined , it can be readily appreciated that such independent gsh pools would afford an elegant mechanism for specific control of redox - sensitive metabolic processes , the failure of which will have significant implications for endothelial pathobiology . one of the undesired consequences for an organism living in an aerobic environment is an increased potential for oxidative damage by reactive oxygen species ( ros ) . the major intracellular sources of ros , namely , superoxide anion ( o2 ) , hydrogen peroxide ( h2o2 ) , or hydroxyl radical ( ho ) , are derived from mitochondrial respiration , arachidonic acid pathway , and activities of cellular oxidases , such as cytochrome p450 , glucose oxidase , amino acid oxidases , xanthine oxidase , nadh / nadph oxidases , or no synthases [ 17 , 18 ] . elevated ros levels are damaging to cellular macromolecules like proteins , lipids , and dna and will induce a state of oxidative stress and redox imbalance . central to maintaining intracellular redox balance is gsh - dependent ros elimination that includes gsh peroxidase - catalyzed hydroperoxide metabolism , gssg reductase - catalyzed , nadph - dependent gsh regeneration , or gsh s - transferase - catalyzed xenobiotic detoxication . the recognition that ros can serve as important mediators of cell signaling and that signal transduction may be mediated by ros - induced gsh redox imbalance is major conceptual breakthrough in our understanding of gsh - dependent redox signaling [ 20 , 21 ] . significantly , low ros levels participate in the signaling of proliferation , senescence , and apoptosis . the latter posttranslational modification , termed s - glutathiolation ( also known as s - glutathiolation ) , refers to the formation of a mixed disulfide between the cysteine of gsh and a cysteine moiety of a protein . ros - dependent protein cysteine oxidation has been implicated in the redox regulation of a wide range of protein functions including enzyme activity , protein expression and abundance , subcellular protein localization , and interaction with other molecular partners in controlling new patterns of cell signaling and gene expression . gsh exerts profound effects on vascular endothelial function , which include endothelial barrier permeability , cell apoptosis , chemotaxis , angiogenesis [ 26 , 27 ] , constitutive and agonist - induced adhesion molecule expression , leukocyte - endothelial adhesion response , and endothelial dependent vasodilation [ 28 , 30 ] . for instance , gsh was shown to attenuate h2o2-induced decrease in transendothelial electrical resistance via negative regulation of the activation of p38 map kinase . in other roles , reduced gsh acts as a substrate for the detoxication enzymes , gsh peroxidase , and gsh s - transferase . our recent studies showed that gsh served as a cofactor in glyoxalase 1-catalyzed detoxication of methylglyoxal and prevented carbonyl stress - induced brain endothelial barrier dysfunction ( figure 1 ) . a large body of evidence supports a role for s - glutathiolation in redox regulation of vascular function , ranging from cell signaling , apoptosis , protein folding , to cytoskeletal reorganization . in hypertensive vessels ,
the thiolation of endothelial nitric oxide synthase ( enos ) is pivotal in the redox control of vascular tone . the bioactive nitric oxide ( no ) molecule plays a crucial role in normal endothelial function , including modulation of vascular dilator tone , inhibition of platelet activation , inhibition of leukocyte adhesion and migration , and inhibition of smooth muscle cell migration and proliferation . therefore , altered no production , such as during oxidative stress , would compromise vascular homeostasis . oxidative stress has been shown to mediate s - glutathiolation of enos that was associated with decreased nos activity , attenuated no production , increased o2 generation , and impaired endothelium - dependent vasodilation , dysregulated processes that were restored by thiol - specific reducing agents . during diamide - induced oxidative stress , activation of endothelial ca signaling
was associated with s - glutathiolation of the inositol-1,4,5-trisphosphate ( ip3 ) receptor ( ip3r ) and the plasmalemmal ca - atpase pump , which promoted ca release from ip3-sensitive internal ca stores and elevated basal [ ca]i in the absence of extracellular ca . the activities of several s - glutathiolated members of the glucose - related protein ( grp ) family of proteins including grp78 , heat shock protein 60 ( hsp60 ) , heat shock cognate 71-kda protein , and hsp90 were similarly increased by s - glutathiolation in diamide - treated endothelial cells . notably , actin - glutathiolation also occurred under conditions of oxidative stress ; in this instance , intracellular actin disassembly or disrupted actin - junctional protein interactions would likely mediate the loss of endothelial barrier function . a role for s - glutathiolated annexin a2-actin interaction
similarly , while s - glutathiolation of endothelial -tubulin has been reported , the biological importance of this modification in endothelial barrier function remains to be defined . gcl - catalyzed de novo synthesis is central to the preservation of tissue gsh balance , particularly during oxidative stress . the gclc subunit alone possesses all of the catalytic activities of the enzyme ; however , heterodimerization with the gclm subunit increases gcl activity ( vmax and kcat ) , substrate affinity ( km ) for glutamate and atp , and the ki for gsh feedback inhibition . metabolic regulation of gcl is mediated by protein phosphorylation at serine and threonine moieties , which inhibits enzyme activity and transcriptional control of gcl function is through the expression of the catalytic and modulatory subunits . the promoter of the human gclc gene contains consensus binding sites for ap-1 , nf-b , nrf2 , and for the antioxidant response ( are ) or electrophile responsive ( epre ) elements . significantly , the increase in gcl activity and gsh synthesis via insulin signaling and activation of the pi3k / akt / mtor / nrf2/gclc pathway prevented hyperglycemia - induced endothelial apoptosis . it is abundantly clear that oxidative stress induced by ros such as o2 , ho , or h2o2 can elicit endothelial barrier dysfunction . moreover , oxidative stress also increased intracellular endothelial calcium concentration ( [ ca]i ) [ 59 , 60 ] ; in pulmonary artery endothelial cells , the blockade of ca entry abolished oxidative stress - induced solute permeability , indicating that oxidative stress was linked to elevated [ ca]i , an important modulator of endothelial permeability ( figure 1 ) . this means that oxidant modulation of the cytoskeletal architecture of the endothelial monolayer could be central to the loss of barrier integrity . control of paracellular permeability in the endothelium is a function of the intercellular endothelial adherens junctions ( aj ) and tight junctions ( tj ) , a complex structure comprised of specific junctional proteins . the cadherins , -catenin , and -catenin proteins are components of the aj , while the transmembrane proteins , occludin , claudin , junction adhesion molecule , and the cytoplasmic accessory zonula occludin ( zo-1 , -2 , and -3 ) proteins comprised the tj . h2o2-induced barrier disruption has been shown to occur through rearrangement of endothelial cadherin and -catenin and the disruption of -catenin / cytoskeletal association , but the signaling events are unresolved . however , activation of erk1/erk2 signaling and occludin phosphorylation were shown to mediate the disorganization of occludin and the disruption of occludin - zo-1 interactions on endothelial cell surfaces . ros activation of signaling pathways , such as pkc , may further regulate the phosphorylation state of other aj and tj proteins . in this regard
, a reversal of thrombin - induced loss of the cadherin junctional proteins , -catenin , -catenin , and p120 , by pkc inhibitor has been described . other evidence revealed that the modification of 20s proteasome by mg decreased proteasomal chymotrypsin - like activity and impaired the chip and chaperone - dependent quality control of the protein , leading to the accumulation of toxic aggregates and endothelial cell death . similarly , mg - derived ros mediated the oxidative and hyperglycemic stress - induced impairment of endothelium - dependent vasorelaxation . our study further revealed that the endothelial gsh status is a determinant of barrier integrity by facilitating glyoxalase i - catalyzed mg metabolism and thereby decreasing the availability of free mg ( li and aw , unpublished data , figure 1 ) . moreover , gsh depletion significantly promoted mg - induced endothelial oxidative stress and cell apoptosis [ 25 , 83 ] . the mammalian cell cycle is characterized by a quiescent g0 phase of nondividing cells followed by cell entry into the cell cycle at g1 and progression through the s , g2 , and m phases in response to environmental or cellular cues that overcome the biological constraint of a mitotic block . similarly , growth - factor - mediated ros production and redox regulation of p16 , p27 , and cyclin d1 , which drove terminally differentiated cells into cell cycle [ 91 , 92 ] , governed the reentry of quiescent cells into the cell cycle [ 91 , 92 ] . as discussed in section 1.2.1 , specificity of redox signaling and independent redox regulation of the functions of single proteins or protein sets are in part attributed to the existence of distinct compartments of gsh within the subcellular organelles . recent evidence suggests that the dynamic cytosol - to - nuclear gsh distribution was a crucial factor in cell cycle progression in that nuclear gsh accumulation provided an intranuclear redox environment that enabled proper regulation of redox signaling events during the various stages of the cell cycle . cell transition through g2/m and the dissolution of the nuclear envelope during mitosis enabled the reequilibration of the cytosolic and nuclear gsh pools , and this return to a pre - cell cycle nuclear - to - cytosolic gsh ratio of 1 to 1 was maintained in non - proliferating cells at g0/g1 . additionally , dna synthesis and replication could be further facilitated by gsh - dependent reorganization of the nuclear matrix and chromatin structure . the details of gsh control of cell cycle checkpoints during endothelial cell proliferation are sketchy and are the subjects of current investigation in our laboratory . given the location of the bbb at the interface between brain parenchyma and systemic blood , the endothelial monolayer is easily exposed to the oxidizing conditions of elevated ros or rcs associated with various pathological states ( section 4 below ) . additionally , an often decreased tissue or systemic gsh level under these diseased states would enhance oxidative damage to the vascular endothelium and the consequent loss of vascular integrity will have important implications for cerebral homeostasis . it is noteworthy that -gt levels were lowest in the more primitive regions of the brain and highest in the more specialized regions of the brain , the reason of which is yet unknown . importantly , within the brain , the microvesicular fractions exhibited significantly higher -gt activity than the neuronal or glial fractions , consistent with a microvesicular localization of the enzyme . equally notable was the finding that membrane - associated -gt activity in the endothelium of capillaries was higher than that in larger vessels in the brain , implying that cerebral small vessel endothelial monolayers will likely be more sensitive to fluctuations in the plasma gsh levels in terms of both susceptibility to injury and efficiency of repair . our recent studies in human microvascular endothelial cells showed that inhibition of gsh synthesis and gsh depletion elicited a delayed s - to - g2 transition reflected in a lengthening of the cell cycle s - phase resident time ( busu and aw , unpublished ) , in agreement with previous observations . what is clear , however , is that through perturbing cell cycle events , a disruption in cellular gsh such as that occuring during oxidative or carbonyl stress could delay endothelial proliferation and tissue repair following oxidative damage to the endothelium , a deleterious scenario for brain function in cerebrovascular and neurodegenerative disorders . interestingly , the receptor for age ( rage ) was upregulated during diabetes , suggesting that increased plasma - to - cellular mg uptake and enhanced gsh - dependent intracellular mg catabolism could provide a means to attenuate the elevated systemic mg levels associated with the diabetic state . it is well known that diabetes is associated with hyperglycemia , elevated oxidative and carbonyl stress , and low tissue and plasma levels of gsh [ 115120 ] , conditions that complicate the diabetic state , which would lead to further exacerbation of gsh loss . thus , mechanisms that promote neurovascular gsh status or those that attenuate oxidative and/or carbonyl stress could preserve endothelial barrier function . furthermore , increasing gsh protection of redox sensitive thiols of membrane proteins , including those of the aj or tj , could preserve the functional integrity of the endothelium . stroke is a cerebrovascular disorder wherein a blood clot or interrupted blood flow to a region of the brain leads to a rapid loss of brain function . significantly , a lack or delayed flux of oxygen and glucose to the brain will result in neuronal death and brain damage . importantly , acute ischemic stroke was associated with elevated oxidative stress , a major contributor to immediate and delayed ischemic brain injury and changes in the parenchymal gsh redox status [ 124 , 125 ] . the proliferation of endothelial cells adjacent to the lesion or injury site is a pivotal step . given the role of gsh in cell proliferation ( section 3 ) , maintaining cellular gsh balance is therefore essential for postdamage endothelial repair and wound healing . s - nitrosoglutathione ( gsno ) is an important physiological metabolite produced by the reaction of no with gsh that is involved in no storage and release through the function of -gt . however , recent studies demonstrated that , at least in plasma , gsno levels are likely to be higher than previously reported due to the presence of exogenous -gt , further underscoring the significance of the enzyme in modulating gsno levels and bioactivity . gsno - mediated cns protection against inflammation appeared to be through suppressing inos induction and promoting enos expression , and maintaining cerebral blood flow . this notwithstanding , it remains unclear whether therapeutic strategies involving exogenous gsh and/or no supplementation during neurovascular inflammatory conditions , such as stroke , would be clinically efficacious in the short term in attenuating the oxidative burden and protecting the bbb , or in the long term in reducing brain edema and tissue damage . ros- and/or rcs - induced endothelial dysregulation is an underlying concern in barrier failure , and , as such , much research has focused on the use of antioxidants as a strategy to attenuate oxidative or carbonyl stress and restore monolayer function . the finding that gsh , a major cellular antioxidant , is able to afford cytoprotection supports the notion that antioxidant therapy is important in endothelial barrier preservation . in past years ,
more recent conceptual advances in redox cell biology have uncovered a fundamental role of gsh in signal transduction and redox signaling in cellular functions . moreover , the finding that distinct pools of gsh exist in subcellular organelles that allow for independent redox regulation has revolutionized our thinking of gsh - dependent redox mechanisms in controlling metabolic processes . the notion that cell proliferation and growth can be a relevant biological process for monolayer repair / restitution following endothelial injury in much the same way as epithelial cell restitution / proliferation restores postinjured epithelium suggests exciting new avenues for future research in endothelial biology . importantly , an understanding of gsh control of endothelial cell proliferative potential under different oxidizing conditions and plasma gsh levels will expand our perspective for future development of therapeutic strategies . targeting endothelial restoration after oxidative insult and tissue damage is likely to be clinically relevant to the neurovascular disorders of diabetes and stroke and additionally could have broader implications for neurodegenerative and neurological disorders as well . | [
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widespread use of prostate - specific antigen ( psa ) screening and increased number of transrectal prostate biopsy encounters have led to an increased diagnosis of prostate cancer ( pca ) along with stage migration toward early - stage , organ - confined disease .
it is estimated that 233,000 new pcas will be diagnosed in the us in 2014 .
recently , the us preventive services task force attributed grade d to psa screening for detecting pca , implying that psa screening may cause harm to the patients .
but , the random nature of the verification test following the psa test , the trus biopsy , may be partly to blame for the poor screening yield of psa .
only about one in four men subjected to trus biopsy reveal cancer , and many of them are indolent and low grade . on the other hand , many significant tumors in anterior gland ( prostate evasive anterior tumor ) , midline and at the apex go undetected because of the random nature of trus biopsy .
multiparametric magnetic resonance imaging ( mp - mri ) of the prostate combining t2-weighted imaging with diffusion - weighted imaging ( dwi ) and perfusion imaging has been extensively studied in recent years .
it has been shown to have a high sensitivity , particularly in detecting clinically significant cancers .
the localizing strength of mp - mri of the prostate has opened up opportunities for targeted diagnosis and treatment strategies .
several studies have shown that there is undergrading of the gleason score on trus biopsy when compared with radical prostatectomy specimens .
the effectiveness of mp - mri used along with psa , followed by targeted biopsy of the mri visible lesion , is now accepted as a better alternative to systematic trus biopsy
recently published a meta - analysis of 16 studies , where the authors concluded that mri - guided targeted biopsy had a higher detection rate for significant cancer and lower rate of detection of insignificant cancer and therefore benefits the diagnosis of pca .
targeted biopsy of the mri visible lesion can be performed by two different techniques , either in - bore mri - guided biopsy of the visible target or registration of mr images with trus images using fusion software for biopsy under trus guidance ( out of bore ) .
both approaches use the advantage of tumor delineation on mp - mri for accurate localization and sampling . apart from the role in risk stratification of pca
, the localizing ability of mp - mri has also opened up opportunities for its focal treatment . at present , men diagnosed with pca are offered one of two options either active surveillance or a radical form of treatment ( surgery / radiation therapy ) .
significant deterioration of urinary and sexual function remain prevalent side - effects of radical therapies .
men with low or low - intermediate risk disease may be candidates for active surveillance .
however , there is usually low adherence to active surveillance with the perceived possibility of disease progression and missing the opportunity for cure .
focal therapy is an intuitive new organ - sparing technique that aims to selectively ablate locally confined , clinically significant index lesions while sparing the majority of the prostate gland and the surrounding delicate neurovascular bundles and urinary sphincter .
although pca is often multifocal , evidence suggests that the natural history of the disease is predominantly determined by the largest lesion , which is most often also the highest grade , the so - called index lesion .
evidence supporting the theory that the largest volume tumor is the index tumor includes the observations that recurrence after radiation therapy nearly always occurs at this site and extracapsular extension arises from the largest tumor in 90% of cases .
the index lesion if clinically significant will often be visualized on mp - mri , can be confirmed on biopsy and targeted for ablation .
various experts have proposed different ablative templates for focal therapy [ figure 1 ] , but in our opinion
focal therapy is somewhat of a misnomer for hemiablation and subtotal zonal ablation templates .
the goal of focal therapy should be to perform targeted ablation of the mri visible index tumor with the aim to eradicate the index lesion and minimizing side - effects of treatment , thereby providing the best balance between oncologic control and maintenance of quality of life ( qol ) .
visualization of tumor allows in - bore mr - guided focal treatment to be more suitable for targeted ablation and accurate targeting without any misregistration concerns . in - bore
treatment also offers the advantage of mr thermometry , which provides real - time monitoring of the thermal map during the treatment to ensure selective and adequate tumor ablation and preservation of sensitive surrounding structures .
contrast - enhanced scan at the end of the procedure shows the non - perfused volume and helps confirm adequate treatment coverage before the patient is taken off the table .
magnetic resonance - guided in - bore ablation is ideally suited for targeted ablation this article reviews the current status of mri - guided prostate biopsies as well as minimally invasive in - bore targeted treatment of organ - confined focal pca .
ongoing clinical trials for in - bore focal pca treatment via the transperineal , transrectal and transurethral routes using different energy modalities for the ablation are discussed .
focal therapy and prostate biopsy performed under trus guidance , with or without use of the newer mri - trus fusion technologies , are beyond the scope of this review and are therefore not included .
mri - guided prostate biopsy was initially reported in 2000 when investigators reported its success via a transperineal route using an open - configuration 0.5 t strength mri .
although the open - bore mri configuration provides easier patient access , it is limited by its low signal to noise ratio , which leads to non- or poor visualization of the target lesion at the time of biopsy on the low - strength ( typically 0.5 t ) open - bore magnets .
the pre - biopsy diagnostic images must therefore be registered to the real - time images at time of biopsy in low - strength mri scanners . in 2005 ,
beyersdorff et al . reported transrectal mr - guided biopsy in a cohort of 12 patients using a robotic device in a closed - bore 1.5 t magnet . since then , several studies have reported successful mr - guided prostate biopsy within closed - bore higher strength magnets ( 1.5 t and 3 t ) , most using the transrectal approach in prone position .
the reported cancer detection rates in these studies ranged from 8% to 70% . in the
largest published series on in - bore prostate biopsy , 265 patients with psa over 4.0 ng / ml and at least one previous negative trus biopsy were sampled following mp - mri in a 3 t magnet .
cancer was detected in 33% of the sites called on mri , and 87% of these were clinically significant .
using dwi for mr - guided in - bore biopsy , hambrock et al . reported correct representation of the gleason grade with prostatectomy specimens in 34 patients , and therefore representing the pre - treatment risk stratification .
utilization of t2-weighted turbo spin echo ( tse ) , t1 weighted spolied gradient echo , ultrafast gradient echo and t2-weighted true fast imaging with steady - state precession sequences ( bssfp ) enabling good visualization of the needle during the procedure have been reported in most studies .
several mr - compatible biopsy devices , registered to the mr images using a coordinate system , have been used along with complex software to guide the needle .
although most of the reported transrectal prostate needle guidance devices have been in the prone position , schwab et al .
recently reported the results of mr - guided biopsy preformed in wide - bore 1.5 t and 3 t magnets in the more comfortable supine lithotomy position in 50 patients .
they used a modified version of a previously used biopsy device with a custom bed allowing elevation of the pelvis and legs and leaving a gap for the biopsy device .
more recently , cepek et al . described a mr - compatible mechatronic ( robotic ) system for in - bore needle guidance via a transperineal route , which could be used for prostate biopsies and focal ablation .
they demonstrated a reliable method for accurate needle placement in a short needle delivery time .
the transperineal approach for prostate intervention is associated with decreased risk of urosepsis , which may occur in about 4% of patients following transrectal prostate biopsies .
the reported procedure time for transrectal mr - guided biopsy varies from 30 to 68 min , which is substantially longer than those reported for trus biopsy . repeatedly moving the table to bring the patient out of the scanner to provide access to the needle for advancing or adjusting
the biopsy needle , followed by repeat imaging , is one of the main reasons that prolongs the procedure time of in - bore procedures , including biopsy .
some investigators have therefore looked at mr - compatible robotic or mechatronic devices using rectal , gluteal and perineal approaches .
all investigators concluded that robot - assisted prostate procedures were feasible and can be performed safely and accurately , although in at least one of these studies the procedure time did not decrease with use of the robotic device when compared with the manual technique .
reported needle delivery time to the target of 9 min using their transperineal mechatronic device , although this assessment was performed at the time of focal laser ablation ( fla ) and not for biopsy and did not take into account the time needed to set up the device at the start of the procedure .
this brings into question the cost - effectiveness of mr - guided in - bore prostate biopsy .
de rooij et al . compared the qol and health care costs for the blind trus - guided biopsy strategy to the imaging - based strategy where mri and directed mr - guided biopsies were performed , modeled to include the cascading effects for a period of 10 years following initial referral for biopsy .
their results suggested comparable healthcare costs in the two strategies but an improved qol in the imaging arm .
the benefit in qol is derived from decrease in overdiagnosis and overtreatment in the imaging arm .
the benefits of mp - mri in the diagnostic pathway of pca include the potential of performing less number of biopsies , based on the negative predictive value of mp - mri , and therefore decreasing the incidence of complications from biopsies ( multiresistant sepsis ) , increased sampling efficiency , decreased histopathology costs , better characterization of gleason grade and fewer missed clinically significant cancers .
mri - trus fusion biopsy exploits the high accuracy of mp - mri with the real - time capability and ease of trus biopsy .
it is less expensive than mr - guided biopsy and can also be performed in a shorter time , although there have not been any published studies directly comparing the two techniques for targeted prostate biopsy .
recent results of mri - trus fusion biopsy with different available software platforms have shown promising results and therefore this technique is likely to provide an alternative targeted biopsy method to overcome the costs of in - bore prostate .
laser is one of the energy modalities currently being evaluated for mr - guided focal therapy of pca .
laser - induced thermal therapy utilizes laser light to deposit high - energy photons to generate coagulation through rapid heating .
the thermal effect depends on the amount of heat energy delivered and on the depth of light distribution , which is regulated by the wavelength of the laser fiber .
improvements in the design of diode laser sources currently used for interstitial therapy have made them smaller , portable and less expensive .
the diameter of the optical fibers for light delivery vary from 300 to 600 um ( typically 400 um for the 15w fiber and 600 um for the 30w fiber ) , with the length of the cylindrical diffuser tip varying from 10 to 40 mm , but most commonly 10 mm and 15 mm for prostate application .
laser ablation is mri compatible , predictable and precise with sharply defined margins , as has been shown in some pre - clinical and phase i clinical studies , and therefore is ideally suited for targeted ablation as opposed to hemi - ablation or zonal ablation .
the intensity of the energy can also be controlled by the operator in real time based on the thermal feedback at the time of treatment .
the procedure is performed under deep sedation in the mri suite in supine position with the legs elevated on support to provide access to the perinuem .
t2-weighted turbo spin echo ( tse ) , t1-weighted spoiled gradient echo , ultrafast gradient echo and t2-weighted true fast imaging with bssfp sequences have been used for visualization of the needle during the procedure [ figure 2 ] .
our group has previously reported on concentration of the contrast agent to be used for filling up the catheter for better visualization on spoiled gradient echo sequences .
real - time thermal maps are obtained using the proton resonance frequency ( prf ) shift [ figure 2 ] .
3d thermal mapping can also be obtained , which helps to show the ablation zone in relation to important surrounding structures .
pre - clinical trials using 980 nm diode laser ( visualase , houston , tx , usa ) as a thermal energy source for mrgfla have shown precise and accurate ablation zones .
linder et al . compared whole mount histology with mri in four patients who underwent fla followed by radical prostatectomy and found that fla created confluent ablation and that post - ablation mri was accurate in determining the ablated area .
two separate phase 1 trials using the transperineal approach on a cohort of 38 and nine patients , respectively , were reported in 2013 , demonstrating its feasibility with minimal or no side - effects of the treatment .
both studies showed near - identical results with no residual disease at the treated site in approximately 75% of the patients , and most of the patients with residual disease had low - volume low - grade disease not visible on mp - mri at 46 months post - treatment . ongoing phase ii clinical trial results at our institute and the university of chicago are expected in 2015 .
our investigators are also presently using a mr - compatible mechatronic device for needle guidance and ablation . in their initial evaluation of 37 needle insertions using the mechatronic device in the course of 10 fla treatments , the median needle guidance error was 3.5 mm and the needle delivery time was 9 min .
used a transrectal approach for mrgfla with the patient in the prone position and reported their experience in 23 patients .
although these initial results are encouraging , larger series with longer follow - up will be needed for validation . magnetic resonance - guided focal laser ablation treatment . a 54-year - old male with biopsy - confirmed gleason 7 ( 3 + 4 ) prostate carcinoma .
( a ) pre - treatment axial apparent diffusion coefficient map ( adc ) shows a well - demarcated magnetic resonance imaging ( mri ) visible lesion ( outlined in red ) in the left transition zone at the level of mid gland .
( b ) intra - operative axial balanced steady - state precession sequence mri scan confirming final position of two transperineally advanced cannulas with gadolinium markers ( arrow ) prior to initiating power .
( c ) thermal map image during treatment showing areas of heat deposition color coded ( orange ) overlaid on tumor outline ( in red ) .
( d ) immediate post - treatment axial post - contrast gd - dtpa ( magnevist , bayer healthcare ) enhanced subtraction image highlights the devascularized ablated volume ( arrow ) , showing no damage to the rectal mucosa or neurovascular bundle or even the adjacent urethra , which is outlined by foleys catheter . ( e )
post - treatment axial t2-weighted image at 6 months shows devascularized cystic area at the site of treatment .
all six samples obtained from the site at the 6-month follow - up biopsy were negative focused ultrasound is a completely non - invasive treatment method of tissue ablation and has been proved effective in a wide variety of benign and malignant tumors .
the high - energy ultrasound waves are converted to thermal energy , resulting in raised temperature and tissue coagulation from protein denaturation . as with other energy modes for focal treatment of pca , integration of focused ultrasound with mri ( mrgfus ) provides for accurate targeting and closed - loop real - time monitoring of temperature by mri thermography thus also allowing for a safer ablation procedure .
focused ultrasound has been shown to ablate prostate tissue with sharply demarcated transition between the coagulated zone and the surrounding gland .
two different systems , one for transrectal and the other for transurethral ablation , have been developed and are being presently evaluated in phase 1 clinical trials .
the exablate 2100 prostate ( insightec inc . , haifa , israel ) is a transrectal mrgfus system .
the system consists of an endorectal transducer made of 990 elements and filled with degassed water at 140c to eliminate air in the beam path and to cool the rectum during treatment .
the ultrasound beam can be steered to the desired location in the prostate [ figure 3 ] .
a proof - of - principle study published in 2013 demonstrated extensive coagulative necrosis without any residual tumor in the ablated area in five patients who underwent radical prostatectomy within 2 weeks of the mrgfus treatment .
our group initially reported the feasibility of transrectal mrgfus treatment for pca with the exablate system , and have recently reported the results with > 18 months follow - up on the first four patients treated with mrgfus system at our institution in an ongoing phase i trial .
all six treatment sites were clear on mri at 6 months , and five of the six target lesions ( 83% ) were free of disease on follow - up biopsy at 6 months post - treatment , while low - risk mr - invisible disease was seen at one of the six treated sites at the 6-month biopsy .
the median treated volume was 3.55 cc and the median procedure time was 215 min , although this was predominantly from system error during one of the treatments leading to increase in procedure time .
there was no significant difference in the pre- and post - treatment qol , iief-15 ( international index of erectile function ) and ipss ( international prostate symptom score ) scores .
intra - operative axial adc map prior to heating shows the endorectal focused ultrasound device steered to the direction of the tumor ( arrow ) the transurethral device ( pad 105 , profound medical inc . , toronto , canada ) includes a transurethral ultrasound device and a rotational positioning system , which allows the transducer to rotate along a defined angular sector of the gland .
the device is inserted into the urethra over a guidewire and positioned in place in the prostatic urethra under mri guidance .
pre - clinical studies have shown the device to be safe , capable of producing highly accurate volumes of ablation and treat entire prostate volumes in a short time .
a suprapubic catheter is placed prior to the procedure for continuous bladder drainage during the procedure .
the first proof - of - principle study demonstrating the safety and feasibility of transurethral prostate ablation in eight human subjects was reported by chopra et al .
they treated a 1800 angular sector of the gland along the posterior half of the gland in this study .
ongoing phase 1 multi - center trial across three sites to evaluate mr - guided transurethral whole gland ablation has completed enrollment of 30 patients , and the 12-month results are due in 2015 .
preliminary results show it to be a safe and feasible technique with low side - effect profile .
transrectal ultrasound - guided percutaneous prostate cryoablation was first described by onik et al . in 1993 . however , the ice ball is not visualized under ultrasound guidance because of the shadowing artifact from the posterior margin of the ice ball once the cooling process is initiated .
procedure and increases the potential for complications such as urtheral or rectal fistula . in - bore
mri - guided focal cryoablation allows real - time visualization of ice ball formation with the added benefit of the spatial resolution provided by mri , which is paramount to identify the sensitive surrounding structures , such as rectal wall , ureters , urethra and external urinary sphincter .
a hyperintense rim can be seen progressing at the margin of the ice ball , which is caused by shortening of t1 and seen in areas cooled to < 20c but as yet not frozen .
this allows monitoring of the ice ball growth in near real time with t1-weighted gradient echo mri .
mr thermography information is less accurate from cooling tissue and hence not used for real - time monitoring of the thermal damage .
also , susceptibility artifacts are seen at the interface between the ice ball and normal tissue , which hinders thermal mapping .
t2w - blade sequence , with its reduced sensitivity to movement , was used to monitor the ice ball in one study . as in other in - bore prostate interventional procedures , t2-weighted true fast imaging with bssfp can be used for confirming the final position of the cryoprobes .
three studies have been recently published documenting feasibility of mr - guided cryoablation in a different cohort of patients .
assessed the feasibility of whole gland cryoablation in 11 patients , eight with newly diagnosed pca and three for salvage therapy following radiation therapy .
they placed four to seven cryoprobes transperineally under mri guidance with an intent to ablate the entire gland .
urethral and rectal warmers were used in most of the cases and two freeze thaw cycles were performed in all treatments .
the reported procedure time was from 2 to 4.5 h and the mean hospital stay was 5 days .
one of the initial patients in the study in whom the rectal warmer was not used developed a recto urethral fistula , which healed in 3 months .
they concluded that mri allows precise positioning of the cryoprobes and excellent 3d monitoring of the ice ball growth , thereby overcoming the two limitations of ultrasound guidance . in another study ,
treated 18 patients with local recurrence after radical prostatectomy ( six of these patients had salvage radiation therapy and subsequent recurrence ) in a 1.5 t wide - bore mr magnet .
they reported better oncologic results in the group who were treated with three freeze thaw cycles .
reported the feasibility of mr - guided cryoablation in 10 patients with locally recurrent pca after radiation therapy .
unlike the other two studies cited above , they evaluated focal treatment , ablating only the lesion with margins in order to keep complications at a minimum .
the median focal tumor size was 20 mm and the treatment time was 210.5 min , very similar to our own initial study that treated four patients with transrectal mrgfus .
the investigators reported a steep learning curve and that the last two procedures in their study took substantially less time .
three patients had recurrent / residual disease at the margin of the treated area in the first 12 months and were retreated .
apart from the treatment modalities described above , there are other techniques such as microwave ablation and photodynamic therapy , which have used mri for planning and follow - up for pca therapy , but the procedures have been performed under ultrasound guidance in most studies . more recently
, early studies on focal irreversible electrophoration for pca under ultrasound guidance have been performed using mri imaging for planning and follow - up .
given the excellent spatial resolution and the multiplanar capability of mri , the feasibility of irreversible electroporation techniques for pca therapy may also be assessed in - bore , in a similar manner to cryoablation , to increase the procedure safety .
the increased costs associated with performing focal therapy in the mri magnet remain a disadvantage .
other limitations include need for mr - compatible materials , time availability on the mri machine and the limited amount of working space for the physician when performing these procedures in the mr system .
use of newer wider bore mri systems and dedicated mri interventional suites in the future would make it easier for the physicians to work in the mri environment . also , use of mri - compatible robotic / mechatronic systems , as highlighted in some of the recent studies , may also help to speed up the treatment and thereby decrease the overall costs of the treatment .
mri - guided prostate biopsy was initially reported in 2000 when investigators reported its success via a transperineal route using an open - configuration 0.5 t strength mri .
although the open - bore mri configuration provides easier patient access , it is limited by its low signal to noise ratio , which leads to non- or poor visualization of the target lesion at the time of biopsy on the low - strength ( typically 0.5 t ) open - bore magnets .
the pre - biopsy diagnostic images must therefore be registered to the real - time images at time of biopsy in low - strength mri scanners . in 2005 ,
beyersdorff et al . reported transrectal mr - guided biopsy in a cohort of 12 patients using a robotic device in a closed - bore 1.5 t magnet . since then , several studies have reported successful mr - guided prostate biopsy within closed - bore higher strength magnets ( 1.5 t and 3 t ) , most using the transrectal approach in prone position .
the reported cancer detection rates in these studies ranged from 8% to 70% . in the
largest published series on in - bore prostate biopsy , 265 patients with psa over 4.0 ng / ml and at least one previous negative trus biopsy were sampled following mp - mri in a 3 t magnet .
cancer was detected in 33% of the sites called on mri , and 87% of these were clinically significant .
using dwi for mr - guided in - bore biopsy , hambrock et al . reported correct representation of the gleason grade with prostatectomy specimens in 34 patients , and therefore representing the pre - treatment risk stratification .
utilization of t2-weighted turbo spin echo ( tse ) , t1 weighted spolied gradient echo , ultrafast gradient echo and t2-weighted true fast imaging with steady - state precession sequences ( bssfp ) enabling good visualization of the needle during the procedure have been reported in most studies .
several mr - compatible biopsy devices , registered to the mr images using a coordinate system , have been used along with complex software to guide the needle .
although most of the reported transrectal prostate needle guidance devices have been in the prone position , schwab et al .
recently reported the results of mr - guided biopsy preformed in wide - bore 1.5 t and 3 t magnets in the more comfortable supine lithotomy position in 50 patients .
they used a modified version of a previously used biopsy device with a custom bed allowing elevation of the pelvis and legs and leaving a gap for the biopsy device .
more recently , cepek et al . described a mr - compatible mechatronic ( robotic ) system for in - bore needle guidance via a transperineal route , which could be used for prostate biopsies and focal ablation .
they demonstrated a reliable method for accurate needle placement in a short needle delivery time .
the transperineal approach for prostate intervention is associated with decreased risk of urosepsis , which may occur in about 4% of patients following transrectal prostate biopsies .
the reported procedure time for transrectal mr - guided biopsy varies from 30 to 68 min , which is substantially longer than those reported for trus biopsy . repeatedly moving the table to bring the patient out of the scanner to provide access to
the needle for advancing or adjusting the biopsy needle , followed by repeat imaging , is one of the main reasons that prolongs the procedure time of in - bore procedures , including biopsy .
some investigators have therefore looked at mr - compatible robotic or mechatronic devices using rectal , gluteal and perineal approaches .
all investigators concluded that robot - assisted prostate procedures were feasible and can be performed safely and accurately , although in at least one of these studies the procedure time did not decrease with use of the robotic device when compared with the manual technique .
reported needle delivery time to the target of 9 min using their transperineal mechatronic device , although this assessment was performed at the time of focal laser ablation ( fla ) and not for biopsy and did not take into account the time needed to set up the device at the start of the procedure .
this brings into question the cost - effectiveness of mr - guided in - bore prostate biopsy .
de rooij et al . compared the qol and health care costs for the blind trus - guided biopsy strategy to the imaging - based strategy where mri and directed mr - guided biopsies were performed , modeled to include the cascading effects for a period of 10 years following initial referral for biopsy .
their results suggested comparable healthcare costs in the two strategies but an improved qol in the imaging arm .
the benefit in qol is derived from decrease in overdiagnosis and overtreatment in the imaging arm .
the benefits of mp - mri in the diagnostic pathway of pca include the potential of performing less number of biopsies , based on the negative predictive value of mp - mri , and therefore decreasing the incidence of complications from biopsies ( multiresistant sepsis ) , increased sampling efficiency , decreased histopathology costs , better characterization of gleason grade and fewer missed clinically significant cancers .
mri - trus fusion biopsy exploits the high accuracy of mp - mri with the real - time capability and ease of trus biopsy .
it is less expensive than mr - guided biopsy and can also be performed in a shorter time , although there have not been any published studies directly comparing the two techniques for targeted prostate biopsy .
recent results of mri - trus fusion biopsy with different available software platforms have shown promising results and therefore this technique is likely to provide an alternative targeted biopsy method to overcome the costs of in - bore prostate .
laser is one of the energy modalities currently being evaluated for mr - guided focal therapy of pca .
laser - induced thermal therapy utilizes laser light to deposit high - energy photons to generate coagulation through rapid heating .
the thermal effect depends on the amount of heat energy delivered and on the depth of light distribution , which is regulated by the wavelength of the laser fiber .
improvements in the design of diode laser sources currently used for interstitial therapy have made them smaller , portable and less expensive .
the diameter of the optical fibers for light delivery vary from 300 to 600 um ( typically 400 um for the 15w fiber and 600 um for the 30w fiber ) , with the length of the cylindrical diffuser tip varying from 10 to 40 mm , but most commonly 10 mm and 15 mm for prostate application .
laser ablation is mri compatible , predictable and precise with sharply defined margins , as has been shown in some pre - clinical and phase i clinical studies , and therefore is ideally suited for targeted ablation as opposed to hemi - ablation or zonal ablation .
the intensity of the energy can also be controlled by the operator in real time based on the thermal feedback at the time of treatment .
the procedure is performed under deep sedation in the mri suite in supine position with the legs elevated on support to provide access to the perinuem .
t2-weighted turbo spin echo ( tse ) , t1-weighted spoiled gradient echo , ultrafast gradient echo and t2-weighted true fast imaging with bssfp sequences have been used for visualization of the needle during the procedure [ figure 2 ] .
our group has previously reported on concentration of the contrast agent to be used for filling up the catheter for better visualization on spoiled gradient echo sequences .
real - time thermal maps are obtained using the proton resonance frequency ( prf ) shift [ figure 2 ] .
3d thermal mapping can also be obtained , which helps to show the ablation zone in relation to important surrounding structures .
pre - clinical trials using 980 nm diode laser ( visualase , houston , tx , usa ) as a thermal energy source for mrgfla have shown precise and accurate ablation zones .
linder et al . compared whole mount histology with mri in four patients who underwent fla followed by radical prostatectomy and found that fla created confluent ablation and that post - ablation mri was accurate in determining the ablated area .
two separate phase 1 trials using the transperineal approach on a cohort of 38 and nine patients , respectively , were reported in 2013 , demonstrating its feasibility with minimal or no side - effects of the treatment .
both studies showed near - identical results with no residual disease at the treated site in approximately 75% of the patients , and most of the patients with residual disease had low - volume low - grade disease not visible on mp - mri at 46 months post - treatment .
ongoing phase ii clinical trial results at our institute and the university of chicago are expected in 2015 .
our investigators are also presently using a mr - compatible mechatronic device for needle guidance and ablation . in their initial evaluation of 37 needle insertions using the mechatronic device in the course of 10 fla treatments , the median needle guidance error was 3.5 mm and the needle delivery time was 9 min .
used a transrectal approach for mrgfla with the patient in the prone position and reported their experience in 23 patients .
although these initial results are encouraging , larger series with longer follow - up will be needed for validation . magnetic resonance - guided focal laser ablation treatment . a 54-year - old male with biopsy - confirmed gleason 7 ( 3 + 4 ) prostate carcinoma .
( a ) pre - treatment axial apparent diffusion coefficient map ( adc ) shows a well - demarcated magnetic resonance imaging ( mri ) visible lesion ( outlined in red ) in the left transition zone at the level of mid gland .
( b ) intra - operative axial balanced steady - state precession sequence mri scan confirming final position of two transperineally advanced cannulas with gadolinium markers ( arrow ) prior to initiating power .
( c ) thermal map image during treatment showing areas of heat deposition color coded ( orange ) overlaid on tumor outline ( in red ) .
( d ) immediate post - treatment axial post - contrast gd - dtpa ( magnevist , bayer healthcare )
enhanced subtraction image highlights the devascularized ablated volume ( arrow ) , showing no damage to the rectal mucosa or neurovascular bundle or even the adjacent urethra , which is outlined by foleys catheter .
( e ) post - treatment axial t2-weighted image at 6 months shows devascularized cystic area at the site of treatment .
all six samples obtained from the site at the 6-month follow - up biopsy were negative
focused ultrasound is a completely non - invasive treatment method of tissue ablation and has been proved effective in a wide variety of benign and malignant tumors .
the high - energy ultrasound waves are converted to thermal energy , resulting in raised temperature and tissue coagulation from protein denaturation . as with other energy modes for focal treatment of pca , integration of focused ultrasound with mri ( mrgfus ) provides for accurate targeting and closed - loop real - time monitoring of temperature by mri thermography thus also allowing for a safer ablation procedure .
focused ultrasound has been shown to ablate prostate tissue with sharply demarcated transition between the coagulated zone and the surrounding gland .
two different systems , one for transrectal and the other for transurethral ablation , have been developed and are being presently evaluated in phase 1 clinical trials .
the exablate 2100 prostate ( insightec inc . , haifa , israel ) is a transrectal mrgfus system .
the system consists of an endorectal transducer made of 990 elements and filled with degassed water at 140c to eliminate air in the beam path and to cool the rectum during treatment .
the ultrasound beam can be steered to the desired location in the prostate [ figure 3 ] .
a proof - of - principle study published in 2013 demonstrated extensive coagulative necrosis without any residual tumor in the ablated area in five patients who underwent radical prostatectomy within 2 weeks of the mrgfus treatment .
our group initially reported the feasibility of transrectal mrgfus treatment for pca with the exablate system , and have recently reported the results with > 18 months follow - up on the first four patients treated with mrgfus system at our institution in an ongoing phase i trial .
all six treatment sites were clear on mri at 6 months , and five of the six target lesions ( 83% ) were free of disease on follow - up biopsy at 6 months post - treatment , while low - risk mr - invisible disease was seen at one of the six treated sites at the 6-month biopsy .
the median treated volume was 3.55 cc and the median procedure time was 215 min , although this was predominantly from system error during one of the treatments leading to increase in procedure time .
there was no significant difference in the pre- and post - treatment qol , iief-15 ( international index of erectile function ) and ipss ( international prostate symptom score ) scores .
intra - operative axial adc map prior to heating shows the endorectal focused ultrasound device steered to the direction of the tumor ( arrow )
the transurethral device ( pad 105 , profound medical inc . , toronto , canada ) includes a transurethral ultrasound device and a rotational positioning system , which allows the transducer to rotate along a defined angular sector of the gland .
the device is inserted into the urethra over a guidewire and positioned in place in the prostatic urethra under mri guidance .
pre - clinical studies have shown the device to be safe , capable of producing highly accurate volumes of ablation and treat entire prostate volumes in a short time .
a suprapubic catheter is placed prior to the procedure for continuous bladder drainage during the procedure .
the first proof - of - principle study demonstrating the safety and feasibility of transurethral prostate ablation in eight human subjects was reported by chopra et al .
they treated a 1800 angular sector of the gland along the posterior half of the gland in this study .
ongoing phase 1 multi - center trial across three sites to evaluate mr - guided transurethral whole gland ablation has completed enrollment of 30 patients , and the 12-month results are due in 2015 .
preliminary results show it to be a safe and feasible technique with low side - effect profile .
transrectal ultrasound - guided percutaneous prostate cryoablation was first described by onik et al . in 1993 . however , the ice ball is not visualized under ultrasound guidance because of the shadowing artifact from the posterior margin of the ice ball once the cooling process is initiated .
procedure and increases the potential for complications such as urtheral or rectal fistula . in - bore
mri - guided focal cryoablation allows real - time visualization of ice ball formation with the added benefit of the spatial resolution provided by mri , which is paramount to identify the sensitive surrounding structures , such as rectal wall , ureters , urethra and external urinary sphincter .
a hyperintense rim can be seen progressing at the margin of the ice ball , which is caused by shortening of t1 and seen in areas cooled to < 20c but as yet not frozen .
this allows monitoring of the ice ball growth in near real time with t1-weighted gradient echo mri .
mr thermography information is less accurate from cooling tissue and hence not used for real - time monitoring of the thermal damage .
also , susceptibility artifacts are seen at the interface between the ice ball and normal tissue , which hinders thermal mapping .
t2w - blade sequence , with its reduced sensitivity to movement , was used to monitor the ice ball in one study . as in other in - bore prostate interventional procedures , t2-weighted true fast imaging with bssfp can be used for confirming the final position of the cryoprobes .
three studies have been recently published documenting feasibility of mr - guided cryoablation in a different cohort of patients .
gangi et al . assessed the feasibility of whole gland cryoablation in 11 patients , eight with newly diagnosed pca and three for salvage therapy following radiation therapy .
they placed four to seven cryoprobes transperineally under mri guidance with an intent to ablate the entire gland .
urethral and rectal warmers were used in most of the cases and two freeze thaw cycles were performed in all treatments .
the reported procedure time was from 2 to 4.5 h and the mean hospital stay was 5 days .
one of the initial patients in the study in whom the rectal warmer was not used developed a recto urethral fistula , which healed in 3 months .
they concluded that mri allows precise positioning of the cryoprobes and excellent 3d monitoring of the ice ball growth , thereby overcoming the two limitations of ultrasound guidance . in another study ,
treated 18 patients with local recurrence after radical prostatectomy ( six of these patients had salvage radiation therapy and subsequent recurrence ) in a 1.5 t wide - bore mr magnet .
they reported better oncologic results in the group who were treated with three freeze thaw cycles .
reported the feasibility of mr - guided cryoablation in 10 patients with locally recurrent pca after radiation therapy .
unlike the other two studies cited above , they evaluated focal treatment , ablating only the lesion with margins in order to keep complications at a minimum .
the median focal tumor size was 20 mm and the treatment time was 210.5 min , very similar to our own initial study that treated four patients with transrectal mrgfus .
the investigators reported a steep learning curve and that the last two procedures in their study took substantially less time .
three patients had recurrent / residual disease at the margin of the treated area in the first 12 months and were retreated .
apart from the treatment modalities described above , there are other techniques such as microwave ablation and photodynamic therapy , which have used mri for planning and follow - up for pca therapy , but the procedures have been performed under ultrasound guidance in most studies .
more recently , early studies on focal irreversible electrophoration for pca under ultrasound guidance have been performed using mri imaging for planning and follow - up .
given the excellent spatial resolution and the multiplanar capability of mri , the feasibility of irreversible electroporation techniques for pca therapy may also be assessed in - bore , in a similar manner to cryoablation , to increase the procedure safety .
the increased costs associated with performing focal therapy in the mri magnet remain a disadvantage .
other limitations include need for mr - compatible materials , time availability on the mri machine and the limited amount of working space for the physician when performing these procedures in the mr system .
use of newer wider bore mri systems and dedicated mri interventional suites in the future would make it easier for the physicians to work in the mri environment . also , use of mri - compatible robotic / mechatronic systems , as highlighted in some of the recent studies , may also help to speed up the treatment and thereby decrease the overall costs of the treatment .
advances in prostate mri have opened up opportunities for targeted diagnosis and treatment of pca .
real - time mri thermometry feedback during treatment and the spatial resolution provided by mri are added advantages of treating in the mri suite , making it ideally suited for targeted therapeutics . at this time
, mrgfla and mrgfus techniques appear to be the most promising of the available in - bore techniques for the primary treatment of focal pca . while mrgfla has the advantage of being precise and interstitial with lower cost of the integrated system , mrgfus is a non - invasive technique .
initial phase 1 in - bore studies for focal therapy of pca have shown promising results , although larger phase ii multi - center trial results are awaited for assessing widespread clinical application . | recent advances in multiparametric magnetic resonance imaging ( mp - mri ) have led to a paradigm shift in the diagnosis and management of prostate cancer ( pca ) .
its sensitivity in detecting clinically significant cancer and the ability to localize the tumor within the prostate gland has opened up discussion on targeted diagnosis and therapy in pca .
use of mp - mri in conjunction with prostate - specific antigen followed by targeted biopsy allows for a better diagnostic pathway than transrectal ultrasound ( trus ) biopsy and improves the diagnosis of pca .
improved detection of pca by mp - mri has also opened up opportunities for focal therapy within the organ while reducing the incidence of side - effects associated with the radical treatment methods for pca .
this review discusses the evidence and techniques for in - bore mri - guided prostate biopsy and provides an update on the status of mri - guided targeted focal therapy in pca . | INTRODUCTION
MRI-guided prostate biopsy
MR-guided focal laser ablation
MR-guided focused ultrasound therapy
Transrectal MRgFUS
MR-guided transurethral focused ultrasound therapy
MR-guided focal cryoablation
CONCLUSION | widespread use of prostate - specific antigen ( psa ) screening and increased number of transrectal prostate biopsy encounters have led to an increased diagnosis of prostate cancer ( pca ) along with stage migration toward early - stage , organ - confined disease . multiparametric magnetic resonance imaging ( mp - mri ) of the prostate combining t2-weighted imaging with diffusion - weighted imaging ( dwi ) and perfusion imaging has been extensively studied in recent years . it has been shown to have a high sensitivity , particularly in detecting clinically significant cancers . the localizing strength of mp - mri of the prostate has opened up opportunities for targeted diagnosis and treatment strategies . the effectiveness of mp - mri used along with psa , followed by targeted biopsy of the mri visible lesion , is now accepted as a better alternative to systematic trus biopsy
recently published a meta - analysis of 16 studies , where the authors concluded that mri - guided targeted biopsy had a higher detection rate for significant cancer and lower rate of detection of insignificant cancer and therefore benefits the diagnosis of pca . targeted biopsy of the mri visible lesion can be performed by two different techniques , either in - bore mri - guided biopsy of the visible target or registration of mr images with trus images using fusion software for biopsy under trus guidance ( out of bore ) . both approaches use the advantage of tumor delineation on mp - mri for accurate localization and sampling . apart from the role in risk stratification of pca
, the localizing ability of mp - mri has also opened up opportunities for its focal treatment . significant deterioration of urinary and sexual function remain prevalent side - effects of radical therapies . focal therapy is an intuitive new organ - sparing technique that aims to selectively ablate locally confined , clinically significant index lesions while sparing the majority of the prostate gland and the surrounding delicate neurovascular bundles and urinary sphincter . the index lesion if clinically significant will often be visualized on mp - mri , can be confirmed on biopsy and targeted for ablation . various experts have proposed different ablative templates for focal therapy [ figure 1 ] , but in our opinion
focal therapy is somewhat of a misnomer for hemiablation and subtotal zonal ablation templates . the goal of focal therapy should be to perform targeted ablation of the mri visible index tumor with the aim to eradicate the index lesion and minimizing side - effects of treatment , thereby providing the best balance between oncologic control and maintenance of quality of life ( qol ) . visualization of tumor allows in - bore mr - guided focal treatment to be more suitable for targeted ablation and accurate targeting without any misregistration concerns . in - bore
treatment also offers the advantage of mr thermometry , which provides real - time monitoring of the thermal map during the treatment to ensure selective and adequate tumor ablation and preservation of sensitive surrounding structures . magnetic resonance - guided in - bore ablation is ideally suited for targeted ablation this article reviews the current status of mri - guided prostate biopsies as well as minimally invasive in - bore targeted treatment of organ - confined focal pca . ongoing clinical trials for in - bore focal pca treatment via the transperineal , transrectal and transurethral routes using different energy modalities for the ablation are discussed . focal therapy and prostate biopsy performed under trus guidance , with or without use of the newer mri - trus fusion technologies , are beyond the scope of this review and are therefore not included . mri - guided prostate biopsy was initially reported in 2000 when investigators reported its success via a transperineal route using an open - configuration 0.5 t strength mri . although the open - bore mri configuration provides easier patient access , it is limited by its low signal to noise ratio , which leads to non- or poor visualization of the target lesion at the time of biopsy on the low - strength ( typically 0.5 t ) open - bore magnets . reported transrectal mr - guided biopsy in a cohort of 12 patients using a robotic device in a closed - bore 1.5 t magnet . since then , several studies have reported successful mr - guided prostate biopsy within closed - bore higher strength magnets ( 1.5 t and 3 t ) , most using the transrectal approach in prone position . in the
largest published series on in - bore prostate biopsy , 265 patients with psa over 4.0 ng / ml and at least one previous negative trus biopsy were sampled following mp - mri in a 3 t magnet . using dwi for mr - guided in - bore biopsy , hambrock et al . recently reported the results of mr - guided biopsy preformed in wide - bore 1.5 t and 3 t magnets in the more comfortable supine lithotomy position in 50 patients . described a mr - compatible mechatronic ( robotic ) system for in - bore needle guidance via a transperineal route , which could be used for prostate biopsies and focal ablation . repeatedly moving the table to bring the patient out of the scanner to provide access to the needle for advancing or adjusting
the biopsy needle , followed by repeat imaging , is one of the main reasons that prolongs the procedure time of in - bore procedures , including biopsy . all investigators concluded that robot - assisted prostate procedures were feasible and can be performed safely and accurately , although in at least one of these studies the procedure time did not decrease with use of the robotic device when compared with the manual technique . this brings into question the cost - effectiveness of mr - guided in - bore prostate biopsy . compared the qol and health care costs for the blind trus - guided biopsy strategy to the imaging - based strategy where mri and directed mr - guided biopsies were performed , modeled to include the cascading effects for a period of 10 years following initial referral for biopsy . the benefits of mp - mri in the diagnostic pathway of pca include the potential of performing less number of biopsies , based on the negative predictive value of mp - mri , and therefore decreasing the incidence of complications from biopsies ( multiresistant sepsis ) , increased sampling efficiency , decreased histopathology costs , better characterization of gleason grade and fewer missed clinically significant cancers . mri - trus fusion biopsy exploits the high accuracy of mp - mri with the real - time capability and ease of trus biopsy . it is less expensive than mr - guided biopsy and can also be performed in a shorter time , although there have not been any published studies directly comparing the two techniques for targeted prostate biopsy . recent results of mri - trus fusion biopsy with different available software platforms have shown promising results and therefore this technique is likely to provide an alternative targeted biopsy method to overcome the costs of in - bore prostate . laser is one of the energy modalities currently being evaluated for mr - guided focal therapy of pca . the procedure is performed under deep sedation in the mri suite in supine position with the legs elevated on support to provide access to the perinuem . compared whole mount histology with mri in four patients who underwent fla followed by radical prostatectomy and found that fla created confluent ablation and that post - ablation mri was accurate in determining the ablated area . two separate phase 1 trials using the transperineal approach on a cohort of 38 and nine patients , respectively , were reported in 2013 , demonstrating its feasibility with minimal or no side - effects of the treatment . both studies showed near - identical results with no residual disease at the treated site in approximately 75% of the patients , and most of the patients with residual disease had low - volume low - grade disease not visible on mp - mri at 46 months post - treatment . in their initial evaluation of 37 needle insertions using the mechatronic device in the course of 10 fla treatments , the median needle guidance error was 3.5 mm and the needle delivery time was 9 min . used a transrectal approach for mrgfla with the patient in the prone position and reported their experience in 23 patients . magnetic resonance - guided focal laser ablation treatment . ( a ) pre - treatment axial apparent diffusion coefficient map ( adc ) shows a well - demarcated magnetic resonance imaging ( mri ) visible lesion ( outlined in red ) in the left transition zone at the level of mid gland . as with other energy modes for focal treatment of pca , integration of focused ultrasound with mri ( mrgfus ) provides for accurate targeting and closed - loop real - time monitoring of temperature by mri thermography thus also allowing for a safer ablation procedure . the ultrasound beam can be steered to the desired location in the prostate [ figure 3 ] . our group initially reported the feasibility of transrectal mrgfus treatment for pca with the exablate system , and have recently reported the results with > 18 months follow - up on the first four patients treated with mrgfus system at our institution in an ongoing phase i trial . ongoing phase 1 multi - center trial across three sites to evaluate mr - guided transurethral whole gland ablation has completed enrollment of 30 patients , and the 12-month results are due in 2015 . transrectal ultrasound - guided percutaneous prostate cryoablation was first described by onik et al . in - bore
mri - guided focal cryoablation allows real - time visualization of ice ball formation with the added benefit of the spatial resolution provided by mri , which is paramount to identify the sensitive surrounding structures , such as rectal wall , ureters , urethra and external urinary sphincter . as in other in - bore prostate interventional procedures , t2-weighted true fast imaging with bssfp can be used for confirming the final position of the cryoprobes . in another study ,
treated 18 patients with local recurrence after radical prostatectomy ( six of these patients had salvage radiation therapy and subsequent recurrence ) in a 1.5 t wide - bore mr magnet . reported the feasibility of mr - guided cryoablation in 10 patients with locally recurrent pca after radiation therapy . given the excellent spatial resolution and the multiplanar capability of mri , the feasibility of irreversible electroporation techniques for pca therapy may also be assessed in - bore , in a similar manner to cryoablation , to increase the procedure safety . the increased costs associated with performing focal therapy in the mri magnet remain a disadvantage . other limitations include need for mr - compatible materials , time availability on the mri machine and the limited amount of working space for the physician when performing these procedures in the mr system . use of newer wider bore mri systems and dedicated mri interventional suites in the future would make it easier for the physicians to work in the mri environment . also , use of mri - compatible robotic / mechatronic systems , as highlighted in some of the recent studies , may also help to speed up the treatment and thereby decrease the overall costs of the treatment . mri - guided prostate biopsy was initially reported in 2000 when investigators reported its success via a transperineal route using an open - configuration 0.5 t strength mri . although the open - bore mri configuration provides easier patient access , it is limited by its low signal to noise ratio , which leads to non- or poor visualization of the target lesion at the time of biopsy on the low - strength ( typically 0.5 t ) open - bore magnets . reported transrectal mr - guided biopsy in a cohort of 12 patients using a robotic device in a closed - bore 1.5 t magnet . since then , several studies have reported successful mr - guided prostate biopsy within closed - bore higher strength magnets ( 1.5 t and 3 t ) , most using the transrectal approach in prone position . in the
largest published series on in - bore prostate biopsy , 265 patients with psa over 4.0 ng / ml and at least one previous negative trus biopsy were sampled following mp - mri in a 3 t magnet . using dwi for mr - guided in - bore biopsy , hambrock et al . recently reported the results of mr - guided biopsy preformed in wide - bore 1.5 t and 3 t magnets in the more comfortable supine lithotomy position in 50 patients . described a mr - compatible mechatronic ( robotic ) system for in - bore needle guidance via a transperineal route , which could be used for prostate biopsies and focal ablation . the transperineal approach for prostate intervention is associated with decreased risk of urosepsis , which may occur in about 4% of patients following transrectal prostate biopsies . repeatedly moving the table to bring the patient out of the scanner to provide access to
the needle for advancing or adjusting the biopsy needle , followed by repeat imaging , is one of the main reasons that prolongs the procedure time of in - bore procedures , including biopsy . all investigators concluded that robot - assisted prostate procedures were feasible and can be performed safely and accurately , although in at least one of these studies the procedure time did not decrease with use of the robotic device when compared with the manual technique . this brings into question the cost - effectiveness of mr - guided in - bore prostate biopsy . compared the qol and health care costs for the blind trus - guided biopsy strategy to the imaging - based strategy where mri and directed mr - guided biopsies were performed , modeled to include the cascading effects for a period of 10 years following initial referral for biopsy . the benefits of mp - mri in the diagnostic pathway of pca include the potential of performing less number of biopsies , based on the negative predictive value of mp - mri , and therefore decreasing the incidence of complications from biopsies ( multiresistant sepsis ) , increased sampling efficiency , decreased histopathology costs , better characterization of gleason grade and fewer missed clinically significant cancers . mri - trus fusion biopsy exploits the high accuracy of mp - mri with the real - time capability and ease of trus biopsy . it is less expensive than mr - guided biopsy and can also be performed in a shorter time , although there have not been any published studies directly comparing the two techniques for targeted prostate biopsy . recent results of mri - trus fusion biopsy with different available software platforms have shown promising results and therefore this technique is likely to provide an alternative targeted biopsy method to overcome the costs of in - bore prostate . laser is one of the energy modalities currently being evaluated for mr - guided focal therapy of pca . the procedure is performed under deep sedation in the mri suite in supine position with the legs elevated on support to provide access to the perinuem . compared whole mount histology with mri in four patients who underwent fla followed by radical prostatectomy and found that fla created confluent ablation and that post - ablation mri was accurate in determining the ablated area . two separate phase 1 trials using the transperineal approach on a cohort of 38 and nine patients , respectively , were reported in 2013 , demonstrating its feasibility with minimal or no side - effects of the treatment . both studies showed near - identical results with no residual disease at the treated site in approximately 75% of the patients , and most of the patients with residual disease had low - volume low - grade disease not visible on mp - mri at 46 months post - treatment . in their initial evaluation of 37 needle insertions using the mechatronic device in the course of 10 fla treatments , the median needle guidance error was 3.5 mm and the needle delivery time was 9 min . used a transrectal approach for mrgfla with the patient in the prone position and reported their experience in 23 patients . magnetic resonance - guided focal laser ablation treatment . ( a ) pre - treatment axial apparent diffusion coefficient map ( adc ) shows a well - demarcated magnetic resonance imaging ( mri ) visible lesion ( outlined in red ) in the left transition zone at the level of mid gland . as with other energy modes for focal treatment of pca , integration of focused ultrasound with mri ( mrgfus ) provides for accurate targeting and closed - loop real - time monitoring of temperature by mri thermography thus also allowing for a safer ablation procedure . two different systems , one for transrectal and the other for transurethral ablation , have been developed and are being presently evaluated in phase 1 clinical trials . the ultrasound beam can be steered to the desired location in the prostate [ figure 3 ] . our group initially reported the feasibility of transrectal mrgfus treatment for pca with the exablate system , and have recently reported the results with > 18 months follow - up on the first four patients treated with mrgfus system at our institution in an ongoing phase i trial . ongoing phase 1 multi - center trial across three sites to evaluate mr - guided transurethral whole gland ablation has completed enrollment of 30 patients , and the 12-month results are due in 2015 . transrectal ultrasound - guided percutaneous prostate cryoablation was first described by onik et al . in - bore
mri - guided focal cryoablation allows real - time visualization of ice ball formation with the added benefit of the spatial resolution provided by mri , which is paramount to identify the sensitive surrounding structures , such as rectal wall , ureters , urethra and external urinary sphincter . as in other in - bore prostate interventional procedures , t2-weighted true fast imaging with bssfp can be used for confirming the final position of the cryoprobes . three studies have been recently published documenting feasibility of mr - guided cryoablation in a different cohort of patients . the reported procedure time was from 2 to 4.5 h and the mean hospital stay was 5 days . they reported better oncologic results in the group who were treated with three freeze thaw cycles . given the excellent spatial resolution and the multiplanar capability of mri , the feasibility of irreversible electroporation techniques for pca therapy may also be assessed in - bore , in a similar manner to cryoablation , to increase the procedure safety . the increased costs associated with performing focal therapy in the mri magnet remain a disadvantage . other limitations include need for mr - compatible materials , time availability on the mri machine and the limited amount of working space for the physician when performing these procedures in the mr system . use of newer wider bore mri systems and dedicated mri interventional suites in the future would make it easier for the physicians to work in the mri environment . also , use of mri - compatible robotic / mechatronic systems , as highlighted in some of the recent studies , may also help to speed up the treatment and thereby decrease the overall costs of the treatment . advances in prostate mri have opened up opportunities for targeted diagnosis and treatment of pca . real - time mri thermometry feedback during treatment and the spatial resolution provided by mri are added advantages of treating in the mri suite , making it ideally suited for targeted therapeutics . at this time
, mrgfla and mrgfus techniques appear to be the most promising of the available in - bore techniques for the primary treatment of focal pca . initial phase 1 in - bore studies for focal therapy of pca have shown promising results , although larger phase ii multi - center trial results are awaited for assessing widespread clinical application . | [
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] |
intestinal ischemic reperfusion ( i / r ) injury is a common pathophysiological process and can be caused by major vascular surgery , mesenteric artery occlusion , small bowel transplantation , cardiopulmonary bypass , hemodialysis , strangulated hernias , trauma , and shock [ 1 , 2 ] . acute intestinal ischemia is a life - threatening vascular emergency ; however , the improvement in recent years has been minimal , and its in - hospital mortality rate remains high at approximately 60%80% [ 3 , 4 ] .
although many studies have focused on i / r , the mechanisms have not been fully elucidated .
many cytokines , such as tnf- , il-6 , il-1 , and icam-1 , have been reported to be involved in the process of intestinal i / r injury , and their expression can be regulated by nuclear factor - kappa b ( nf-b ) [ 57 ] .
these cytokines may increase capillary permeability and damage the intestinal microcirculation , which in turn may cause the intestinal mucosal membrane to lose its resistance to bacteria .
this results in bacterial translocation and endotoxemia , ultimately leading to the systemic inflammatory response syndrome ( sirs ) and multiple organ dysfunction ( modf ) .
it has been well demonstrated that the nf-b pathway plays an important role in this cytokine - induced intestinal barrier dysfunction [ 8 , 9 ] .
the application of nf-b inhibitors was found to significantly attenuate the expression of inflammatory cytokines and to alleviate intestinal i / r injury .
therefore , understanding the nf-b activation mechanism will provide new means for the clinical treatment of intestinal i / r injury and reduce the mortality of severe i / r - injured patients .
bone morphogenic protein ( bmp ) is a key member of the transforming growth factor- ( tgf- ) super family .
more than 30 bmp proteins have been identified , and they can be further classified into several subgroups , including the bmp2/4 group , the bmp5/6/7/8 group ( op-1 [ osteogenic protein-1 ] group ) , the growth and differentiation factor- ( gdf- ) 5/6/7 group , and the bmp9/10 group .
after these receptors combine with their ligands , the bmp proteins function via either typical or atypical pathways .
the canonical pathway activates the phosphorylation of smad 1 , 5 , and 8 , while the noncanonical pathway activates the nf-b pathway .
our previous studies have shown that the expression of bmp2/4 in intestinal epithelial cells increased in tpn mice , the erk1/2 pathway was activated , and the proliferation of intestinal epithelial cells was weakened .
these effects may lead to weaker intestinal barrier function and thereby increase the possibility of endogenous infection .
bmps have recently been thought to influence inflammatory processes in adults due to their chemotactic activity on fibroblasts , myocytes , and inflammatory cells .
it has been shown that bmp2 can induce inflammatory reactions in endothelial cells , fibroblasts , preosteoblasts , and soft tissues .
. found that bmp4 plays an inflammatory role in the early steps of atherogenesis by initiating an inflammatory cascade in an nf-b - dependent manner through the stimulation of icam-1 surface expression in activated endothelial cells .
the activation of nf-b in human embryonic kidney ( hek ) cells also depends on the bmp signaling pathway .
therefore , we hypothesized that the bmp pathway would be upregulated and play an important role , through activation of the nf-b signaling pathway , in the damage caused to the intestinal barrier function by i / r injury .
our data indicate that bmp2/4 expression is increased in both an early cell hypoxia model and a male sprague - dawley ( sd ) rat i / r model .
bmp2/4 is able to directly increase the expression of the cytokines tnf- and il-6 and decrease the expression of the tight junction protein occludin by activating nf-b signaling .
anti - p - p65 , anti - p65 , anti - p - erk1/2 , anti - erk1/2 , anti - p - akt , and anti - akt were purchased from cell signaling technology ( boston , ma , usa ) .
anti - gapdh antibody was purchased from the proteintech group ( chicago , il , usa ) .
anti - bmp2 , anti - bmp4 , anti - bmpria , anti - bmprii , anti - bmprib , and anti - occludin antibodies were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) .
recombinant bmp2 , bmp4 , and noggin were purchased from peprotech ( nj , usa ) .
the inhibitor of nf-b , pdtc , was purchased from beyotime ( wuhan , china ) .
iec-6 intestinal epithelial cells were purchased from the american type culture collection ( atcc , manassas , va ) and grown in dulbecco 's modified eagle 's medium ( dmem , hyclone ) supplemented with 10% fetal bovine serum ( gibco ) , 100 iu / ml penicillin , and 100 mg / ml streptomycin .
the iec-6 cells were cultured at 37c in either normoxic ( 5% co2 and 20% o2 ) or hypoxic ( 5% co2 and 1% o2 in a hypoxia chamber ) conditions ( thermo fisher scientific , ohio , usa ) . for western blot analysis ,
bmp2 , bmp4 , and noggin were added to the medium for 6 h or for a time gradient from 0 min to 120 min . for real - time pcr analysis , bmp2 , bmp4 , and noggin
were added to the medium for 3 h. for immunofluorescence analysis , bmp2 , bmp4 , and noggin were added to the medium for 30 min .
the cells were washed twice with phosphate - buffered saline ( pbs ) before lysis in cold ripa buffer ( 50 mm tris , 150 mm nacl , 1% triton x-100 , 1% sodium deoxycholate , 0.1% sds , and 2 mm sodium pyrophosphate ) .
proteins were loaded onto 810% sds - page gels at 100 v for 2 h. after electrophoresis , the proteins were electroblotted onto nc membranes at 200 ma for 2 h. nonspecific binding was blocked by incubation in tris - buffered saline ( tbs ) containing 0.1% tween 20 ( tbs - t ) and 5% skim milk .
the transferred membranes were incubated overnight at 4c with primary antibody as follows : anti - p - p65 ( 1 : 1000 ) , anti - p65 ( 1 : 1000 ) , anti - p - erk ( 1 : 500 ) , anti - erk ( 1 : 500 ) , anti - p - akt ( 1 : 1000 ) , anti - akt ( 1 : 1000 ) , anti - bmp2 ( 1 : 500 ) , anti - bmp4 ( 1 : 500 ) , anti - bmpria ( 1 : 200 ) , anti - bmprii ( 1 : 750 ) , anti - bmprib ( 1 : 200 ) , and anti - gapdh ( 1 : 5000 ) . after washing three times in tbs - t
china ) conjugated to horseradish peroxidase at a dilution of 1 : 3000 in tbs - t containing 5% skim milk for 1 h at 37 c .
after three additional washes with tbs - t , the signals were visualized using the super signal west pico trial kit ( pierce , usa ) and detected with image station 4000r ( kodak ) .
male sprague - dawley ( sd ) rats weighing 200250 g were obtained from our university 's laboratory animals .
after the rats were fasted for 12 hours , their abdomens were opened via a midline incision under sodium pentobarbital anesthesia .
the rats were randomly assigned to three groups : ( 1 ) the i / r group , in which the superior mesenteric artery ( sma ) was occluded for 30 minutes followed by defined times of reperfusion ; ( 2 ) the noggin+ i / r group , in which an intraperitoneal injection of 4 g / kg noggin was given 30 min before i / r , followed by ischemia , reperfusion , and sacrifice of the rats at defined times ; and ( 3 ) the sham group , which included animals subjected to anesthesia and laparotomy without ischemia .
the tissue was cut along the longitudinal axis , washed in physiological saline , and immediately frozen in liquid nitrogen and stored at 70c for future use .
all animal experiments were performed in compliance with our university 's guidelines for the care and use of laboratory animals . for the current study
after fixation in 4% formaldehyde for 20 min , the sections were incubated in 3% h2o2 for 30 min to quench endogenous peroxidases .
nonspecific binding was blocked with 5% bsa in phosphate buffered saline for 30 min at room temperature .
sections were incubated overnight at 4c in 3% bsa in pbs with primary antibodies as follows : anti - bmp2 , anti - bmp4 , anti - bmpria , anti - bmpr2 ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) in a 1 : 50 dilution , and anti - p65 ( cell signaling biotechnology ) in a 1 : 100 dilution .
sections were washed three times in bsa in pbs and incubated with alexa 488-conjugated goat anti - rabbit antibody for 1 h at rt .
after the nuclei were stained with dapi , images were analyzed and collected with a leica tcssp confocal imaging system ( leica , heidelberg , germany ) .
iec-6 monolayers were washed three times with pbs before fixation in 4% paraformaldehyde for 20 min at room temperature .
triton x-100 for 10 min and blocked in 5% bsa in pbs at room temperature for 30 min .
then , the iec-6 cells were incubated overnight at 4c in 5% bsa in pbs with anti - p65 at a 1 : 50 dilution .
monolayers were washed with pbs and incubated with fitc green - conjugated goat anti - rabbit secondary antibodies ( 1 : 100 ; zhongshan ) for 1 h in dark conditions .
louis , mo ) was used to stain the nuclei , and the cells were imaged using a laser scanning fluorescence microscope ( leica , heidelberg , germany ) .
total cellular rna was isolated with trizol reagent and used for first strand cdna synthesis with the reverse transcription system .
quantification of gene transcripts was performed with a 7500 real - time pcr system ( applied biosystems , foster city , california , usa ) using power sybr green ( applied biosystems , foster city , california , usa ) .
pcr conditions were one cycle of 94c for 2 min followed by 50 cycles of 94c for 10 s , a specified annealing temperature for 15 s and 72c for 15 s. amplification was followed by melting curve analysis , which used the following program : one cycle at 65c for 1 s , 94c for 2 s , and 37c for 5 s. the housekeeping gene -actin was used as an endogenous reference gene to which the expression of the other genes was normalized using the comparative cycle of threshold value .
the following pcr primers were used : tnf - a forward primer : 5-gcgtgttcatccgttctcta-3 , reverse primer 5-cgtctcgtgtgtttctgagc-3 ; il-6 forward primer : 5-agttgccttcttgggactga-3 , reverse primer : 5-actggtctgttgtgggtggt-3 ; and -actin forward primer : 5-cccatctatgagggttacgc-3 , reverse primer : 5-tttaatgtcacgcacgatttc-3. statistical analyses were performed using spss 13.0 software .
comparisons among 3 or more groups were made by analysis of variance ( anova ) , and 2 groups were compared by student 's t - test .
if not otherwise stated , all experiments were repeated for at least 3 individual experiments to ensure reproducibility .
we found that the expression level of bmp2 and bmp4 was upregulated 2.5-fold ( figure 1(a ) ) and 3.1-fold ( figure 1(b ) ) , respectively , in iec-6 cells after 6 h of hypoxia .
meanwhile , we detected the expression of bmp2 and bmp4 in intestinal epithelial cells in an i / r rat model .
if analysis showed that these proteins were also significantly increased along the crypt / villus axis after 1 h of i / r , consistent with the significantly increased bmp2 and bmp4 levels in intestinal epithelial cells under hypoxia . normally , bmp2 and bmp4 are expressed in both the epithelial and mesenchymal compartments , but bmp4 is highly expressed and enriched in the mesenchyme [ 13 , 16 ] . in the present study ,
the bmp2 level significantly increased in the mid - to - distal villus region after 1 h of i / r , while the bmp4 level increased significantly in both the villi and mesenchyme in the i / r rat ( figure 1(c ) ) .
the main bmp receptors include the type ii bmp receptor ( bmprii ) and the following type i receptors : the bmpri group ( bmpria and bmprib ; also denoted as alk-3 and alk-6 , resp . ) , the alk-1 group ( alk-1 and alk-2 ) , and the tbr - i group ( alk-4/actr - ib , alk-5/tbr - i , and alk-7 ) .
typically , bmp2 and bmp4 bind to bmpria and bmprib , but bmpria has a high - affinity binding site for bmp2 . to investigate
whether the greater abundance of bmp2/4 led to an increase in intracellular bmp signaling , we evaluated the expression of bmprii and bmpr - ia in epithelial cells under hypoxia and i / r . at 6 h after hypoxia , bmpria and bmprii expression levels were both significantly increased ( figures 2(a ) and 2(b ) ) .
we also detected the expression of bmp receptors in the rat i / r model .
sections of the small intestine were collected to detect changes in bmpria and bmprii expression via immunofluorescence analysis .
immunofluorescence staining showed that the expression levels of the transmembrane receptors bmpria and bmprii were significantly increased in the villi but had lower expression levels in the matrix ( figure 2(c ) ) .
we used western blotting to determine the effect of bmp2 and bmp4 on nf-b transcriptional activity .
bmp2 and bmp4 increased nf-b transcriptional activity 3.5-fold and 3.4-fold , respectively , while bmp2/4 combined with noggin resulted in lower levels of nf-b transcriptional activity ( figure 3(a ) ) .
, nf-b translates to the nucleus to trigger the transcription of genes involved in inflammatory cellular responses and other types of signals .
we used if to detect the expression of nf-b after stimulation by exogenous bmp2/4 for 30 min .
the fluorescence intensity was greater compared to the control group , and blockade of bmp2/4 signaling by noggin completely reversed the nuclear localization of nf-b induced by bmp2 and bmp4 ( figure 3(b ) ) .
meanwhile , the in vivo results showed that nf-b signaling was obviously activated in intestinal epithelial cells after 1 h of i / r treatment .
in contrast , after intraperitoneal injection of 4 g / kg noggin 30 min before i / r , the expression of nf-b was significantly inhibited ( figure 3(c ) ) .
because mitogen - activated protein kinase ( mapk ) is known as an upstream regulator of nf-b , we evaluated the three common proteins of the mapk pathway , erk , p38 , and jnk .
exogenous bmp2 and bmp4 activated erk ( figure 3(d ) ) but not p38 or jnk ( data not shown ) .
these results are consistent with the results from s. o. kim and m. r. kim , who found that treatment with an erk - specific blocking agent completely inhibited nf-b activity .
these results indicate that nf-b may be activated by bmp2 and bmp4 via an increase in erk phosphorylation .
nf-b plays a central role in regulating the transcription of cytokines , adhesion molecules , and other mediators involved in acute respiratory distress syndrome ( ards ) , sepsis , and multiple organ dysfunction syndrome ( mods ) . to test whether the activation of nf-b induced by bmp2 and bmp4 resulted in an increase in inflammatory cytokines , we used rt - pcr to detect the expression of tnf- mrna and il-6 mrna in iec-6 cells after treatment with bmp2 and bmp4 for 3 h. treatment of iec-6 cells with 100 ng / ml bmp2 caused the level of tnf- mrna to increase 6.3-fold compared to the control group ( figure 4(a ) ) , while the effect of bmp4 in inducing the expression of tnf- mrna was weaker ( figure 4(b ) ) .
tumor necrosis factor is one of the most powerful inducers and promoters of inflammation , and nf-b both is activated by cytokines and induces the expression of inflammatory cytokines .
this gives rise to the potential for nf-b activation to spread from cell to cell within a tissue and beyond .
bmp2 and bmp4 both increased the expression of il-6 mrna , and these effects were decreased by noggin ( figures 4(c ) and 4(d ) ) .
the activation of nf-b by ifn- enhances the permeability of t84 cells and decreases the levels of intercellular tight junction proteins , whereas the inhibition of nf-b will block the increase in t84tj permeability and alter occludin expression [ 8 , 19 ] .
we asked whether bmp2/4 would disrupt the intestinal mucosal barrier function via the activation of nf-b .
we used recombinant bmp2 and bmp4 to stimulate intestinal epithelial cells for 24 h , and the tight junction protein occludin was detected by western blotting .
the expression of occludin decreased after treatment with bmp2 and bmp4 , while this effect was abolished by noggin or the nf-b inhibitor pdtc ( figure 5(a ) ) .
these results may indicate that bmp2 and bmp4 disrupt the integrity of the intestinal mucosal barrier via the activation of nf-b and that pdtc can reverse the decrease in tj proteins .
boivin et al . have shown that pi3-k / akt activation was required for the activation of nf-b pathways in the modulation of the tj barrier by treatment with ifn- .
we also detected the expression of akt at the indicated times after stimulation of the iec-6 cells with bmp2 and bmp4 .
the phosphorylation of akt progressively increased in a time - dependent manner ( figure 5(b ) ) .
previous research has focused on the roles of the bmp pathway in early intestinal development and in the proliferation and differentiation of intestinal epithelial cells [ 2022 ] .
however , the relationship between the bmp pathway and intestinal mucosal barrier dysfunction caused by i / r has rarely been examined .
shen et al . found that , in rats after acetabular surgery , treatment with 4 mg / ml bmp2 protein significantly induced local inflammation , including an early and pronounced polymorphonuclear cell infiltration accompanied by the increased expression of tnf- and il-6 .
intestinal i / r results in the release of abundant inflammatory factors , the generation of oxygen free radicals ( ros ) , and the activation of nf-b .
these factors lead to enhanced permeability in the intestinal barrier and systemic inflammatory reactions [ 24 , 25 ] . because of the critical role of bmp in inflammatory reactions
, we hypothesized that the bmp signaling pathway would contribute to the mechanisms involved in promoting i / r - associated intestinal mucosal barrier injury .
our present study shows that , with hypoxia and i / r , intestinal epithelial cells produce abundant bmp2 and bmp4 and that bmpria and bmprii expressions are also enhanced .
western blotting and if showed that recombinant bmp2 and bmp4 directly activate nf-b in iec-6 cells .
bmp2/4 is able to directly increase the expression of the cytokines tnf- and il-6 and decrease the expression of the tight junction protein occludin by activating nf-b signaling .
this effect was attenuated in part by either the bmp - specific antagonist noggin or the nf-b inhibitor pdtc .
our study may provide new evidence that the activation of nf-b in intestinal i / r injury involves the bmp signaling pathway .
it is interesting to note that bmp signaling is complex ; different bmp subgroups can mediate antagonistic effects , or the same ligand can produce different effects in similar tissues .
for example , in an in vivo model , bmp4 inhibits liver proliferation , and the bmp4 antagonist noggin enhances regeneration , whereas another report demonstrates that bmp7 is an endocrine factor expressed in the kidney that enhances liver regeneration .
our findings demonstrate that hypoxia and i / r increased intestinal epithelial cell bmp2/4 signaling , thereby activating nf-b signaling .
these changes led to increased expression of inflammatory factors , such as tnf- and il-6 , and decreased expression of the tight junction protein occludin , which could result in disruption of the intestinal barrier .
that , after 6 h in injured airways , bmp signaling is activated , e - cadherin expression is downregulated , and migration in normal adult airway epithelial cells is increased .
furthermore , inhibition of bmp activity protects epithelial barrier function in cases of lung injury . however , our results contrast with another report that bmp7 has protective and anti - inflammatory functions in acute ischemic renal injury [ 30 , 31 ] .
in addition , bmp7 administration conferred intestinal mucosal protection and reduced systemic il-6 expression levels in an inflammatory bowel disease model . additionally , bmp7 administration before intestinal i / r injury protects against intestinal mucosal injury and liver injury , preserves intestinal function , and prevents intestinal inflammation .
one possible explanation is that bmp7 preferentially acts through alk2 ( a stimulatory pathway ) , whereas bmp2/4 preferentially acts through alk3 ( an inhibitory pathway ) .
alternative signal transduction mechanisms may also play a role : the canonical bmp pathway regulates gene expression via the smad - dependent pathway , while the noncanonical bmp pathway regulates nf-b via the mek / erk pathway .
our studies have demonstrated that phosphorylated erk1/2 and akt expression levels progressively increased in intestinal epithelial cells upon treatment with bmp2/4 .
thus , we believe that nf-b may be activated by bmp2 and bmp4 via the mek / erk pathway in the hypoxia and intestinal i / r model .
previous studies have shown that nf-b is activated in the process of intestinal i / r injury and that the amount of activated nf-b correlates with the degree of mucosal inflammation [ 34 , 35 ] .
the activation of nf-b could regulate the expression of several inflammatory genes , such as cell factors , adhesion molecules , and enzymes . in turn , the released inflammatory factors ( e.g. , tnf- and il-6 ) can activate nf-b , thereby aggravating the deterioration of intestinal barrier function .
additionally , the upregulated expression of tnf- and il-6 induced in intestinal epithelial cells by bmp2 and bmp4 treatment confirms that bmp2 and bmp4 can mediate the intestinal mucosal injury caused by i / r via the activation of nf-b .
moreover , bmp2 and bmp4 have been shown to activate erk1/2 , while the activation of the erk pathway reduces the proliferation of intestinal epithelial cells ; thus , persistent bmp expression is unfavorable for the repair of intestinal mucosal injury .
in addition to the amount of proinflammatory cytokines released by i / r , ischemia and hypoxia also disrupt the integrity of the intestinal mucosal barrier .
we asked whether bmp2 and bmp4 could decrease the expression of tight junction proteins , and we evaluated changes in the expression of the tight junction protein occludin .
our results show that the expression of occludin decreased after treatment with bmp2 and bmp4 , while these effects were abolished by treatment with noggin or pdtc .
these results are consistent with those of boivin et al . , who used nf-b blockers to prevent the ifn-induced increase in epithelial permeability and the destruction of tight junction proteins .
our previous study also found that ifn- induced intestinal barrier function injury via the nf-b / hif-1a pathway .
there are many types of extracellular bmp antagonists , such as noggin , chordin , cerberus , and follistatin [ 36 , 37 ] . by binding bmp
, these secretory factors act as competitive antagonists and are important negative regulatory factors in the bmp pathway . in mouse intestine ,
transient expression of the bmp antagonist noggin has been observed in both pericryptal mesenchymal cells and intestinal epithelial stem cells , which may contribute to maintaining intestinal stem cell self - renewal by activating wnt signaling and inhibiting bmp signaling of the basal crypt epithelial cells .
our data indicate that bmp2/4 expression increased in both an early intestinal epithelial cell hypoxia model and a rat i / r model .
furthermore , nf-b was directly activated by recombinant bmp2/4 in iec-6 cells by western blotting and immunofluorescence ( if ) .
finally , bmp2/4 induced the expression of intracellular tnf- mrna and il-6 mrna in iec-6 cells , and this effect was abolished by the bmp - specific antagonist noggin .
the in vivo rat model also showed that , with an intraperitoneal injection of noggin before i / r , the activation of nf-b was obviously inhibited .
thus , in future investigations , it will be important to examine the expression of a number of bmp antagonists ( noggin , chordin , gremlin , and follistatin ) in a rat i / r model .
hypoxia - inducible factor 1 ( hif-1 ) is one of the master regulators that orchestrate the cellular responses to hypoxia .
recent studies have found that bmp expression is modulated by hif-1 expression , bmp2 expression was increased in primary chondrocytes under hypoxic conditions , and addition of the hif-1 activator dfo significantly increased bmp2 expression . additionally , hypoxia stimulation increased bmp2 mrna and protein expression levels in osteoblasts via an hif-1-alpha - dependent mechanism involving the activation of the ilk / akt and mtor pathways .
further work is required to better understand the mechanisms guiding the increased bmp expression in the hypoxia and intestinal i / r model . to the best of our knowledge ,
the present study is the first to report that bmp2 and bmp4 can directly activate nf-b , induce the expression of the inflammatory cytokines tnf - a and il-6 in the intestinal epithelial cells , and decrease the expression of the tight junction protein occludin , which could result in disruption of the intestinal barrier .
all of these effects may contribute to the mechanism by which bmp2 and bmp4 mediate intestinal mucosa injury in ischemic reperfusion . | intestinal ischemic reperfusion ( i / r ) can cause dysfunction of the intestinal mucosal barrier ; however , the mechanism of the intestinal mucosal barrier dysfunction caused by i / r remains unclear . in this study , using intestinal epithelial cells under anaerobic cultivation and an in vivo rat intestinal i / r model , we found that hypoxia and i / r increased the expression of bmp2/4 and upregulated bmp type ia receptor and bmp type ii receptor expression .
we also found that exogenous bmp2/4 can activate the erk and akt signaling pathways in rat small intestine ( iec-6 ) cells , thereby activating nf-b signaling , which leads to increased levels of inflammatory factors , such as tnf- and il-6 .
furthermore , recombinant bmp2/4 decreased the expression of the tight junction protein occludin via the activation of the nf-b pathway ; these effects were abolished by treatment with the bmp - specific antagonist noggin or the nf-b inhibitor pyrrolidine dithiocarbamate ( pdtc ) .
all these factors can destroy the intestinal mucosal barrier , thereby leading to weaker barrier function . on the basis of these data ,
we conclude that bmp2/4 may act as the pathogenic basis for intestinal mucosal barrier dysfunction when the intestines suffer an i / r injury .
our results provide background for the development pharmacologic interventions in the management of i / r injury . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion | intestinal ischemic reperfusion ( i / r ) injury is a common pathophysiological process and can be caused by major vascular surgery , mesenteric artery occlusion , small bowel transplantation , cardiopulmonary bypass , hemodialysis , strangulated hernias , trauma , and shock [ 1 , 2 ] . acute intestinal ischemia is a life - threatening vascular emergency ; however , the improvement in recent years has been minimal , and its in - hospital mortality rate remains high at approximately 60%80% [ 3 , 4 ] . although many studies have focused on i / r , the mechanisms have not been fully elucidated . many cytokines , such as tnf- , il-6 , il-1 , and icam-1 , have been reported to be involved in the process of intestinal i / r injury , and their expression can be regulated by nuclear factor - kappa b ( nf-b ) [ 57 ] . these cytokines may increase capillary permeability and damage the intestinal microcirculation , which in turn may cause the intestinal mucosal membrane to lose its resistance to bacteria . this results in bacterial translocation and endotoxemia , ultimately leading to the systemic inflammatory response syndrome ( sirs ) and multiple organ dysfunction ( modf ) . it has been well demonstrated that the nf-b pathway plays an important role in this cytokine - induced intestinal barrier dysfunction [ 8 , 9 ] . the application of nf-b inhibitors was found to significantly attenuate the expression of inflammatory cytokines and to alleviate intestinal i / r injury . therefore , understanding the nf-b activation mechanism will provide new means for the clinical treatment of intestinal i / r injury and reduce the mortality of severe i / r - injured patients . more than 30 bmp proteins have been identified , and they can be further classified into several subgroups , including the bmp2/4 group , the bmp5/6/7/8 group ( op-1 [ osteogenic protein-1 ] group ) , the growth and differentiation factor- ( gdf- ) 5/6/7 group , and the bmp9/10 group . after these receptors combine with their ligands , the bmp proteins function via either typical or atypical pathways . the canonical pathway activates the phosphorylation of smad 1 , 5 , and 8 , while the noncanonical pathway activates the nf-b pathway . our previous studies have shown that the expression of bmp2/4 in intestinal epithelial cells increased in tpn mice , the erk1/2 pathway was activated , and the proliferation of intestinal epithelial cells was weakened . these effects may lead to weaker intestinal barrier function and thereby increase the possibility of endogenous infection . it has been shown that bmp2 can induce inflammatory reactions in endothelial cells , fibroblasts , preosteoblasts , and soft tissues . found that bmp4 plays an inflammatory role in the early steps of atherogenesis by initiating an inflammatory cascade in an nf-b - dependent manner through the stimulation of icam-1 surface expression in activated endothelial cells . the activation of nf-b in human embryonic kidney ( hek ) cells also depends on the bmp signaling pathway . therefore , we hypothesized that the bmp pathway would be upregulated and play an important role , through activation of the nf-b signaling pathway , in the damage caused to the intestinal barrier function by i / r injury . our data indicate that bmp2/4 expression is increased in both an early cell hypoxia model and a male sprague - dawley ( sd ) rat i / r model . bmp2/4 is able to directly increase the expression of the cytokines tnf- and il-6 and decrease the expression of the tight junction protein occludin by activating nf-b signaling . iec-6 intestinal epithelial cells were purchased from the american type culture collection ( atcc , manassas , va ) and grown in dulbecco 's modified eagle 's medium ( dmem , hyclone ) supplemented with 10% fetal bovine serum ( gibco ) , 100 iu / ml penicillin , and 100 mg / ml streptomycin . after three additional washes with tbs - t , the signals were visualized using the super signal west pico trial kit ( pierce , usa ) and detected with image station 4000r ( kodak ) . the rats were randomly assigned to three groups : ( 1 ) the i / r group , in which the superior mesenteric artery ( sma ) was occluded for 30 minutes followed by defined times of reperfusion ; ( 2 ) the noggin+ i / r group , in which an intraperitoneal injection of 4 g / kg noggin was given 30 min before i / r , followed by ischemia , reperfusion , and sacrifice of the rats at defined times ; and ( 3 ) the sham group , which included animals subjected to anesthesia and laparotomy without ischemia . for the current study
after fixation in 4% formaldehyde for 20 min , the sections were incubated in 3% h2o2 for 30 min to quench endogenous peroxidases . then , the iec-6 cells were incubated overnight at 4c in 5% bsa in pbs with anti - p65 at a 1 : 50 dilution . total cellular rna was isolated with trizol reagent and used for first strand cdna synthesis with the reverse transcription system . pcr conditions were one cycle of 94c for 2 min followed by 50 cycles of 94c for 10 s , a specified annealing temperature for 15 s and 72c for 15 s. amplification was followed by melting curve analysis , which used the following program : one cycle at 65c for 1 s , 94c for 2 s , and 37c for 5 s. the housekeeping gene -actin was used as an endogenous reference gene to which the expression of the other genes was normalized using the comparative cycle of threshold value . we found that the expression level of bmp2 and bmp4 was upregulated 2.5-fold ( figure 1(a ) ) and 3.1-fold ( figure 1(b ) ) , respectively , in iec-6 cells after 6 h of hypoxia . meanwhile , we detected the expression of bmp2 and bmp4 in intestinal epithelial cells in an i / r rat model . if analysis showed that these proteins were also significantly increased along the crypt / villus axis after 1 h of i / r , consistent with the significantly increased bmp2 and bmp4 levels in intestinal epithelial cells under hypoxia . normally , bmp2 and bmp4 are expressed in both the epithelial and mesenchymal compartments , but bmp4 is highly expressed and enriched in the mesenchyme [ 13 , 16 ] . in the present study ,
the bmp2 level significantly increased in the mid - to - distal villus region after 1 h of i / r , while the bmp4 level increased significantly in both the villi and mesenchyme in the i / r rat ( figure 1(c ) ) . the main bmp receptors include the type ii bmp receptor ( bmprii ) and the following type i receptors : the bmpri group ( bmpria and bmprib ; also denoted as alk-3 and alk-6 , resp . ) to investigate
whether the greater abundance of bmp2/4 led to an increase in intracellular bmp signaling , we evaluated the expression of bmprii and bmpr - ia in epithelial cells under hypoxia and i / r . we also detected the expression of bmp receptors in the rat i / r model . sections of the small intestine were collected to detect changes in bmpria and bmprii expression via immunofluorescence analysis . immunofluorescence staining showed that the expression levels of the transmembrane receptors bmpria and bmprii were significantly increased in the villi but had lower expression levels in the matrix ( figure 2(c ) ) . we used if to detect the expression of nf-b after stimulation by exogenous bmp2/4 for 30 min . meanwhile , the in vivo results showed that nf-b signaling was obviously activated in intestinal epithelial cells after 1 h of i / r treatment . in contrast , after intraperitoneal injection of 4 g / kg noggin 30 min before i / r , the expression of nf-b was significantly inhibited ( figure 3(c ) ) . because mitogen - activated protein kinase ( mapk ) is known as an upstream regulator of nf-b , we evaluated the three common proteins of the mapk pathway , erk , p38 , and jnk . these results are consistent with the results from s. o. kim and m. r. kim , who found that treatment with an erk - specific blocking agent completely inhibited nf-b activity . to test whether the activation of nf-b induced by bmp2 and bmp4 resulted in an increase in inflammatory cytokines , we used rt - pcr to detect the expression of tnf- mrna and il-6 mrna in iec-6 cells after treatment with bmp2 and bmp4 for 3 h. treatment of iec-6 cells with 100 ng / ml bmp2 caused the level of tnf- mrna to increase 6.3-fold compared to the control group ( figure 4(a ) ) , while the effect of bmp4 in inducing the expression of tnf- mrna was weaker ( figure 4(b ) ) . tumor necrosis factor is one of the most powerful inducers and promoters of inflammation , and nf-b both is activated by cytokines and induces the expression of inflammatory cytokines . bmp2 and bmp4 both increased the expression of il-6 mrna , and these effects were decreased by noggin ( figures 4(c ) and 4(d ) ) . the activation of nf-b by ifn- enhances the permeability of t84 cells and decreases the levels of intercellular tight junction proteins , whereas the inhibition of nf-b will block the increase in t84tj permeability and alter occludin expression [ 8 , 19 ] . we asked whether bmp2/4 would disrupt the intestinal mucosal barrier function via the activation of nf-b . we used recombinant bmp2 and bmp4 to stimulate intestinal epithelial cells for 24 h , and the tight junction protein occludin was detected by western blotting . the expression of occludin decreased after treatment with bmp2 and bmp4 , while this effect was abolished by noggin or the nf-b inhibitor pdtc ( figure 5(a ) ) . these results may indicate that bmp2 and bmp4 disrupt the integrity of the intestinal mucosal barrier via the activation of nf-b and that pdtc can reverse the decrease in tj proteins . have shown that pi3-k / akt activation was required for the activation of nf-b pathways in the modulation of the tj barrier by treatment with ifn- . we also detected the expression of akt at the indicated times after stimulation of the iec-6 cells with bmp2 and bmp4 . previous research has focused on the roles of the bmp pathway in early intestinal development and in the proliferation and differentiation of intestinal epithelial cells [ 2022 ] . however , the relationship between the bmp pathway and intestinal mucosal barrier dysfunction caused by i / r has rarely been examined . found that , in rats after acetabular surgery , treatment with 4 mg / ml bmp2 protein significantly induced local inflammation , including an early and pronounced polymorphonuclear cell infiltration accompanied by the increased expression of tnf- and il-6 . intestinal i / r results in the release of abundant inflammatory factors , the generation of oxygen free radicals ( ros ) , and the activation of nf-b . these factors lead to enhanced permeability in the intestinal barrier and systemic inflammatory reactions [ 24 , 25 ] . because of the critical role of bmp in inflammatory reactions
, we hypothesized that the bmp signaling pathway would contribute to the mechanisms involved in promoting i / r - associated intestinal mucosal barrier injury . our present study shows that , with hypoxia and i / r , intestinal epithelial cells produce abundant bmp2 and bmp4 and that bmpria and bmprii expressions are also enhanced . bmp2/4 is able to directly increase the expression of the cytokines tnf- and il-6 and decrease the expression of the tight junction protein occludin by activating nf-b signaling . this effect was attenuated in part by either the bmp - specific antagonist noggin or the nf-b inhibitor pdtc . our study may provide new evidence that the activation of nf-b in intestinal i / r injury involves the bmp signaling pathway . it is interesting to note that bmp signaling is complex ; different bmp subgroups can mediate antagonistic effects , or the same ligand can produce different effects in similar tissues . for example , in an in vivo model , bmp4 inhibits liver proliferation , and the bmp4 antagonist noggin enhances regeneration , whereas another report demonstrates that bmp7 is an endocrine factor expressed in the kidney that enhances liver regeneration . our findings demonstrate that hypoxia and i / r increased intestinal epithelial cell bmp2/4 signaling , thereby activating nf-b signaling . these changes led to increased expression of inflammatory factors , such as tnf- and il-6 , and decreased expression of the tight junction protein occludin , which could result in disruption of the intestinal barrier . furthermore , inhibition of bmp activity protects epithelial barrier function in cases of lung injury . however , our results contrast with another report that bmp7 has protective and anti - inflammatory functions in acute ischemic renal injury [ 30 , 31 ] . additionally , bmp7 administration before intestinal i / r injury protects against intestinal mucosal injury and liver injury , preserves intestinal function , and prevents intestinal inflammation . alternative signal transduction mechanisms may also play a role : the canonical bmp pathway regulates gene expression via the smad - dependent pathway , while the noncanonical bmp pathway regulates nf-b via the mek / erk pathway . our studies have demonstrated that phosphorylated erk1/2 and akt expression levels progressively increased in intestinal epithelial cells upon treatment with bmp2/4 . thus , we believe that nf-b may be activated by bmp2 and bmp4 via the mek / erk pathway in the hypoxia and intestinal i / r model . previous studies have shown that nf-b is activated in the process of intestinal i / r injury and that the amount of activated nf-b correlates with the degree of mucosal inflammation [ 34 , 35 ] . the activation of nf-b could regulate the expression of several inflammatory genes , such as cell factors , adhesion molecules , and enzymes . in turn , the released inflammatory factors ( e.g. , tnf- and il-6 ) can activate nf-b , thereby aggravating the deterioration of intestinal barrier function . additionally , the upregulated expression of tnf- and il-6 induced in intestinal epithelial cells by bmp2 and bmp4 treatment confirms that bmp2 and bmp4 can mediate the intestinal mucosal injury caused by i / r via the activation of nf-b . moreover , bmp2 and bmp4 have been shown to activate erk1/2 , while the activation of the erk pathway reduces the proliferation of intestinal epithelial cells ; thus , persistent bmp expression is unfavorable for the repair of intestinal mucosal injury . in addition to the amount of proinflammatory cytokines released by i / r , ischemia and hypoxia also disrupt the integrity of the intestinal mucosal barrier . we asked whether bmp2 and bmp4 could decrease the expression of tight junction proteins , and we evaluated changes in the expression of the tight junction protein occludin . our results show that the expression of occludin decreased after treatment with bmp2 and bmp4 , while these effects were abolished by treatment with noggin or pdtc . , who used nf-b blockers to prevent the ifn-induced increase in epithelial permeability and the destruction of tight junction proteins . our previous study also found that ifn- induced intestinal barrier function injury via the nf-b / hif-1a pathway . there are many types of extracellular bmp antagonists , such as noggin , chordin , cerberus , and follistatin [ 36 , 37 ] . by binding bmp
, these secretory factors act as competitive antagonists and are important negative regulatory factors in the bmp pathway . in mouse intestine ,
transient expression of the bmp antagonist noggin has been observed in both pericryptal mesenchymal cells and intestinal epithelial stem cells , which may contribute to maintaining intestinal stem cell self - renewal by activating wnt signaling and inhibiting bmp signaling of the basal crypt epithelial cells . our data indicate that bmp2/4 expression increased in both an early intestinal epithelial cell hypoxia model and a rat i / r model . furthermore , nf-b was directly activated by recombinant bmp2/4 in iec-6 cells by western blotting and immunofluorescence ( if ) . finally , bmp2/4 induced the expression of intracellular tnf- mrna and il-6 mrna in iec-6 cells , and this effect was abolished by the bmp - specific antagonist noggin . the in vivo rat model also showed that , with an intraperitoneal injection of noggin before i / r , the activation of nf-b was obviously inhibited . thus , in future investigations , it will be important to examine the expression of a number of bmp antagonists ( noggin , chordin , gremlin , and follistatin ) in a rat i / r model . recent studies have found that bmp expression is modulated by hif-1 expression , bmp2 expression was increased in primary chondrocytes under hypoxic conditions , and addition of the hif-1 activator dfo significantly increased bmp2 expression . additionally , hypoxia stimulation increased bmp2 mrna and protein expression levels in osteoblasts via an hif-1-alpha - dependent mechanism involving the activation of the ilk / akt and mtor pathways . further work is required to better understand the mechanisms guiding the increased bmp expression in the hypoxia and intestinal i / r model . to the best of our knowledge ,
the present study is the first to report that bmp2 and bmp4 can directly activate nf-b , induce the expression of the inflammatory cytokines tnf - a and il-6 in the intestinal epithelial cells , and decrease the expression of the tight junction protein occludin , which could result in disruption of the intestinal barrier . all of these effects may contribute to the mechanism by which bmp2 and bmp4 mediate intestinal mucosa injury in ischemic reperfusion . | [
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individual patients and controls who contributed these dna samples provided written informed consent via representatives from the coriell institute , university of michigan als patient biorepository , or individually to a member of the research group .
dna samples for repeat - primed pcr and genomic pcr were from the following sources : 1 g of genomic dna from 199 patients with als and 136 healthy controls from the university of michigan als patient biorepository . patients with als met the revised el escorial criteria and were recruited from the university of michigan als clinic ; controls were recruited via the university of michigan clinical trials web site ( https://umclinicalstudies.org/ ) .
two hundred fifty nanograms of genomic dna from 86 patients with sporadic als from coriell cell repository panel # ndpt026 ( coriell institute ) was used for determination of c9orf72 repeat status only .
one hundred micrograms of genomic dna from a patient with als4 and 1 non - als sibling from a previously reported large pedigree was extracted from 4 ml of whole blood using a commercial dna isolation kit ( dneasy blood & tissue kit ; qiagen , netherlands ) .
genomic dna from a second published als4 case with a family history was extracted from patient fibroblast cells obtained from a collaborator 's laboratory using the same kit .
we performed a blast search ( national center for biotechnology information ) against the human genome for g4c2 repeat sequences using a sequence of 5 g4c2 pure repeats ( ggggccggggccggggccggggccggggcc ) as a start query .
the identified repeat loci were overlaid with published genetic loci associated with als or ftd ( see a recent review and table 1 ) .
we constrained our analysis to repeat loci within 2 mega base pairs ( mb ) of either the mapped critical region for an als or ftd candidate gene / locus or with single - nucleotide polymorphisms ( snps ) that achieved statistical significance on gwas in sporadic als cohorts .
additional candidate repeats located more than 2 mb outside of disease - associated loci were identified by requiring at least 3 pure repeats in a gene with abundant neuronal expression in brain based on biogps and proteomic db database analysis .
three additional candidate genes with g4c2 repeats within the previously identified critical region of als4 but missed by our initial in silico analysis were added after we obtained access to case samples . g4c2 repeat loci evaluated in this study g4c2 repeat numbers in the longer allele were determined by repeat - primed pcr as previously reported , followed by capillary electrophoresis and fragment analysis .
the individual reverse primers for each candidate gene were designed using primer 3 tools ( http://biotools.umassmed.edu/bioapps/primer3_www.cgi ) and were labeled with either 6-fam or 5-hex fluorescent dye .
the pcr products were diluted in highly deionized formamide ( hidi formamide ; thermofisher scientific , waltham , wa ) containing size standard rox-1000 ( bioventure , tn ) .
fragment analysis was performed at the university of michigan sequencing core facility on an abi 3730 sequencer .
the data were analyzed using genemarker ( softgenetics , pa ) to determine the maximal repeat number . for samples with fewer than 35 g4c2 repeats in c9orf72 , dna flanking
the repeat regions was pcr - amplified with the following primers : c9orf72 forward : 5-ccg cag cct gta gca agc-3 and c9orf72 reverse : 5-agt cgc tag agg cga aag c-3 using the same thermal cycling program as the repeat - primed pcr .
the pcr products were gel purified and subjected to sanger sequencing at the university of michigan sequencing core facility to determine the exact repeat number . for samples with a 31-nucleotide insertion in the vav2 gene , dna flanking the repeat region was pcr - amplified with the following primers : vav2 forward : 5-gcc cag gac agg agg cct cag ca-3 and vav2 reverse : 5- ctc agg gcc ggg agg aag cac ct-3 using the same thermal cycling program as the repeat - primed pcr .
the pcr products were gel - purified and subjected to sanger sequencing as described above . for repeats at huwe1 and rgs14 ,
pcr primers flanking the repeat regions were used to determine specific repeat sizes ( see supplemental data ) . for southern blot analysis , 25-g aliquots of lymphoblast genomic dna
two additional lymphoblast cell lines were obtained from the coriell institute and were grown up to 15 million cells , and approximately 100 g of lymphoblast genomic dna was extracted using a dna isolation kit .
ten fibroblast cell lines with g4c2 repeat expansion and 2 control fibroblast lines ( 1 als sample without expansion and 1 control ) were obtained from the university of michigan als patient biorepository and 100 g of fibroblast genomic dna was extracted from 15 million cells .
two - tailed fisher exact tests or tests were performed to test for association of repeat length with als and to verify genotype frequencies in the vav2 gene . for rgs14 repeat length ,
a 2-tailed nonparametric t test was performed to compare the median repeat size differences . for the correlation of c9orf72 repeat size and patient age at onset of als ,
individual patients and controls who contributed these dna samples provided written informed consent via representatives from the coriell institute , university of michigan als patient biorepository , or individually to a member of the research group .
dna samples for repeat - primed pcr and genomic pcr were from the following sources : 1 g of genomic dna from 199 patients with als and 136 healthy controls from the university of michigan als patient biorepository . patients with als met the revised el escorial criteria and were recruited from the university of michigan als clinic ; controls were recruited via the university of michigan clinical trials web site ( https://umclinicalstudies.org/ ) .
two hundred fifty nanograms of genomic dna from 86 patients with sporadic als from coriell cell repository panel # ndpt026 ( coriell institute ) was used for determination of c9orf72 repeat status only .
one hundred micrograms of genomic dna from a patient with als4 and 1 non - als sibling from a previously reported large pedigree was extracted from 4 ml of whole blood using a commercial dna isolation kit ( dneasy blood & tissue kit ; qiagen , netherlands ) .
genomic dna from a second published als4 case with a family history was extracted from patient fibroblast cells obtained from a collaborator 's laboratory using the same kit .
we performed a blast search ( national center for biotechnology information ) against the human genome for g4c2 repeat sequences using a sequence of 5 g4c2 pure repeats ( ggggccggggccggggccggggccggggcc ) as a start query .
the identified repeat loci were overlaid with published genetic loci associated with als or ftd ( see a recent review and table 1 ) .
we constrained our analysis to repeat loci within 2 mega base pairs ( mb ) of either the mapped critical region for an als or ftd candidate gene / locus or with single - nucleotide polymorphisms ( snps ) that achieved statistical significance on gwas in sporadic als cohorts .
additional candidate repeats located more than 2 mb outside of disease - associated loci were identified by requiring at least 3 pure repeats in a gene with abundant neuronal expression in brain based on biogps and proteomic db database analysis .
three additional candidate genes with g4c2 repeats within the previously identified critical region of als4 but missed by our initial in silico analysis were added after we obtained access to case samples . g4c2
g4c2 repeat numbers in the longer allele were determined by repeat - primed pcr as previously reported , followed by capillary electrophoresis and fragment analysis .
the individual reverse primers for each candidate gene were designed using primer 3 tools ( http://biotools.umassmed.edu/bioapps/primer3_www.cgi ) and were labeled with either 6-fam or 5-hex fluorescent dye .
the pcr products were diluted in highly deionized formamide ( hidi formamide ; thermofisher scientific , waltham , wa ) containing size standard rox-1000 ( bioventure , tn ) .
fragment analysis was performed at the university of michigan sequencing core facility on an abi 3730 sequencer .
the data were analyzed using genemarker ( softgenetics , pa ) to determine the maximal repeat number .
for samples with fewer than 35 g4c2 repeats in c9orf72 , dna flanking the repeat regions was pcr - amplified with the following primers : c9orf72 forward : 5-ccg cag cct gta gca agc-3 and c9orf72 reverse : 5-agt cgc tag agg cga aag c-3 using the same thermal cycling program as the repeat - primed pcr .
the pcr products were gel purified and subjected to sanger sequencing at the university of michigan sequencing core facility to determine the exact repeat number . for samples with a 31-nucleotide insertion in the vav2 gene , dna flanking the repeat region was pcr - amplified with the following primers : vav2 forward : 5-gcc cag gac agg agg cct cag ca-3 and vav2 reverse : 5- ctc agg gcc ggg agg aag cac ct-3 using the same thermal cycling program as the repeat - primed pcr .
the pcr products were gel - purified and subjected to sanger sequencing as described above . for repeats at huwe1 and rgs14 , pcr primers flanking the repeat regions were used to determine specific repeat sizes ( see supplemental data ) .
for southern blot analysis , 25-g aliquots of lymphoblast genomic dna were obtained for 5 selected coriell samples .
two additional lymphoblast cell lines were obtained from the coriell institute and were grown up to 15 million cells , and approximately 100 g of lymphoblast genomic dna was extracted using a dna isolation kit .
ten fibroblast cell lines with g4c2 repeat expansion and 2 control fibroblast lines ( 1 als sample without expansion and 1 control ) were obtained from the university of michigan als patient biorepository and 100 g of fibroblast genomic dna was extracted from 15 million cells .
two - tailed fisher exact tests or tests were performed to test for association of repeat length with als and to verify genotype frequencies in the vav2 gene . for rgs14 repeat length ,
a 2-tailed nonparametric t test was performed to compare the median repeat size differences . for the correlation of c9orf72 repeat size and patient age at onset of als ,
we first conducted an in silico experiment to identify all human genes containing at least 2 g4c2 repeats .
our rationale was that transcribed repeats at such loci could become unstable and lead to disease - causing expansions , unless the chromosomal context of the c9orf72 repeat was critical to disease pathogenesis .
our initial analysis revealed that short g4c2 repeats are quite common in the human genome , with 344 identified as intragenic repeats . to narrow our window of potential sites of repeat expansion to a number that we could readily assay , we compared this in silico list of repeat loci with genetic loci implicated in als or ftd , either through critical region mapping in familial forms or by gwas .
we reasoned that utilization of gwas conducted in patient populations similar to our cohort as well as known genetic loci in rare families ( which have subsequently been shown to contribute to a percentage of sporadic cases ) would identify most repeat expansions that confer a substantial contribution to the genetic burden of als in the united states .
consistent with this idea , the c9orf72 locus was identified on multiple gwas surveys in als and ftd in sporadic populations prior to identification of the causative repeat . using this approach
, we identified 21 candidate genes , including c9orf72 , containing at least 2 g4c2 repeats that are located within 2 mb of known als - ftd loci or loci identified in gwas by at least 2 associated snps ( figure 1a ) .
( b h ) graphs show the distribution of repeat sizes from als cases and controls for 7 genes . *
significant ( p < 0.05 ) difference in the distribution of repeats larger than 33 times between cases and controls by fisher exact test .
we developed a modified fluorescent fragment analysis after repeat - primed pcr to evaluate instability at each of these loci , as previously described for c9orf72 repeat expansions .
these assays were tested on control samples , and the actual repeat sizes were confirmed by standard pcr and sanger sequencing . all 21 assays demonstrated accurate detection of repeat size , based on comparison with sanger sequencing ( data not shown ) .
this assay readily detected g4c2 expansions at c9orf72 in samples from als cases known to harbor the repeat .
we applied these assays to a sample collection from the university of michigan als patient repository containing 199 als cases , of which 17 had a family history of als , and 136 healthy controls ( university of michigan ) .
of note , there were no differences in the age at sample collection , sex composition , or racial / ethnic makeup between als cases and controls , and both cases and controls had similar frequencies of a family history of a non - als neurodegenerative condition ( table e-1 ) .
in addition , we used samples from coriell cell repository panel ndpt026 , containing 86 sporadic als cases , for additional screening for repeat expansions in c9orf72 ( coriell institute ) .
consistent with published findings , we observed a wide variation of g4c2 repeat length at c9orf72 in both cases and controls ( table 1 , table e-3 , figure 1b ) .
pathogenic repeat expansions were observed in 47% of the familial cases and 8.8% ( 17/182 ) of sporadic als cases in the university of michigan als patient repository ( figure 1b and table e-3 ) . c9orf72
repeat expansions were observed at a similar frequency ( 6/86 ; 7.0% ) in als samples from the coriell institute . to validate these findings and determine the actual repeat expansion size in cases with low repeat numbers , genomic
one sample contained exactly 32 repeats in the longer allele , which was confirmed by sanger sequencing .
other samples exhibited a single band by genomic pcr reflecting the shorter , pcr - amplifiable allele .
southern blotting on genomic dna extracted from patient - derived fibroblasts or lymphocytes in a subset of cases demonstrated a wide range of pathologic repeat expansion sizes from 200 to more than 1,500 repeats ( figure e-1 ) .
repeat expansion size from peripheral blood samples or cultured cells did not correlate with clinical age at onset ( r = 0.173 in pearson correlation test , p = 0.572 , figure e-2 ) , consistent with previous reports .
in contrast to c9orf72 , g4c2 repeats assayed at other loci exhibited little or no variance in repeat number in the university of michigan als patient biorepository samples .
of the 20 novel repeats analyzed , 16 showed no variance at all across the cohort ( 80% ) .
four genes displayed small variations in repeat number ( table 1 ) , but none of these cases contained expansions of more than 2 repeats greater than the prevalent alleles .
the degree of repeat length variation was not significantly different between control and als groups , demonstrating a lack of association between repeat number variation and als / ftd in our cohort ( figure 1 , b
e , p < 0.001 for c9orf72 , p = 0.76 for gpr123 , p = 0.69 for huwe1 , p = 0.374 for itpr3 ; test for c9orf72 and itpr3 , and fisher exact test for gpr123 and huwe1 used for the rest for different allele frequencies ) . to expand the initial screening we selected 4 additional candidate genes that are highly expressed in brain and contain at least 3 g4c2 repeats in controls . in these candidate genes , no large repeat expansions were found in als or control groups ( table 1 , figure 1 , f and g ) .
however , an intronic repeat in rgs14 displayed a much larger variation than those in other loci studied , with between 4 and 15 repeats present in both controls and als cases .
the distribution of repeat lengths in rgs14 was independent of clinical als ( figure 1f ) or c9orf72 expansion status ( control 8.49 1.74 vs als 8.1 1.93 , p = 0.082 , 2-tailed student t test ) .
as a third approach , we examined additional loci harboring g4c2 repeats located near the als4 locus in patients with this disorder .
als4 is a form of juvenile als with very slow progression that is thought to result from dominant mutations in setx .
the mechanism of these dominant mutations is unclear , because recessive mutations in setx cause a distinct neurodegenerative disorder , ataxia with oculomotor apraxia type 2 , which does not include motor neuron degeneration . within the critical region defined by linkage analysis , there are 4 genes containing short g4c2 repeats : prrx2 , pccr2b , surf4 , and vav2 ( figure 2a )
. to investigate whether a cryptic repeat expansion might provide an alternative explanation for the als4 phenotype , we analyzed repeat numbers in 2 patients with als4 and an unaffected control from 2 previously described als4 families .
only 1 ( prrc2b ) was included in our initial repeat screen in the university of michigan als patient repository .
assessment of the additional 3 loci in this larger cohort identified no repeat expansions , only a minor repeat length variance in gene prrx2 ( table 1 , figure 1h ) .
we did observe an amplicon length variant of 31 bp in vav2 by fragment analysis in the als4 family ( figure 2b ) .
sanger sequencing identified it as a 31-bp intronic insertion in the longer allele that contains an imperfect duplication of intronic sequence ( gccggggccgtgtggccctcacgcagtgacc ) .
this insertion was common in controls and als cases in the university of michigan als patient biorepository panel ( figure 2c ) , and appears to be nonpathogenic ( p = 0.398 , test ) .
( a ) schematic of the als4 locus defined by chromosome markers d9s1831 and d9s164 ( black boxes ) drawn to scale showing the location of 4 g4c2 repeat - containing genes ( red boxes ) and the proposed disease - causing gene setx ( blue box ) .
( b ) chromatograms showing the g4c2 repeat peaks in the 4 g4c2 repeat - containing genes from an affected als4 case and a control sibling .
one control had a 31-bp insertion in the vav2 gene ( double peak marked with an arrow ) .
( c ) genotypes of controls and als cases in the university of michigan als patient biorepository for the 31-bp insertion in gene vav2 .
als = amyotrophic lateral sclerosis ; cen = centromere ; g4c2 = ggggcc ; nt = nucleotide ; ter = telomere ; wt = wild type .
g4c2 repeat expansions in c9orf72 are the most common known cause of als and ftd . here
we tested whether g4c2 repeat instability at other loci could be a common cause of als .
our results confirmed that g4c2 repeat expansions and instability are common at the c9orf72 locus in both familial and sporadic als cases in 2 patient cohorts ( university of michigan als patient repository and the ndpt026 als collection from coriell ) .
however , for 27 other genomic loci that harbor g4c2 repeats , no pathogenic g4c2 expansions were identified .
thus , repeat instability at these alternative loci are unlikely to be major contributors to the genetic burden in als in the united states .
this study is not exhaustive in the number of repeat loci assayed or the number of patient and familial cohorts examined .
therefore , we can not rule out g4c2 repeat expansions as rare causes of als . for practical reasons , we assayed fewer than 10% of the g4c2 repeats located near protein - coding genes and a much smaller fraction of the gc - rich repeats in the human genome . specifically , we focused our analysis on repeats located adjacent to loci already implicated in als or ftd .
our rationale for this choice was that utilization of gwas conducted in similar patient populations as well as known genetic loci in rare families ( which have subsequently been shown to contribute to a percentage of sporadic cases ) would colocalize with repeat expansions that confer a substantial contribution to the genetic burden of als in the united states .
consistent with this idea , the c9orf72 locus was identified on multiple gwas surveys in als and ftd in sporadic populations prior to identification of the causative repeat .
moreover , we reasoned that cryptic repeat expansions might have escaped identification during the initial analyses of these loci , several of which lack an identified causative gene or a clear pathogenic mechanism for the currently implicated gene .
as such , our findings that none of these alternative loci harbor repeat expansions in 199 als cases suggest that g4c2 repeat expansions at non - c9orf72 loci are unlikely to be a common cause of als in our patient population , given that most common highly penetrant mutations would be detected by this approach .
however , it is important to acknowledge that for highly penetrant loci identified in familial als cases , it might be necessary to examine the original published cohorts , which might carry rare or private mutations .
we took this step for als4 and found no g4c2 repeat instability at 4 neighboring loci .
application of this approach to other isolated pedigrees could more definitively rule out expansions at these genetically implicated loci .
the lack of g4c2 repeat expansions at alternative gene loci suggests that there could be something about the c9orf72 locus that promotes instability . the vast majority ( 75% ) of the other hexanucleotide repeats studied here exhibited no variation among 335 individuals , and variance in repeat size was largely absent from the 1,000-genome database for the 321 intragenic g4c2 repeat loci not assayed ( data not shown ) . with one exception , the repeat size variants observed were limited to 1 or 2 repeats .
studies in vitro suggest that stretches of g4c2 as short as 4 repeats are capable of forming intramolecular g quadruplexes , which might contribute to repeat instability through activation of dna mismatch repair pathways and mistemplating . in general , our data are consistent with this threshold , since only genes with at least 4 repeats had variation of more than 1 repeat . however , some of the g4c2 repeats we studied were larger than this threshold but were invariant , suggesting that the length of the repeat is insufficient to explain instability .
while future work will be needed to determine which factors drive selective instability , it may reflect specific aspects of the gc - rich sequence surrounding the repeat at the c9orf72 loci .
this concept is supported by the finding that all pathologic repeat expansions at c9orf72 observed to date have occurred on a single haplotype background .
however , g4c2 repeat expansions in genes other than c9orf72 may be enriched in neurologic disorders other than als .
phenotypes of polyglutamine disorders associated with cag repeat expansions , for example , are not all the same . in this context
, it is interesting to note that the c9orf72 repeat expansion exhibits neurologic symptoms in addition to motor neuron disease and frontotemporal cognitive dysfunction , which are still being explored .
we observed noteworthy repeat instability in both cases and controls in one other gene : a g4c2 intronic repeat in rgs14 with a mean repeat length of 9 and a range of 414 repeats .
rgs14 encodes a member of the regulator of g - protein signaling family of proteins .
it is highly expressed in the developing brain and has known functions in hippocampal learning and synaptic plasticity .
although we observed no association between the size of this repeat and clinical als , our study can not rule out a role for expansion in rare als families or other racial / ethnic populations .
as such , reevaluation of this locus may be worthy of further study in familial als , ftd , and other neurodegenerative disorders .
other repeat expansions are implicated in causing or modifying motor neuron disease phenotypes . a ggcctg repeat expansion in nop56 causes
moderate cag repeat expansions ( encoding polyglutamine ) in atxn2 and gcg repeat expansions ( encoding polyalanine ) in nip1a are associated with an increased risk of als . however , other known pathogenic cag and cgg repeat expansions do not influence the risk of als or ftd , suggesting that the observed effects may be gene specific rather than repeat sequence specific .
we screened 28 loci harboring g4c2 repeats near known als / ftd loci in 199 patients with als and did not find pathogenic repeat expansions other than that previously reported in c9orf72 .
while future studies in larger cohorts and select families are still needed , our results suggest that g4c2 repeat expansions outside the c9orf72 gene are not a common cause of als , supporting the importance of identifying cis elements that explain the genetic instability at the c9orf72 locus .
grants from the centers for disease control and prevention ( 200 - 2013 - 56856 ) and the a. alfred taubman medical research institute to e.l.f . ; and grants from the michigan alzheimer 's disease center at the university of michigan , nih r01ns08681001 , and the department of veterans affairs blrd 1i21bx001841 to p.k.t .
the funders played no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript .
figueroa - romero has received research support from the katherine rayner fund and the a. alfred taubman medical research institute .
feldman receives funding support from novo nordisk , the nih , the juvenile diabetes research foundation , the us - israel binational science foundation , the american diabetes association , and the a. alfred taubman medical research institute .
goutman receives research funding support from neuralstem , inc ; the centers for disease control and prevention / agency for toxic substances and disease registry ; and the agency for toxic substances and disease registry ( contract # 200 - 2013 - 56856 ) .
callaghan receives funding support from impeto medical inc and the nih , has received travel / speaker honoraria from the american academy of neurology and the world federation of neurology , performs medical consultations for advance medical , consults for a pcori grant , has received honoraria from the british medical journal , has worked with the als association , and has acted as a consultant for medical legal cases .
dr . todd receives funding support from the nih , the veterans administration , the national ataxia foundation , the national fragile x foundation , and the muscular dystrophy association ; serves as a paid consultant for denali therapeutics and for an nih grant ; serves as an unpaid consultant for the national fragile x foundation ; has received travel / speaker honoraria from the american academy of neurology , the national fragile x foundation , and the japanese society of neurochemistry ; has received publishing royalties from uptodate ; and has received license fee payments for antibodies developed by his group from millipore for distribution to scientists and commercial groups , and for vectors and antibodies developed by his group from denali therapeutics . | objective : to determine whether ggggcc ( g4c2 ) repeat expansions at loci other than c9orf72 serve as common causes of amyotrophic lateral sclerosis ( als).methods : we assessed g4c2 repeat number in 28 genes near known als and frontotemporal dementia ( ftd ) loci by repeat - primed pcr coupled with fluorescent fragment analysis in 199 patients with als ( 17 familial , 182 sporadic ) and 136 healthy controls .
we also obtained blood from patients with als4 for evaluation of repeats surrounding the setx gene locus .
c9orf72 expansions were evaluated in parallel.results:expansions of g4c2 repeats in c9orf72 explained 8.8% of sporadic and 47% of familial als cases analyzed .
repeat variance was observed at one other locus , rgs14 , but no large expansions were observed , and repeat sizes were not different between cases and controls .
no g4c2 repeat expansions were identified at other als or ftd risk loci or in als4 cases.conclusions:g4c2 expansions near known als and ftd loci other than c9orf72 are not a common cause of als . | METHODS
Standard protocol approvals, registrations, and patient consents.
ALS and control patient cohorts and DNA isolation.
Candidate gene selection.
G
Sanger sequencing.
Southern blot confirmation of repeat expansions at
Statistical analysis.
RESULTS
DISCUSSION
Supplementary Material
AUTHOR CONTRIBUTIONS
STUDY FUNDING
DISCLOSURE | dna samples for repeat - primed pcr and genomic pcr were from the following sources : 1 g of genomic dna from 199 patients with als and 136 healthy controls from the university of michigan als patient biorepository . patients with als met the revised el escorial criteria and were recruited from the university of michigan als clinic ; controls were recruited via the university of michigan clinical trials web site ( https://umclinicalstudies.org/ ) . one hundred micrograms of genomic dna from a patient with als4 and 1 non - als sibling from a previously reported large pedigree was extracted from 4 ml of whole blood using a commercial dna isolation kit ( dneasy blood & tissue kit ; qiagen , netherlands ) . the identified repeat loci were overlaid with published genetic loci associated with als or ftd ( see a recent review and table 1 ) . we constrained our analysis to repeat loci within 2 mega base pairs ( mb ) of either the mapped critical region for an als or ftd candidate gene / locus or with single - nucleotide polymorphisms ( snps ) that achieved statistical significance on gwas in sporadic als cohorts . additional candidate repeats located more than 2 mb outside of disease - associated loci were identified by requiring at least 3 pure repeats in a gene with abundant neuronal expression in brain based on biogps and proteomic db database analysis . g4c2 repeat loci evaluated in this study g4c2 repeat numbers in the longer allele were determined by repeat - primed pcr as previously reported , followed by capillary electrophoresis and fragment analysis . the data were analyzed using genemarker ( softgenetics , pa ) to determine the maximal repeat number . for samples with fewer than 35 g4c2 repeats in c9orf72 , dna flanking
the repeat regions was pcr - amplified with the following primers : c9orf72 forward : 5-ccg cag cct gta gca agc-3 and c9orf72 reverse : 5-agt cgc tag agg cga aag c-3 using the same thermal cycling program as the repeat - primed pcr . the pcr products were gel purified and subjected to sanger sequencing at the university of michigan sequencing core facility to determine the exact repeat number . for samples with a 31-nucleotide insertion in the vav2 gene , dna flanking the repeat region was pcr - amplified with the following primers : vav2 forward : 5-gcc cag gac agg agg cct cag ca-3 and vav2 reverse : 5- ctc agg gcc ggg agg aag cac ct-3 using the same thermal cycling program as the repeat - primed pcr . for repeats at huwe1 and rgs14 ,
pcr primers flanking the repeat regions were used to determine specific repeat sizes ( see supplemental data ) . for southern blot analysis , 25-g aliquots of lymphoblast genomic dna
two additional lymphoblast cell lines were obtained from the coriell institute and were grown up to 15 million cells , and approximately 100 g of lymphoblast genomic dna was extracted using a dna isolation kit . two - tailed fisher exact tests or tests were performed to test for association of repeat length with als and to verify genotype frequencies in the vav2 gene . for the correlation of c9orf72 repeat size and patient age at onset of als ,
individual patients and controls who contributed these dna samples provided written informed consent via representatives from the coriell institute , university of michigan als patient biorepository , or individually to a member of the research group . dna samples for repeat - primed pcr and genomic pcr were from the following sources : 1 g of genomic dna from 199 patients with als and 136 healthy controls from the university of michigan als patient biorepository . patients with als met the revised el escorial criteria and were recruited from the university of michigan als clinic ; controls were recruited via the university of michigan clinical trials web site ( https://umclinicalstudies.org/ ) . we performed a blast search ( national center for biotechnology information ) against the human genome for g4c2 repeat sequences using a sequence of 5 g4c2 pure repeats ( ggggccggggccggggccggggccggggcc ) as a start query . the identified repeat loci were overlaid with published genetic loci associated with als or ftd ( see a recent review and table 1 ) . we constrained our analysis to repeat loci within 2 mega base pairs ( mb ) of either the mapped critical region for an als or ftd candidate gene / locus or with single - nucleotide polymorphisms ( snps ) that achieved statistical significance on gwas in sporadic als cohorts . additional candidate repeats located more than 2 mb outside of disease - associated loci were identified by requiring at least 3 pure repeats in a gene with abundant neuronal expression in brain based on biogps and proteomic db database analysis . g4c2
g4c2 repeat numbers in the longer allele were determined by repeat - primed pcr as previously reported , followed by capillary electrophoresis and fragment analysis . the individual reverse primers for each candidate gene were designed using primer 3 tools ( http://biotools.umassmed.edu/bioapps/primer3_www.cgi ) and were labeled with either 6-fam or 5-hex fluorescent dye . the data were analyzed using genemarker ( softgenetics , pa ) to determine the maximal repeat number . for samples with fewer than 35 g4c2 repeats in c9orf72 , dna flanking the repeat regions was pcr - amplified with the following primers : c9orf72 forward : 5-ccg cag cct gta gca agc-3 and c9orf72 reverse : 5-agt cgc tag agg cga aag c-3 using the same thermal cycling program as the repeat - primed pcr . the pcr products were gel purified and subjected to sanger sequencing at the university of michigan sequencing core facility to determine the exact repeat number . for samples with a 31-nucleotide insertion in the vav2 gene , dna flanking the repeat region was pcr - amplified with the following primers : vav2 forward : 5-gcc cag gac agg agg cct cag ca-3 and vav2 reverse : 5- ctc agg gcc ggg agg aag cac ct-3 using the same thermal cycling program as the repeat - primed pcr . for repeats at huwe1 and rgs14 , pcr primers flanking the repeat regions were used to determine specific repeat sizes ( see supplemental data ) . two additional lymphoblast cell lines were obtained from the coriell institute and were grown up to 15 million cells , and approximately 100 g of lymphoblast genomic dna was extracted using a dna isolation kit . two - tailed fisher exact tests or tests were performed to test for association of repeat length with als and to verify genotype frequencies in the vav2 gene . for the correlation of c9orf72 repeat size and patient age at onset of als ,
we first conducted an in silico experiment to identify all human genes containing at least 2 g4c2 repeats . to narrow our window of potential sites of repeat expansion to a number that we could readily assay , we compared this in silico list of repeat loci with genetic loci implicated in als or ftd , either through critical region mapping in familial forms or by gwas . we reasoned that utilization of gwas conducted in patient populations similar to our cohort as well as known genetic loci in rare families ( which have subsequently been shown to contribute to a percentage of sporadic cases ) would identify most repeat expansions that confer a substantial contribution to the genetic burden of als in the united states . consistent with this idea , the c9orf72 locus was identified on multiple gwas surveys in als and ftd in sporadic populations prior to identification of the causative repeat . using this approach
, we identified 21 candidate genes , including c9orf72 , containing at least 2 g4c2 repeats that are located within 2 mb of known als - ftd loci or loci identified in gwas by at least 2 associated snps ( figure 1a ) . ( b h ) graphs show the distribution of repeat sizes from als cases and controls for 7 genes . *
significant ( p < 0.05 ) difference in the distribution of repeats larger than 33 times between cases and controls by fisher exact test . we developed a modified fluorescent fragment analysis after repeat - primed pcr to evaluate instability at each of these loci , as previously described for c9orf72 repeat expansions . these assays were tested on control samples , and the actual repeat sizes were confirmed by standard pcr and sanger sequencing . this assay readily detected g4c2 expansions at c9orf72 in samples from als cases known to harbor the repeat . we applied these assays to a sample collection from the university of michigan als patient repository containing 199 als cases , of which 17 had a family history of als , and 136 healthy controls ( university of michigan ) . of note , there were no differences in the age at sample collection , sex composition , or racial / ethnic makeup between als cases and controls , and both cases and controls had similar frequencies of a family history of a non - als neurodegenerative condition ( table e-1 ) . in addition , we used samples from coriell cell repository panel ndpt026 , containing 86 sporadic als cases , for additional screening for repeat expansions in c9orf72 ( coriell institute ) . consistent with published findings , we observed a wide variation of g4c2 repeat length at c9orf72 in both cases and controls ( table 1 , table e-3 , figure 1b ) . pathogenic repeat expansions were observed in 47% of the familial cases and 8.8% ( 17/182 ) of sporadic als cases in the university of michigan als patient repository ( figure 1b and table e-3 ) . c9orf72
repeat expansions were observed at a similar frequency ( 6/86 ; 7.0% ) in als samples from the coriell institute . in contrast to c9orf72 , g4c2 repeats assayed at other loci exhibited little or no variance in repeat number in the university of michigan als patient biorepository samples . four genes displayed small variations in repeat number ( table 1 ) , but none of these cases contained expansions of more than 2 repeats greater than the prevalent alleles . the degree of repeat length variation was not significantly different between control and als groups , demonstrating a lack of association between repeat number variation and als / ftd in our cohort ( figure 1 , b
e , p < 0.001 for c9orf72 , p = 0.76 for gpr123 , p = 0.69 for huwe1 , p = 0.374 for itpr3 ; test for c9orf72 and itpr3 , and fisher exact test for gpr123 and huwe1 used for the rest for different allele frequencies ) . to expand the initial screening we selected 4 additional candidate genes that are highly expressed in brain and contain at least 3 g4c2 repeats in controls . in these candidate genes , no large repeat expansions were found in als or control groups ( table 1 , figure 1 , f and g ) . however , an intronic repeat in rgs14 displayed a much larger variation than those in other loci studied , with between 4 and 15 repeats present in both controls and als cases . the distribution of repeat lengths in rgs14 was independent of clinical als ( figure 1f ) or c9orf72 expansion status ( control 8.49 1.74 vs als 8.1 1.93 , p = 0.082 , 2-tailed student t test ) . as a third approach , we examined additional loci harboring g4c2 repeats located near the als4 locus in patients with this disorder . within the critical region defined by linkage analysis , there are 4 genes containing short g4c2 repeats : prrx2 , pccr2b , surf4 , and vav2 ( figure 2a )
. to investigate whether a cryptic repeat expansion might provide an alternative explanation for the als4 phenotype , we analyzed repeat numbers in 2 patients with als4 and an unaffected control from 2 previously described als4 families . we did observe an amplicon length variant of 31 bp in vav2 by fragment analysis in the als4 family ( figure 2b ) . this insertion was common in controls and als cases in the university of michigan als patient biorepository panel ( figure 2c ) , and appears to be nonpathogenic ( p = 0.398 , test ) . ( a ) schematic of the als4 locus defined by chromosome markers d9s1831 and d9s164 ( black boxes ) drawn to scale showing the location of 4 g4c2 repeat - containing genes ( red boxes ) and the proposed disease - causing gene setx ( blue box ) . ( b ) chromatograms showing the g4c2 repeat peaks in the 4 g4c2 repeat - containing genes from an affected als4 case and a control sibling . als = amyotrophic lateral sclerosis ; cen = centromere ; g4c2 = ggggcc ; nt = nucleotide ; ter = telomere ; wt = wild type . g4c2 repeat expansions in c9orf72 are the most common known cause of als and ftd . here
we tested whether g4c2 repeat instability at other loci could be a common cause of als . our results confirmed that g4c2 repeat expansions and instability are common at the c9orf72 locus in both familial and sporadic als cases in 2 patient cohorts ( university of michigan als patient repository and the ndpt026 als collection from coriell ) . however , for 27 other genomic loci that harbor g4c2 repeats , no pathogenic g4c2 expansions were identified . therefore , we can not rule out g4c2 repeat expansions as rare causes of als . for practical reasons , we assayed fewer than 10% of the g4c2 repeats located near protein - coding genes and a much smaller fraction of the gc - rich repeats in the human genome . specifically , we focused our analysis on repeats located adjacent to loci already implicated in als or ftd . our rationale for this choice was that utilization of gwas conducted in similar patient populations as well as known genetic loci in rare families ( which have subsequently been shown to contribute to a percentage of sporadic cases ) would colocalize with repeat expansions that confer a substantial contribution to the genetic burden of als in the united states . consistent with this idea , the c9orf72 locus was identified on multiple gwas surveys in als and ftd in sporadic populations prior to identification of the causative repeat . as such , our findings that none of these alternative loci harbor repeat expansions in 199 als cases suggest that g4c2 repeat expansions at non - c9orf72 loci are unlikely to be a common cause of als in our patient population , given that most common highly penetrant mutations would be detected by this approach . however , it is important to acknowledge that for highly penetrant loci identified in familial als cases , it might be necessary to examine the original published cohorts , which might carry rare or private mutations . we took this step for als4 and found no g4c2 repeat instability at 4 neighboring loci . the lack of g4c2 repeat expansions at alternative gene loci suggests that there could be something about the c9orf72 locus that promotes instability . the vast majority ( 75% ) of the other hexanucleotide repeats studied here exhibited no variation among 335 individuals , and variance in repeat size was largely absent from the 1,000-genome database for the 321 intragenic g4c2 repeat loci not assayed ( data not shown ) . however , some of the g4c2 repeats we studied were larger than this threshold but were invariant , suggesting that the length of the repeat is insufficient to explain instability . while future work will be needed to determine which factors drive selective instability , it may reflect specific aspects of the gc - rich sequence surrounding the repeat at the c9orf72 loci . this concept is supported by the finding that all pathologic repeat expansions at c9orf72 observed to date have occurred on a single haplotype background . however , g4c2 repeat expansions in genes other than c9orf72 may be enriched in neurologic disorders other than als . phenotypes of polyglutamine disorders associated with cag repeat expansions , for example , are not all the same . we observed noteworthy repeat instability in both cases and controls in one other gene : a g4c2 intronic repeat in rgs14 with a mean repeat length of 9 and a range of 414 repeats . as such , reevaluation of this locus may be worthy of further study in familial als , ftd , and other neurodegenerative disorders . a ggcctg repeat expansion in nop56 causes
moderate cag repeat expansions ( encoding polyglutamine ) in atxn2 and gcg repeat expansions ( encoding polyalanine ) in nip1a are associated with an increased risk of als . however , other known pathogenic cag and cgg repeat expansions do not influence the risk of als or ftd , suggesting that the observed effects may be gene specific rather than repeat sequence specific . we screened 28 loci harboring g4c2 repeats near known als / ftd loci in 199 patients with als and did not find pathogenic repeat expansions other than that previously reported in c9orf72 . while future studies in larger cohorts and select families are still needed , our results suggest that g4c2 repeat expansions outside the c9orf72 gene are not a common cause of als , supporting the importance of identifying cis elements that explain the genetic instability at the c9orf72 locus . ; and grants from the michigan alzheimer 's disease center at the university of michigan , nih r01ns08681001 , and the department of veterans affairs blrd 1i21bx001841 to p.k.t . feldman receives funding support from novo nordisk , the nih , the juvenile diabetes research foundation , the us - israel binational science foundation , the american diabetes association , and the a. alfred taubman medical research institute . todd receives funding support from the nih , the veterans administration , the national ataxia foundation , the national fragile x foundation , and the muscular dystrophy association ; serves as a paid consultant for denali therapeutics and for an nih grant ; serves as an unpaid consultant for the national fragile x foundation ; has received travel / speaker honoraria from the american academy of neurology , the national fragile x foundation , and the japanese society of neurochemistry ; has received publishing royalties from uptodate ; and has received license fee payments for antibodies developed by his group from millipore for distribution to scientists and commercial groups , and for vectors and antibodies developed by his group from denali therapeutics . | [
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the incidence of atrial fibrillation ( af ) and af - related strokes increase with age. warfarin has been clearly shown to effectively prevent thromboembolic events in patients with af , especially in elderly patients. however , warfarin therapy is often underutilized in elderly patients due to a higher risk of bleeding and poor tolerability . recently ,
non - vitamin k antagonist oral anticoagulants ( noacs ) such as dabigatran and rivaroxaban have been shown to be non - inferior or even superior to warfarin for long - term stroke prevention in patients with non - valvular af .
, a recent meta - analysis reported that noacs did not cause excess bleeding and were associated with equal or greater efficacy than warfarin therapy in elderly patients ( 75 years ) . moreover , subgroup analysis of major clinical trials in asian populations showed even better results for noacs in terms of bleeding complications .
, therefore , it is expected that noacs would be an effective alternative to warfarin , or even a better option , in patients with a higher risk of bleeding . as societies worldwide have aged ,
the efficacy and safety of noacs in extremely elderly patients or octogenarians have emerged as an important area of research . as renal
function decreases with age and creatinine levels poorly reflect renal function in elderly patients , the bleeding risk of noacs could be substantial in this extreme age group .
our present study aimed to evaluate the efficacy ( prevention of stroke or systemic embolism ) and safety ( risk of major bleeding ) of noacs and warfarin in korean octogenarian patients in a real - world setting .
we performed a systematic computerized search of our hospital 's database ( asan biomedical research environment ) , and selected patients who were 80 years old with non - valvular af ( 418 patients on noacs and 232 patients on warfarin ) at asan medical center between january 2011 and september 2014 .
patients receiving anticoagulant therapy for the purpose of primary or secondary prevention of ischemic stroke or systemic embolism were solely selected .
non - valvular af was defined as rhythm disturbance occurring in the absence of rheumatic mitral valve disease , a prosthetic heart valve , or mitral valve repair .
patients were excluded if they were taking anticoagulants for other purposes including prevention or treatment of pulmonary thromboembolism or deep vein thrombosis ; if they had bridging therapy with noac or pretreatment before cardioversion ; if they did not have at least two months of follow - up after medication ; if they had a creatinine clearance ( crcl ) of < 30 ml / min .
finally , 148 patients were enrolled into the noacs group and 145 patients were enrolled into the warfarin group , respectively ( figure 1 ) .
all patients were treated with noacs ( 148 patients , 50.5% ) or warfarin [ target international normalized ratio ( inr ) : 2.03.0 ; 145 patients , 49.5% ] according to the patient 's or physician 's preference .
of the 148 patients that received noacs , 52 ( 35.1% ) received dabigatran [ 48 on a low - dose ( 110 mg twice a day ) and 4 on a common dose ( 150 mg twice a day ) ] and 96 ( 64.9% ) received rivaroxaban [ 32 on a low dose ( 15 mg once daily ) and 64 on a common dose ( 20 mg once daily ) ] .
apixaban was not included in our analysis because it was not available in our hospital during the study period .
types ( dabigatran or rivaroxaban ) and doses ( low dose or common dose ) of noacs were prescribed at the discretion of the attending physicians .
stroke was defined as the sudden onset of a focal neurological deficit lasting at least 24 h. systemic embolism was defined as acute vascular occlusion of an extremity or major organ documented at the time of autopsy , angiography , or vascular imaging .
major bleeding was defined as ( 1 ) fatal bleeding , ( 2 ) symptomatic bleeding in a critical area or organ , such as intracranial , intraspinal , intraocular , retroperitoneal , intraarticular , or pericardial , or intramuscular with compartment syndrome , and/or ( 3 ) bleeding causing a fall in hemoglobin level of 2 g / dl or leading to the transfusion of 2 units of whole blood or red cells .
two investigators ( kwon ch and kim ms ) reviewed all medical records of the study patients during follow - up and adjudicated all stroke , systemic embolism , and bleeding events that contributed to the pre - specified outcomes .
event rates are presented as event numbers per 100 patient - years of follow - up .
, chicago , il ) , and data are expressed as the mean sd ( continuous variables ) or as frequency ( categorical variables ) . when comparing baseline variables between the noacs and warfarin groups , continuous variables
were compared using the student 's t test and mann - whitney u test , and categorical variables were compared using the chi - square test or fisher 's exact test .
hazard ratios ( hrs ) and p values were calculated by the use of multivariable cox 's regression analysis that included sex , anticoagulant type , concomitant antiplatelet use , and has - bled score .
the kaplan - meier method was used to estimate the survival - free rate of major bleeding between the treatment groups . additionally , the survival rates were compared using the log - rank test .
we performed a systematic computerized search of our hospital 's database ( asan biomedical research environment ) , and selected patients who were 80 years old with non - valvular af ( 418 patients on noacs and 232 patients on warfarin ) at asan medical center between january 2011 and september 2014 .
patients receiving anticoagulant therapy for the purpose of primary or secondary prevention of ischemic stroke or systemic embolism were solely selected .
non - valvular af was defined as rhythm disturbance occurring in the absence of rheumatic mitral valve disease , a prosthetic heart valve , or mitral valve repair .
patients were excluded if they were taking anticoagulants for other purposes including prevention or treatment of pulmonary thromboembolism or deep vein thrombosis ; if they had bridging therapy with noac or pretreatment before cardioversion ; if they did not have at least two months of follow - up after medication ; if they had a creatinine clearance ( crcl ) of < 30 ml / min .
finally , 148 patients were enrolled into the noacs group and 145 patients were enrolled into the warfarin group , respectively ( figure 1 ) .
all patients were treated with noacs ( 148 patients , 50.5% ) or warfarin [ target international normalized ratio ( inr ) : 2.03.0 ; 145 patients , 49.5% ] according to the patient 's or physician 's preference .
of the 148 patients that received noacs , 52 ( 35.1% ) received dabigatran [ 48 on a low - dose ( 110 mg twice a day ) and 4 on a common dose ( 150 mg twice a day ) ] and 96 ( 64.9% ) received rivaroxaban [ 32 on a low dose ( 15 mg once daily ) and 64 on a common dose ( 20 mg once daily ) ] .
apixaban was not included in our analysis because it was not available in our hospital during the study period .
types ( dabigatran or rivaroxaban ) and doses ( low dose or common dose ) of noacs were prescribed at the discretion of the attending physicians .
stroke was defined as the sudden onset of a focal neurological deficit lasting at least 24 h. systemic embolism was defined as acute vascular occlusion of an extremity or major organ documented at the time of autopsy , angiography , or vascular imaging .
major bleeding was defined as ( 1 ) fatal bleeding , ( 2 ) symptomatic bleeding in a critical area or organ , such as intracranial , intraspinal , intraocular , retroperitoneal , intraarticular , or pericardial , or intramuscular with compartment syndrome , and/or ( 3 ) bleeding causing a fall in hemoglobin level of 2 g / dl or leading to the transfusion of 2 units of whole blood or red cells .
two investigators ( kwon ch and kim ms ) reviewed all medical records of the study patients during follow - up and adjudicated all stroke , systemic embolism , and bleeding events that contributed to the pre - specified outcomes .
event rates are presented as event numbers per 100 patient - years of follow - up .
, chicago , il ) , and data are expressed as the mean sd ( continuous variables ) or as frequency ( categorical variables ) . when comparing baseline variables between the noacs and warfarin groups , continuous variables were compared using the student 's t test and mann - whitney u test , and categorical variables were compared using the chi - square test or fisher 's exact test .
hazard ratios ( hrs ) and p values were calculated by the use of multivariable cox 's regression analysis that included sex , anticoagulant type , concomitant antiplatelet use , and has - bled score .
the kaplan - meier method was used to estimate the survival - free rate of major bleeding between the treatment groups .
additionally , the survival rates were compared using the log - rank test . a p value < 0.05 was considered statistically significant .
in addition , the has - bled scores as determined by previous bleeding history , hypertension , abnormal renal / liver function , stroke , bleeding history or predisposition , labile international normalized ratio , elderly , and drug or alcohol use were significantly higher in the patients in the noacs group than in those in the warfarin group .
the median follow - up duration was 10.9 months ( interquartile range , 5.221.2 months ) for the noacs group and 12.8 months ( interquartile range , 5.324.9 months ) for the warfarin group .
a total of 29 patients ( 9.9% ) had concomitant antiplatelet agent with none on dual antiplatelet therapy .
there was no significant difference in number of patients on concomitant antiplatelet agents between noacs group ( n = 15 , 10.1% ) and warfarin group ( n = 14 , 9.7% ) .
mean inr of patients in the warfarin group was measured 2.1 1.5 at the last follow - up .
the efficacy and safety outcomes according to the treatment group are presented in table 2 and figure 2 .
the incidence rates of stroke or systemic embolic events were low in both anticoagulant groups and there were no significant differences in stroke or systemic embolic events between the treatment groups . however , major bleeding events occurred in a significant numbers of patients treated with both noacs and warfarin .
major bleeding events tended to be lower in the low dose noacs group than the common dose noacs or warfarin groups , but the difference was not statistically significant ( figure 2b ) .
values are expressed as the mean sd , median ( interquartile range ) , or n ( % ) .
cad : coronary artery disease ; cha2ds2 vasc score : cumulative score of congestive heart failure , hypertension , age ( 75 years or 65 years ) , diabetes , stroke , vascular disease , and sex ( female ) ; egfr : estimated glomerular filtration rate ; has - bled score : cumulative score of hypertension , abnormal renal and liver function , stroke , bleeding , labile inrs , elderly ( e.g. , age > 65 years ) , and drugs or alcohol ; mi : myocardial infarction ; noacs : non - vitamin k antagonist oral anticoagulants ; se : systemic embolism .
p value between treatment groups was achieved for the overall follow - up duration by univariable cox regression model .
major bleeding occurred in 15 patients ( 10.1% ) in the noacs group and 24 patients ( 16.4% ) in the warfarin group ( figure 3 ) .
gastrointestinal bleeding was the most common cause of major bleeding in both groups [ 6 patients ( 4.1% ) in the noac group vs. 10 patients ( 6.8% ) in the warfarin group ] .
intracranial bleeding occurred in one patient ( 0.7% ) in the noac group and three patients ( 2.1% ) in the warfarin group .
major bleeding events tended to increase according to has - bled scores but no statistically significant differences were shown ( online data figure s1 ) . likewise
, no significant difference was found between the patients with vs .. without concomitant antiplatelet agents ( 5/29 , 17.2% vs. 34/264 , 12.9% ) .
concomitant antiplatelet agent was not associated with an increased incidence of major bleeding in cox regression analysis ( hrs = 1.273 , 95% ci : 0.4973.263 , p = 0.615 ) . among the 29 patients on antiplatelet therapy , major bleeding occurred in 2 out of 14 patients in the warfarin , 3 of 11 patients in the low dose noacs , and no one ( 0/4 patients ) in common dose noacs group , respectively ( log rank , p = 0.572 ) . on the other hand , major bleeding events significantly increased in patients with decreased renal function ( crcl : 3044 ml / min ) ( figure 4 ) .
meier analysis revealed a significant difference in the incidence of major bleeding according to renal function ( figure 5 ) .
has - bled : cumulative score of hypertension , abnormal renal and liver function , stroke , bleeding , labile inrs , elderly ( e.g. , age > 65 years ) , and drugs or alcohol ; noacs : non - vitamin k antagonist oral anticoagulants .
in addition , the has - bled scores as determined by previous bleeding history , hypertension , abnormal renal / liver function , stroke , bleeding history or predisposition , labile international normalized ratio , elderly , and drug or alcohol use were significantly higher in the patients in the noacs group than in those in the warfarin group .
the median follow - up duration was 10.9 months ( interquartile range , 5.221.2 months ) for the noacs group and 12.8 months ( interquartile range , 5.324.9 months ) for the warfarin group .
a total of 29 patients ( 9.9% ) had concomitant antiplatelet agent with none on dual antiplatelet therapy .
there was no significant difference in number of patients on concomitant antiplatelet agents between noacs group ( n = 15 , 10.1% ) and warfarin group ( n = 14 , 9.7% ) .
mean inr of patients in the warfarin group was measured 2.1 1.5 at the last follow - up .
the efficacy and safety outcomes according to the treatment group are presented in table 2 and figure 2 .
the incidence rates of stroke or systemic embolic events were low in both anticoagulant groups and there were no significant differences in stroke or systemic embolic events between the treatment groups . however , major bleeding events occurred in a significant numbers of patients treated with both noacs and warfarin .
major bleeding events tended to be lower in the low dose noacs group than the common dose noacs or warfarin groups , but the difference was not statistically significant ( figure 2b ) .
values are expressed as the mean sd , median ( interquartile range ) , or n ( % ) .
cad : coronary artery disease ; cha2ds2 vasc score : cumulative score of congestive heart failure , hypertension , age ( 75 years or 65 years ) , diabetes , stroke , vascular disease , and sex ( female ) ; egfr : estimated glomerular filtration rate ; has - bled score : cumulative score of hypertension , abnormal renal and liver function , stroke , bleeding , labile inrs , elderly ( e.g. , age > 65 years ) , and drugs or alcohol ; mi : myocardial infarction ; noacs : non - vitamin k antagonist oral anticoagulants ; se : systemic embolism .
p value between treatment groups was achieved for the overall follow - up duration by univariable cox regression model .
major bleeding occurred in 15 patients ( 10.1% ) in the noacs group and 24 patients ( 16.4% ) in the warfarin group ( figure 3 ) .
gastrointestinal bleeding was the most common cause of major bleeding in both groups [ 6 patients ( 4.1% ) in the noac group vs. 10 patients ( 6.8% ) in the warfarin group ] .
intracranial bleeding occurred in one patient ( 0.7% ) in the noac group and three patients ( 2.1% ) in the warfarin group .
major bleeding events tended to increase according to has - bled scores but no statistically significant differences were shown ( online data figure s1 ) .
likewise , no significant difference was found between the patients with vs .. without concomitant antiplatelet agents ( 5/29 , 17.2% vs. 34/264 , 12.9% ) .
concomitant antiplatelet agent was not associated with an increased incidence of major bleeding in cox regression analysis ( hrs = 1.273 , 95% ci : 0.4973.263 , p = 0.615 ) . among the 29 patients on antiplatelet therapy , major bleeding occurred in 2 out of 14 patients in the warfarin , 3 of 11 patients in the low dose noacs , and no one ( 0/4 patients ) in common dose noacs group , respectively ( log rank , p = 0.572 ) . on the other hand ,
major bleeding events significantly increased in patients with decreased renal function ( crcl : 3044 ml / min ) ( figure 4 ) . kaplan
meier analysis revealed a significant difference in the incidence of major bleeding according to renal function ( figure 5 ) .
has - bled : cumulative score of hypertension , abnormal renal and liver function , stroke , bleeding , labile inrs , elderly ( e.g. , age > 65 years ) , and drugs or alcohol ; noacs : non - vitamin k antagonist oral anticoagulants .
our present study is a retrospective analysis to compare noacs with warfarin therapy for stroke prevention in asian octogenarian patients with non - valvular af .
the major findings of our analyses are as follows : ( 1 ) the incidence rates of stroke or systemic embolism were low in octogenarian patients , and there was no significant difference in the efficacy outcomes ( stroke or systemic embolism ) between the noacs and warfarin treatments ( 1.16% for noacs vs. 2.98% for warfarin per 100 patient - years , p = 0.46 ) ; and ( 2 ) in contrast , major bleeding occurred in a significant number of patients with both noacs ( 8.96 per 100 patient - years ) and warfarin ( 12.46 per 100 patient - years ) treatments , which was not significantly different between the two groups ( p = 0.29 ) . in our present study series , stroke and systemic embolism event rates were low and were not significantly different between the noacs and warfarin groups .
recent studies in asian populations reported similar rates of stroke or systemic embolism , ranging from 1.26% to 2.6% per year with noacs and from 2.61% to 3.4% per year with warfarin .
, , the mean age of the study population of the previous asian studies was around 70 years , whereas the mean age of our study patients was 83.7 years .
in addition , the patients in the noacs group were significantly older than those in the warfarin group in this study .
taking these facts into consideration , noacs appear to be not only non - inferior , but may also be more effective than warfarin at preventing embolic events in very old ( 80 years ) af patients .
moreover , a recent meta - analysis of elderly patients ( 75 years ) that supported this assumption also reported that the incidence of stroke or systemic embolism was significantly lower with noacs than warfarin therapy ( 3.3% vs. 4.7% ; odds ratio = 0.65 ; 95% ci : 0.480.87 ) . in this study , patients treated with low dose noacs experienced numerically fewer stroke or systemic embolism events than those treated with common dose noacs or warfarin .
in the randomized evaluation of long - term anticoagulation therapy ( re - ly ) trial , the risks of stroke or systemic embolism were not statistically different between the two doses ( 110 mg and 150 mg ) of dabigatran in elderly patients aged 75 years . in a japanese trial that used low - dose ( 15 mg once daily ) rivaroxaban ( mean age of study population , 71 years ) , there was a strong trend for a reduction in the rate of stroke / systemic embolism with low dose rivaroxaban vs. warfarin ( hr = 0.49 ; p = 0.050 ) .
a recent chinese study suggested that low dose dabigatran achieved superior stroke risk reduction than warfarin in chinese octogenarian patients with af .
therefore , low dose noacs may be non - inferior than common dose noacs or warfarin at preventing stroke or systemic embolism in east asian octogenarians
. the risks of total bleeding and major bleeding were relatively high in our current study patients .
one study found that the cumulative incidence of major hemorrhage for octogenarians was 13.1 per 100 patient - years , which was consistent with our present findings .
however , an italian prospective observational study reported a low rate of major bleeding ( 1.87 per 100 patient - years ) in octogenarians on warfarin .
in other recent sub - analyses of randomized trials , the risk of major bleeding was 4.374.40 per 100 patient - years on warfarin in patients aged 75 years .
previous sub - analyses according to old age ( 75 years ) reported that the incidence of major bleeding was 4.435.10 per 100 patient - years on noacs .
, in asian patients ( mean age , about 70 years ) , the risk of major bleeding on noacs was reported to be 2.993.59 per 100 patient - years .
, , compared with these previous results , the risk of major bleeding on noacs was about twice as high in our current study population . given that our result was drawn from the real - world setting , the risk of major bleeding in octogenarian patients seems to be markedly high with both noacs and warfarin .
the risks of major bleeding tended to be lower in patients treated with low dose noacs than in those treated with common dose noacs and warfarin therapy in our analysis .
moreover , the patients in our noacs group were significantly older and had higher has - bled scores and previous bleeding history than those in the warfarin group . based on these results , low dose noacs
may not only be non - inferior , but may be superior to common dose noacs or warfarin for preventing major bleeding in frail patients such as octogenarians . a simple laboratory test with a capability of indicating the effects of different dosages of noacs or gauging the degree of anticoagulation would be of great help to adjust dosage , especially at the beginning of noac treatment . in the sub - analysis
, major bleeding risks tended to be higher in patients with moderate - to - high bleeding risk ( has - bled score 2 ) than in those with low bleeding risk ( has - bled score 1 ) .
however , major bleeding was significantly higher in patients with stage 3b chronic kidney disease ( crcl 3044 ml / min ) than in other less decreased renal function groups . therefore , circumspection is needed when prescribing noacs and warfarin to patients with decreased renal function .
although warfarin is effective for the prevention of stroke and systemic embolism in af , advanced age and asian ethnicity are associated with a greater risk for warfarin - related bleeding , especially intracranial hemorrhage. our current analysis also showed an increased tendency for major bleeding with warfarin compared with noacs .
therefore , noacs would be an attractive alternative to warfarin for achieving fewer thromboembolic and bleeding events in asian octogenarian patients .
second , the number of patients analyzed was relatively small and the follow - up period was relatively short .
thus , we could not compare the efficacy and safety between different noacs . of note , apixaban was not included in our analysis .
considering previous results of apixaban on the superior safety outcomes , bleeding in our patient cohort might have been different if apixaban was available . however , as there has been no head - to - head comparison using different noacs , it is difficult to extrapolate individual noacs results to comparison of different noacs .
fourthly , we could not test patient 's adherence to prescribed anticoagulant regimen . finally , we did not calculate time - in - therapeutic range for the warfarin group , and thus could not evaluate the appropriateness of warfarin therapy .
however , our study largely reflects the current status of real - world anticoagulation practice , and deserves its significance . despite these limitations ,
our findings are meaningful because they reveal the incidence of stroke or systemic embolic events and major bleeding risks in noacs or warfarin groups in a real - world clinical setting for asian octogenarian patients .
the incidence rates of stroke or systemic embolism are low in asian octogenarian af patients treated with warfarin or noacs .
however , major bleeding events can be excessively high in both anticoagulant groups , especially in patients with decreased renal function .
the efficacy and safety outcomes tended to decrease non - significantly in patients with low dose noacs than in those treated with common dose noacs or warfarin therapy . in the future , a well - designed , prospective study on the optimal anticoagulant regimen ( low dose regimen and new criteria for prescription of low dose noacs ) is required in elderly asian population , especially in patients with impaired renal function .
in our present study series , stroke and systemic embolism event rates were low and were not significantly different between the noacs and warfarin groups .
recent studies in asian populations reported similar rates of stroke or systemic embolism , ranging from 1.26% to 2.6% per year with noacs and from 2.61% to 3.4% per year with warfarin .
, , the mean age of the study population of the previous asian studies was around 70 years , whereas the mean age of our study patients was 83.7 years .
in addition , the patients in the noacs group were significantly older than those in the warfarin group in this study .
taking these facts into consideration , noacs appear to be not only non - inferior , but may also be more effective than warfarin at preventing embolic events in very old ( 80 years ) af patients .
moreover , a recent meta - analysis of elderly patients ( 75 years ) that supported this assumption also reported that the incidence of stroke or systemic embolism was significantly lower with noacs than warfarin therapy ( 3.3% vs. 4.7% ; odds ratio = 0.65 ; 95% ci : 0.480.87 ) . in this study , patients treated with low dose noacs experienced numerically fewer stroke or systemic embolism events than those treated with common dose noacs or warfarin .
this result was consistent with those of previous studies . in the randomized evaluation of long - term anticoagulation therapy ( re - ly ) trial , the risks of stroke or systemic embolism were not statistically different between the two doses ( 110 mg and 150 mg ) of dabigatran in elderly patients aged 75 years . in a japanese trial that used low - dose ( 15 mg once daily ) rivaroxaban ( mean age of study population , 71 years ) , there was a strong trend for a reduction in the rate of stroke / systemic embolism with low dose rivaroxaban vs. warfarin ( hr = 0.49 ; p = 0.050 ) .
a recent chinese study suggested that low dose dabigatran achieved superior stroke risk reduction than warfarin in chinese octogenarian patients with af .
therefore , low dose noacs may be non - inferior than common dose noacs or warfarin at preventing stroke or systemic embolism in east asian octogenarians .
the risks of total bleeding and major bleeding were relatively high in our current study patients .
one study found that the cumulative incidence of major hemorrhage for octogenarians was 13.1 per 100 patient - years , which was consistent with our present findings .
however , an italian prospective observational study reported a low rate of major bleeding ( 1.87 per 100 patient - years ) in octogenarians on warfarin .
in other recent sub - analyses of randomized trials , the risk of major bleeding was 4.374.40 per 100 patient - years on warfarin in patients aged 75 years .
previous sub - analyses according to old age ( 75 years ) reported that the incidence of major bleeding was 4.435.10 per 100 patient - years on noacs .
, in asian patients ( mean age , about 70 years ) , the risk of major bleeding on noacs was reported to be 2.993.59 per 100 patient - years .
, , compared with these previous results , the risk of major bleeding on noacs was about twice as high in our current study population . given that our result was drawn from the real - world setting , the risk of major bleeding in octogenarian patients seems to be markedly high with both noacs and warfarin .
the risks of major bleeding tended to be lower in patients treated with low dose noacs than in those treated with common dose noacs and warfarin therapy in our analysis . moreover ,
the patients in our noacs group were significantly older and had higher has - bled scores and previous bleeding history than those in the warfarin group . based on these results , low dose noacs
may not only be non - inferior , but may be superior to common dose noacs or warfarin for preventing major bleeding in frail patients such as octogenarians . a simple laboratory test with a capability of indicating the effects of different dosages of noacs or gauging the degree of anticoagulation would be of great help to adjust dosage , especially at the beginning of noac treatment . in the sub - analysis , major bleeding risks tended to be higher in patients with moderate - to - high bleeding risk ( has - bled score 2 ) than in those with low bleeding risk ( has - bled score 1 ) .
however , major bleeding was significantly higher in patients with stage 3b chronic kidney disease ( crcl 3044 ml / min ) than in other less decreased renal function groups . therefore , circumspection is needed when prescribing noacs and warfarin to patients with decreased renal function .
although warfarin is effective for the prevention of stroke and systemic embolism in af , advanced age and asian ethnicity are associated with a greater risk for warfarin - related bleeding , especially intracranial hemorrhage. our current analysis also showed an increased tendency for major bleeding with warfarin compared with noacs .
therefore , noacs would be an attractive alternative to warfarin for achieving fewer thromboembolic and bleeding events in asian octogenarian patients .
second , the number of patients analyzed was relatively small and the follow - up period was relatively short .
thus , we could not compare the efficacy and safety between different noacs . of note , apixaban was not included in our analysis .
considering previous results of apixaban on the superior safety outcomes , bleeding in our patient cohort might have been different if apixaban was available . however , as there has been no head - to - head comparison using different noacs , it is difficult to extrapolate individual noacs results to comparison of different noacs .
finally , we did not calculate time - in - therapeutic range for the warfarin group , and thus could not evaluate the appropriateness of warfarin therapy .
however , our study largely reflects the current status of real - world anticoagulation practice , and deserves its significance . despite these limitations ,
our findings are meaningful because they reveal the incidence of stroke or systemic embolic events and major bleeding risks in noacs or warfarin groups in a real - world clinical setting for asian octogenarian patients .
the incidence rates of stroke or systemic embolism are low in asian octogenarian af patients treated with warfarin or noacs
. however , major bleeding events can be excessively high in both anticoagulant groups , especially in patients with decreased renal function .
the efficacy and safety outcomes tended to decrease non - significantly in patients with low dose noacs than in those treated with common dose noacs or warfarin therapy . in the future , a well - designed , prospective study on the optimal anticoagulant regimen ( low dose regimen and new criteria for prescription of low dose noacs ) is required in elderly asian population , especially in patients with impaired renal function . | backgroundthe efficacy and safety of non - vitamin k antagonist oral anticoagulants ( noacs ) and warfarin in asian octogenarian atrial fibrillation ( af ) patients have not been established in a real - world setting .
we aimed to evaluate the efficacy and safety of noacs and warfarin in korean octogenarian patients.methodsa total of 293 consecutive patients aged 80 years with non - valvular af who had taken either noacs ( 148 cases , 50.5% ) or warfarin ( 145 cases , 49.5% ) were retrospectively reviewed .
the efficacy outcome was the composite of stroke or systemic embolism .
the safety outcome was major bleeding.resultsthe follow - up duration was 375 patient - years ( 172 patient - years with noacs and 203 patient - years with warfarin ) .
patients on noacs were slightly older ( p = 0.006 ) and had slightly higher has - bled scores ( p = 0.034 ) .
the efficacy of both anticoagulants was high ( 1.16% for noacs vs. 2.98% for warfarin per 100 patient - years , p = 0.46 ) .
the safety outcome was relatively high in both noacs and warfarin groups ( 8.96% vs. 12.46% , p = 0.29 ) .
the efficacy and safety outcomes tended to decrease non - significantly in low dose noacs than in common dose noacs or warfarin ( 0.85% vs. 1.84% vs. 2.98% in efficacy outcome , p = 0.69 ; and 6.97% vs. 13.29% vs. 12.46% in safety outcome , p = 0.34).conclusionsnoacs were highly effective for prevention of stroke or systemic embolism in asian octogenarian af patients . however , major bleeding occurred excessively high in both anticoagulant groups .
further study is required on the optimal anticoagulant regimen in octogenarian population . | Introduction
Methods
Study subjects
Efficacy and safety outcomes
Statistical analysis
Results
Study subjects
Efficacy and safety outcomes
Discussion
Efficacy of NOACs and warfarin
Safety of NOACs and warfarin
Limitations of the study
Conclusions | the incidence of atrial fibrillation ( af ) and af - related strokes increase with age. recently ,
non - vitamin k antagonist oral anticoagulants ( noacs ) such as dabigatran and rivaroxaban have been shown to be non - inferior or even superior to warfarin for long - term stroke prevention in patients with non - valvular af . as societies worldwide have aged ,
the efficacy and safety of noacs in extremely elderly patients or octogenarians have emerged as an important area of research . our present study aimed to evaluate the efficacy ( prevention of stroke or systemic embolism ) and safety ( risk of major bleeding ) of noacs and warfarin in korean octogenarian patients in a real - world setting . we performed a systematic computerized search of our hospital 's database ( asan biomedical research environment ) , and selected patients who were 80 years old with non - valvular af ( 418 patients on noacs and 232 patients on warfarin ) at asan medical center between january 2011 and september 2014 . patients receiving anticoagulant therapy for the purpose of primary or secondary prevention of ischemic stroke or systemic embolism were solely selected . all patients were treated with noacs ( 148 patients , 50.5% ) or warfarin [ target international normalized ratio ( inr ) : 2.03.0 ; 145 patients , 49.5% ] according to the patient 's or physician 's preference . of the 148 patients that received noacs , 52 ( 35.1% ) received dabigatran [ 48 on a low - dose ( 110 mg twice a day ) and 4 on a common dose ( 150 mg twice a day ) ] and 96 ( 64.9% ) received rivaroxaban [ 32 on a low dose ( 15 mg once daily ) and 64 on a common dose ( 20 mg once daily ) ] . types ( dabigatran or rivaroxaban ) and doses ( low dose or common dose ) of noacs were prescribed at the discretion of the attending physicians . event rates are presented as event numbers per 100 patient - years of follow - up . we performed a systematic computerized search of our hospital 's database ( asan biomedical research environment ) , and selected patients who were 80 years old with non - valvular af ( 418 patients on noacs and 232 patients on warfarin ) at asan medical center between january 2011 and september 2014 . patients receiving anticoagulant therapy for the purpose of primary or secondary prevention of ischemic stroke or systemic embolism were solely selected . all patients were treated with noacs ( 148 patients , 50.5% ) or warfarin [ target international normalized ratio ( inr ) : 2.03.0 ; 145 patients , 49.5% ] according to the patient 's or physician 's preference . types ( dabigatran or rivaroxaban ) and doses ( low dose or common dose ) of noacs were prescribed at the discretion of the attending physicians . major bleeding was defined as ( 1 ) fatal bleeding , ( 2 ) symptomatic bleeding in a critical area or organ , such as intracranial , intraspinal , intraocular , retroperitoneal , intraarticular , or pericardial , or intramuscular with compartment syndrome , and/or ( 3 ) bleeding causing a fall in hemoglobin level of 2 g / dl or leading to the transfusion of 2 units of whole blood or red cells . event rates are presented as event numbers per 100 patient - years of follow - up . in addition , the has - bled scores as determined by previous bleeding history , hypertension , abnormal renal / liver function , stroke , bleeding history or predisposition , labile international normalized ratio , elderly , and drug or alcohol use were significantly higher in the patients in the noacs group than in those in the warfarin group . the median follow - up duration was 10.9 months ( interquartile range , 5.221.2 months ) for the noacs group and 12.8 months ( interquartile range , 5.324.9 months ) for the warfarin group . there was no significant difference in number of patients on concomitant antiplatelet agents between noacs group ( n = 15 , 10.1% ) and warfarin group ( n = 14 , 9.7% ) . the efficacy and safety outcomes according to the treatment group are presented in table 2 and figure 2 . the incidence rates of stroke or systemic embolic events were low in both anticoagulant groups and there were no significant differences in stroke or systemic embolic events between the treatment groups . however , major bleeding events occurred in a significant numbers of patients treated with both noacs and warfarin . major bleeding events tended to be lower in the low dose noacs group than the common dose noacs or warfarin groups , but the difference was not statistically significant ( figure 2b ) . , age > 65 years ) , and drugs or alcohol ; mi : myocardial infarction ; noacs : non - vitamin k antagonist oral anticoagulants ; se : systemic embolism . p value between treatment groups was achieved for the overall follow - up duration by univariable cox regression model . major bleeding events tended to increase according to has - bled scores but no statistically significant differences were shown ( online data figure s1 ) . concomitant antiplatelet agent was not associated with an increased incidence of major bleeding in cox regression analysis ( hrs = 1.273 , 95% ci : 0.4973.263 , p = 0.615 ) . among the 29 patients on antiplatelet therapy , major bleeding occurred in 2 out of 14 patients in the warfarin , 3 of 11 patients in the low dose noacs , and no one ( 0/4 patients ) in common dose noacs group , respectively ( log rank , p = 0.572 ) . , age > 65 years ) , and drugs or alcohol ; noacs : non - vitamin k antagonist oral anticoagulants . in addition , the has - bled scores as determined by previous bleeding history , hypertension , abnormal renal / liver function , stroke , bleeding history or predisposition , labile international normalized ratio , elderly , and drug or alcohol use were significantly higher in the patients in the noacs group than in those in the warfarin group . the median follow - up duration was 10.9 months ( interquartile range , 5.221.2 months ) for the noacs group and 12.8 months ( interquartile range , 5.324.9 months ) for the warfarin group . the efficacy and safety outcomes according to the treatment group are presented in table 2 and figure 2 . the incidence rates of stroke or systemic embolic events were low in both anticoagulant groups and there were no significant differences in stroke or systemic embolic events between the treatment groups . however , major bleeding events occurred in a significant numbers of patients treated with both noacs and warfarin . major bleeding events tended to be lower in the low dose noacs group than the common dose noacs or warfarin groups , but the difference was not statistically significant ( figure 2b ) . , age > 65 years ) , and drugs or alcohol ; mi : myocardial infarction ; noacs : non - vitamin k antagonist oral anticoagulants ; se : systemic embolism . p value between treatment groups was achieved for the overall follow - up duration by univariable cox regression model . major bleeding events tended to increase according to has - bled scores but no statistically significant differences were shown ( online data figure s1 ) . concomitant antiplatelet agent was not associated with an increased incidence of major bleeding in cox regression analysis ( hrs = 1.273 , 95% ci : 0.4973.263 , p = 0.615 ) . among the 29 patients on antiplatelet therapy , major bleeding occurred in 2 out of 14 patients in the warfarin , 3 of 11 patients in the low dose noacs , and no one ( 0/4 patients ) in common dose noacs group , respectively ( log rank , p = 0.572 ) . , age > 65 years ) , and drugs or alcohol ; noacs : non - vitamin k antagonist oral anticoagulants . our present study is a retrospective analysis to compare noacs with warfarin therapy for stroke prevention in asian octogenarian patients with non - valvular af . the major findings of our analyses are as follows : ( 1 ) the incidence rates of stroke or systemic embolism were low in octogenarian patients , and there was no significant difference in the efficacy outcomes ( stroke or systemic embolism ) between the noacs and warfarin treatments ( 1.16% for noacs vs. 2.98% for warfarin per 100 patient - years , p = 0.46 ) ; and ( 2 ) in contrast , major bleeding occurred in a significant number of patients with both noacs ( 8.96 per 100 patient - years ) and warfarin ( 12.46 per 100 patient - years ) treatments , which was not significantly different between the two groups ( p = 0.29 ) . in our present study series , stroke and systemic embolism event rates were low and were not significantly different between the noacs and warfarin groups . recent studies in asian populations reported similar rates of stroke or systemic embolism , ranging from 1.26% to 2.6% per year with noacs and from 2.61% to 3.4% per year with warfarin . taking these facts into consideration , noacs appear to be not only non - inferior , but may also be more effective than warfarin at preventing embolic events in very old ( 80 years ) af patients . moreover , a recent meta - analysis of elderly patients ( 75 years ) that supported this assumption also reported that the incidence of stroke or systemic embolism was significantly lower with noacs than warfarin therapy ( 3.3% vs. 4.7% ; odds ratio = 0.65 ; 95% ci : 0.480.87 ) . in this study , patients treated with low dose noacs experienced numerically fewer stroke or systemic embolism events than those treated with common dose noacs or warfarin . in the randomized evaluation of long - term anticoagulation therapy ( re - ly ) trial , the risks of stroke or systemic embolism were not statistically different between the two doses ( 110 mg and 150 mg ) of dabigatran in elderly patients aged 75 years . in a japanese trial that used low - dose ( 15 mg once daily ) rivaroxaban ( mean age of study population , 71 years ) , there was a strong trend for a reduction in the rate of stroke / systemic embolism with low dose rivaroxaban vs. warfarin ( hr = 0.49 ; p = 0.050 ) . a recent chinese study suggested that low dose dabigatran achieved superior stroke risk reduction than warfarin in chinese octogenarian patients with af . therefore , low dose noacs may be non - inferior than common dose noacs or warfarin at preventing stroke or systemic embolism in east asian octogenarians
. one study found that the cumulative incidence of major hemorrhage for octogenarians was 13.1 per 100 patient - years , which was consistent with our present findings . however , an italian prospective observational study reported a low rate of major bleeding ( 1.87 per 100 patient - years ) in octogenarians on warfarin . in other recent sub - analyses of randomized trials , the risk of major bleeding was 4.374.40 per 100 patient - years on warfarin in patients aged 75 years . previous sub - analyses according to old age ( 75 years ) reported that the incidence of major bleeding was 4.435.10 per 100 patient - years on noacs . , in asian patients ( mean age , about 70 years ) , the risk of major bleeding on noacs was reported to be 2.993.59 per 100 patient - years . , , compared with these previous results , the risk of major bleeding on noacs was about twice as high in our current study population . given that our result was drawn from the real - world setting , the risk of major bleeding in octogenarian patients seems to be markedly high with both noacs and warfarin . the risks of major bleeding tended to be lower in patients treated with low dose noacs than in those treated with common dose noacs and warfarin therapy in our analysis . moreover , the patients in our noacs group were significantly older and had higher has - bled scores and previous bleeding history than those in the warfarin group . based on these results , low dose noacs
may not only be non - inferior , but may be superior to common dose noacs or warfarin for preventing major bleeding in frail patients such as octogenarians . in the sub - analysis
, major bleeding risks tended to be higher in patients with moderate - to - high bleeding risk ( has - bled score 2 ) than in those with low bleeding risk ( has - bled score 1 ) . however , major bleeding was significantly higher in patients with stage 3b chronic kidney disease ( crcl 3044 ml / min ) than in other less decreased renal function groups . although warfarin is effective for the prevention of stroke and systemic embolism in af , advanced age and asian ethnicity are associated with a greater risk for warfarin - related bleeding , especially intracranial hemorrhage. thus , we could not compare the efficacy and safety between different noacs . despite these limitations ,
our findings are meaningful because they reveal the incidence of stroke or systemic embolic events and major bleeding risks in noacs or warfarin groups in a real - world clinical setting for asian octogenarian patients . the incidence rates of stroke or systemic embolism are low in asian octogenarian af patients treated with warfarin or noacs . however , major bleeding events can be excessively high in both anticoagulant groups , especially in patients with decreased renal function . the efficacy and safety outcomes tended to decrease non - significantly in patients with low dose noacs than in those treated with common dose noacs or warfarin therapy . in the future , a well - designed , prospective study on the optimal anticoagulant regimen ( low dose regimen and new criteria for prescription of low dose noacs ) is required in elderly asian population , especially in patients with impaired renal function . in our present study series , stroke and systemic embolism event rates were low and were not significantly different between the noacs and warfarin groups . recent studies in asian populations reported similar rates of stroke or systemic embolism , ranging from 1.26% to 2.6% per year with noacs and from 2.61% to 3.4% per year with warfarin . taking these facts into consideration , noacs appear to be not only non - inferior , but may also be more effective than warfarin at preventing embolic events in very old ( 80 years ) af patients . moreover , a recent meta - analysis of elderly patients ( 75 years ) that supported this assumption also reported that the incidence of stroke or systemic embolism was significantly lower with noacs than warfarin therapy ( 3.3% vs. 4.7% ; odds ratio = 0.65 ; 95% ci : 0.480.87 ) . in this study , patients treated with low dose noacs experienced numerically fewer stroke or systemic embolism events than those treated with common dose noacs or warfarin . in the randomized evaluation of long - term anticoagulation therapy ( re - ly ) trial , the risks of stroke or systemic embolism were not statistically different between the two doses ( 110 mg and 150 mg ) of dabigatran in elderly patients aged 75 years . in a japanese trial that used low - dose ( 15 mg once daily ) rivaroxaban ( mean age of study population , 71 years ) , there was a strong trend for a reduction in the rate of stroke / systemic embolism with low dose rivaroxaban vs. warfarin ( hr = 0.49 ; p = 0.050 ) . therefore , low dose noacs may be non - inferior than common dose noacs or warfarin at preventing stroke or systemic embolism in east asian octogenarians . one study found that the cumulative incidence of major hemorrhage for octogenarians was 13.1 per 100 patient - years , which was consistent with our present findings . however , an italian prospective observational study reported a low rate of major bleeding ( 1.87 per 100 patient - years ) in octogenarians on warfarin . in other recent sub - analyses of randomized trials , the risk of major bleeding was 4.374.40 per 100 patient - years on warfarin in patients aged 75 years . previous sub - analyses according to old age ( 75 years ) reported that the incidence of major bleeding was 4.435.10 per 100 patient - years on noacs . , in asian patients ( mean age , about 70 years ) , the risk of major bleeding on noacs was reported to be 2.993.59 per 100 patient - years . given that our result was drawn from the real - world setting , the risk of major bleeding in octogenarian patients seems to be markedly high with both noacs and warfarin . the risks of major bleeding tended to be lower in patients treated with low dose noacs than in those treated with common dose noacs and warfarin therapy in our analysis . moreover ,
the patients in our noacs group were significantly older and had higher has - bled scores and previous bleeding history than those in the warfarin group . based on these results , low dose noacs
may not only be non - inferior , but may be superior to common dose noacs or warfarin for preventing major bleeding in frail patients such as octogenarians . in the sub - analysis , major bleeding risks tended to be higher in patients with moderate - to - high bleeding risk ( has - bled score 2 ) than in those with low bleeding risk ( has - bled score 1 ) . however , major bleeding was significantly higher in patients with stage 3b chronic kidney disease ( crcl 3044 ml / min ) than in other less decreased renal function groups . although warfarin is effective for the prevention of stroke and systemic embolism in af , advanced age and asian ethnicity are associated with a greater risk for warfarin - related bleeding , especially intracranial hemorrhage. despite these limitations ,
our findings are meaningful because they reveal the incidence of stroke or systemic embolic events and major bleeding risks in noacs or warfarin groups in a real - world clinical setting for asian octogenarian patients . the incidence rates of stroke or systemic embolism are low in asian octogenarian af patients treated with warfarin or noacs
. however , major bleeding events can be excessively high in both anticoagulant groups , especially in patients with decreased renal function . the efficacy and safety outcomes tended to decrease non - significantly in patients with low dose noacs than in those treated with common dose noacs or warfarin therapy . in the future , a well - designed , prospective study on the optimal anticoagulant regimen ( low dose regimen and new criteria for prescription of low dose noacs ) is required in elderly asian population , especially in patients with impaired renal function . | [
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] |
the search for new antibacterial compounds is a challenging task as bacteria are continuously developing resistance to antimicrobial compounds ; however , infections due to such bacterial strains are infrequent although potentially fatal [ 13 ] .
this ongoing problem has resulted in the search for newer , more effective antibacterial compounds [ 13 ] .
urea , thiourea 3 ( x = o or s ) , and benzo-1,3-oxazine compounds 5 and 6 ( scheme 1 ) have been shown to possess antibacterial and antifungal properties [ 411 ] .
the benzyl thiourea analogue 3 has been reported to show activity against gram - positive bacteria .
the n - benzoyl-2-hydroxybenzamides are important pharmacophores for antibacterial activity in which the 2-hydroxy group ( hydrogen bonding donor ) contributes to the activity , the imide linker ( preferred ) or urea linker retains activity and free nh is required for high activity .
the topliss method was used in the optimization of salicylic acid derivatives for potential use as antibacterial agents . the employment and analysis of physicochemical parameters and molecular electronic surfaces which highlight the electronic , lipophilic , and steric features may be useful guidelines in the continuous search for new , more effective 3-amino - salicylic acid analogs .
the synthesis of the urea or thiourea product ( 3 , scheme 1 ) was previously achieved from the reaction of benzoylisocyanate or benzoylisothiocyanate 1 ( x = o or s , resp . ) with amines 2 [ 1517 ] .
the substitution r on the aromatic ring could be alkyl , alkoxy , oc = och3 in positions 2 , 3 , 4 , 5 , and 6 , and the r1-n - r2 in product 3 could be aliphatic , aromatic , or cyclic amine substitutions .
limitation associated with this method is that r , the substitution on the aromatic ring , can not be oh or rn , which is desirable for antimicrobial activity , particularly the hydroxy group .
furthermore , the synthesis of 2-amino - substituted-1,3- benzoxazine 5 was achieved through the reaction of the corresponding amine 2 with ethyl 2-cyanobenzoate 4 [ 1921 ] .
however , again the limitation in this method is that the r in product 5 can not be oh or nh2 .
the reaction of substituted 2-thio-1,3-benzoxazine-4-one 6 ( scheme 1 ) with primary and secondary amines showed considerable interest in the literature .
the secondary amine 2 ( dimethyl , diethyl , and cyclic amines ) reaction with 2-thioxo-2,3-substituted 1,3-benzoxazin-4-one 6 gave only 2-amino - substituted-1,3-benzoxazine 5 [ 19 , 20 , 22 , 23 ] .
reactions with primary amines lead to the opening of the oxazine ring and produce thiourea type analogues ( compound 3 r = 2-oh and r1nr2 = hn - alkyl ) while with nh2 ch2ph gave 3 r = 2-oh and r1nr2 = nhch2ph mixed with 2-(benzyl amino)-4h - benz[e]-1,3-oxazin-4-one ( 5 r = h and r1nr2 = nhch2ph [ 19 , 20 ] .
the above reaction produced low yields or a mixture of products 13a and 15a ( scheme 2 ) which was difficult to separate . in this work
, we developed simple general procedures for the synthesize of n-(benzyl carbamothioyl)-2-hydroxybenzamides 13 , 20 , and 21 , n-(benzyl carbamoyl)-2-hydroxy - substituted - benzamides 23 , and 2-(benzyl amino)-substituted-1,3-benzoxazin-4-one 15 and 26 .
the antibacterial and antifungal activity was evaluated for a number of these products with the intension of producing novel products that can be used to eliminate problematic bacteria in the environmental and medical settings .
5-substituted-4,6-dihydroxybenzene-1,3-dicarboxylic acids 16a c , and 2,3-dihydroxybenzene-1,4-dicarboxylic acid 17 were prepared by carboxylation of 2-substituted-1,3-hydroxy - benzene and 1,2-dihydroxy - benzene , respectively , according to the previously reported method .
compound 7 was selectively acetylated in the presence of naoh and acetic anhydride with the reaction maintained at ph 6 - 7 in accordance with the reported procedure [ 2527 ] to give the acid 8a an excellent yield ( 82% ) ( scheme 2 ) .
allowing compound 7 to react with substituted benzaldehyde gave the corresponding schiff base 8b - c ( scheme 2 ) [ 2833 ] .
reduction of schiff base 8b - c with sodium borohydride gave 4-(substituted - benzylamino)-2-hydroxybenzoic acids 9a c with high yield ( 7385% ) .
the structures of the prepared acids were confirmed using mp , ir , h , and cnmr which was in good agreement with the previously reported physical and spectroscopy data [ 2628 ] . the 2-thio - substituted-1,3-benzoxazines 6 , 10 , 11 , 12 ( a r = 8-ch3 , b r = 8-ph , c r = 6-br , d r = 6-och2ch3 , e r = 7-och2ch3 , f r = 8-och2ch3 , g r = 6-och3 , h r = 7-och3 , i r = 8-och3 , j r = 6,8-i , k r = 7-oh , l
r = 7-oh,-8-ch3 , and m r = 8-oh ) , 18 , and 19 were synthesized from the reaction of the substituted 2-hydroxy benzoic acid with freshly prepared ph3p(scn)2 ( schemes 2 and 3 ) following the reported conditions [ 22 , 34 ] with some modifications ( see experimental ) .
it is worth noting that the reaction of 4-benzylideneamino-2-hydroxybenzoic acid 8d formed the corresponding 7-(benzylidene)amino-2-thio-1,3-benzoxazines which were hydrolysed during the isolation of the product and gave 7-amino-2-thio-1,3-benzoxazines 10b .
however , the reaction of 4-amino-2-hydroxybenzoic acid 7 with the freshly prepared ph3p(scn)2 failed to produce the expected product 10b however gave a complex , unidentifiable molecule containing a triphenylphosphene group attached to the oxazine product .
7-(substituted benzylamino)-2-thioxo-2,3-dihydro-4h-1,3-benzoxazin-4-one 11a c were synthesized from the reaction of 4-(substituted - benzylamino)-2-hydroxybenzoic acids 9a c with the freshly prepared ph3p(scn)2 [ 22 , 32 ] ( scheme 2 ) .
the structures of the new products 11a c were confirmed using h , and cnmr and microanalysis .
similarly , the substituted-2-thioxo-1,3-benzoxazin-4-one 6 and 12b n prepared using a previously reported [ 22 , 23 , 33 ] method gave products with identical physical and spectroscopic data .
the benz - bis-(1,3-oxazine ) 18a c and 19 were prepared from the reaction of dihydroxy - dicarboxybenzoic acids 16a c and 17 with the freshly prepared ph3p(scn)2 [ 22 , 32 ] according to the previously reported method for the synthesis of 18c with some modification to improve the yield .
the structures of the new dibenzoxines products 18a.b and 19 were confirmed using h and cnmr and microanalysis ( scheme 4 ) .
2-methylthio - substituted-1,3-oxazines 14a i ( a r = h , b r = 8-ch3 , c r = 8-ph , d r = 7-och3 , e r = 7-och2ch3 , f r = 7-oh , g r = 7-oh,-8-ch3 , h
r = acnh , and i r = nh2 ) and 24 were prepared by the reaction of ch3i in the presence of nahco3 with substituted-1,3-oxazines 6 , 10a , b , 11c , and 12 ( a r = 8-ch3 , b r = 8-ph , e r = 7-och2ch3 , h
r = 7-och3 , k r = 7-oh , and l r = 7-oh,-8-ch3 ) according to the previously reported procedure .
physical properties , ir , h nmr , and c nmr , collected for products 14a i were found to be identical to the reported data . the structures of new product , 14 h , i , and 24 were confirmed with analysis of the ir , h , and c nmr data .
compounds 14a i and 24 were used in the synthesis of compounds 15a i and 26 with no further purification .
initially , the equimolar reaction of compound 6 with benzylamine in dioxane was repeated according to the earlier reported method [ 19 , 20 ] , in which the mixture that was refluxed for 4 hours gave 13a and was a 46% yield ( scheme 3 ) .
however , when the reaction was carried out using solvent free conditions , excess benzylamine was added directly to powered 2-thio-1,3-benzoxazine 6 and the mixture was left at room temperature for 2 days , the benzyl thiourea product 13a had decreased yield ( 19% ) , and none of the cyclic analogue 15a could be isolated .
when 2-thio-1,3-benzoxazine 6 was allowed to react with excess benzylamine ( 4-fold ) and the mixture was then heated to reflux in dioxane for 2 hours , the open product 13a was again isolated in 18% yield . to overcome this low yield and possible mixture formation ,
the reaction procedure was modified in which the benzoxazines 6 , 10a - b , 11d , and 12b m were mixed with nahco3 and suspended in 1 : 1 mixture of methanol , water and the mixture was then heated to 40c for a few minutes .
excess ( 1.5-fold ) benzyl amine was added dropwise at room temperature and left stirring for 4 hours .
products 13a q was isolated according to the general procedure b ( see experimental ) and the yield were moderate to high ( 6987% ) ( scheme 3 ) . with slight modification to the procedure b , by altering the ratio of benzylamine to starting material ( 3 : 1 ) and the reaction time to 16 hours , the bis - oxazines 18a , c and
19 were found to react in a similar fashion to give substituted bis(benzyl carbamothioyl ) analogues 20a , c ( yield 63 , 49% ) and 21 ( yield 63% ) ( scheme 4 ) .
the previously prepared n-(benzyl carbamothioyl)-2-hydroxybenzamide 13a was characterized by comparison of its physical data ( mp , ir , h and c nmr spectra ) with values found in the literature [ 12 , 19 , 20 ] .
the structures of new benzyl thiourea compounds 13b q , 20a , c , and 21 were confirmed using ir , h nmr and c nmr spectroscopy and microanalysis .
the h nmr and ir spectra also showed a high correlation with the previously prepared benzyl thiourea 13a [ 19 , 20 ] . in the h nmr spectra , the ch2 , h-5 of the benzyl amine in compounds 13a q , 20a , c , and 21 appeared as a doublet at ~ 4.9 ppm and the 4-nh , appeared as a triplet at ~ 11.0 ppm in all cases .
assignment of the carbon-13 chemical shifts was made using the previous reported chemical shifts of 13a [ 19 , 20 ] .
the h and c nmr spectra of the parent 2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12 were also used to aid with structural identification .
the simulated h and c nmr spectra using chemdraw v12 ultra were also used as references to aid the analysis of the observed h and c nmr spectra of the new products . as mentioned earlier the reaction of the 2-thio-1,3-benzoxazine 6 with a primary amine
the reaction of 2-methylthio-(substituted)-1,3-benzoxazines 14 ( a r = h , b r = 8-ch3 , c r = 8-ph , d r = 7-och3 , e r = 7-och2ch3 , f r = 7-oh , g r = 7-oh,-8-ch3 , and h r = acnh- ) and 24 with excess ( 5-fold ) benzyl amine according to the general procedure c gave 2-benzyl amino - substituted-1,3-benzoxazines 15a h and 26 with moderate to high yields 6283% ( schemes 3 and 5 ) .
the reaction of benzylamine with 2-methylthio - benzoxazine took place with no trace of the thiourea analogue 13 .
following their successful synthesis , many of the n-(benzyl carbamothioyl)-2-hydroxybenzamides 13a , b , f , and g were then cyclised by refluxing in acetic acid for 2 hours according to the general procedure d ( scheme 3 ) and gave the corresponding 2-benzylamino-1,3-benzoxazines 15a , b , f , and g fair to good yields .
previously prepared 2-benzyl amino-1,3-benzoxazine 15a was characterized by comparison of the physical data ( mp , ir , and h and c nmr spectra ) with that found in the literature [ 19 , 20 ] .
the structures of new 2-benzyl amino-1,3-benzoxazine compounds 15b g were confirmed using ir , h nmr , and c nmr spectroscopy and microanalysis . in the h spectra the ch2 of the benzyl amine appears as a doublet at ~ 4.5 ppm .
the previously analysed h and c nmr spectra of the parent 2-methylthio-1,3-benzoxazines 14 were used to aid in the analysis of the new products 15b
g s. 2-methylthio - substituted - benzoxazines 14a , b , and d g were allowed to react with 10% hcl according to the general procedure e and gave 2-dione-1,3-benzoxazines 22a h with good to excellent yield ( scheme 5 ) ; however , product 22h was produce if 40% hcl was used in the hydrolysis of 14h .
similarly , the 2,8-dioxo product 25 was prepared from the hydrochloric acid hydrolysis of the corresponding 2,8-dimethylthio - analogue 24 ( scheme 5 ) .
the synthesis of the substituted - n-(benzyl carbamoyl)-2-hydroxybenzamides 23a g was achieved using the reaction of the relevant benzoxazine-2 , 4-di - one 22a
h with excess benzylamine in dioxane with refluxed according to the general procedure f.
structure elucidation of substituted - n-(benzyl carbamoyl)-2-hydroxybenzamides
23a g .
the structures of the newly prepared substituted urea compounds 23a g were confirmed using ir and h and c nmr spectroscopy and microanalysis .
the h nmr and ir spectra supported the proposed structures and showed some correlation with the previously prepared benzyl thiourea 13a q , 20a , c , and 21 .
in the h spectra , the methylene ch2 ( h-11 of compounds 23a g ) of the benzyl amine appears to shift up field as a doublet at ~ 4.3 ppm .
the nh ( h-10 of compounds 23a g ) appears as a broad triplet at ~ 8.5 ppm .
the h and c nmr spectra of the parent 2-oxo-1,3-benzoxazines 22 and 25 . in this study , some of the newly prepared compounds were tested and showed antimicrobial activity against 8 different bacterial strains and 4 cultures of fungi .
the bacterial species investigated were p. aeruginosa , b. subtilis , s. aureus , a. baumannii , e. coli , s. agalactiae m. smegmatis , and m. chlorophenolicum . the antifungal evaluation was determined against a. niger , a. corymbifera , r. oryzae , and a. alternata .
the minimal inhibitory concentrations ( mics ) and minimal fungicidal concentrations ( mfcs ) , defined as the lowest concentration of drug that inhibits the growth of bacteria or fungi in the inoculums ' , were determined using the broth dilution methods .
the compounds which demonstrated mic and mfc values less than 300 and 200 g ml , respectively , are listed in tables 1 and 2 . according to , antimicrobial agents are effective on a range of bacterial species at low concentrations ,
. therefore , we conducted our mic experiments using concentrations as high as 300 g ml .
we also selected a range of bacterial and fungal species to test our newly synthesised compounds .
some of the species are potentially pathogenic to humans and animals and others are problematic in an environmental setting .
furthermore , the bacterial species selected have different cell wall compositions , that is , some are gram - negative and some are gram - positive strains . some antimicrobial agents inhibit bacteria by interacting with components of the cell wall that are absent in gram - negative bacteria ; therefore , the selection of strains was carefully selected with the possibility that an inhibitory compound would also hint to its mechanism of action . based on the results obtained
it is clear that the gram - negative strains , that is , p. aeruginosa , e. coli , and a. baumannii were least affected by the compounds and when inhibition was observed it was at high levels 200 g ml or higher .
interestingly , compound 9d seemed to have a more dramatic effect gram - positive strains with the exception of m. smegmatis .
despite the effects that some of the compounds had on the bacterial strains , it appears that these compounds are not so effective when tested on the four fungal cultures chosen with the exception of 9d on a. corymbifera . based on the results obtained for each of the newly synthesised compounds ,
it is evident that compound 9d has more potent effect when compared to the others .
this reveals that despite the fact that most of the compounds do not seem to have a noteworthy effect on the strains , compound 9d is of interest and further investigation is required .
disc diffusion susceptibility testing was performed on compounds with poor solubility in broth dilution susceptibility testing .
compounds that inhibited certain bacteria or fungi are summarized in ( tables 3 and 4 ) .
the control data is used to determine if the bacterial strains are resistant ( r ) or sensitive ( s ) to the prepared compounds tested .
the disc diffusion assay was used as a preliminary guide for all compounds and used in correspondence with the broth dilution method for determining mic / mfc values .
this method is particularly useful when mic / mfc values are unable to be determined using the broth dilution method due to the compounds insolubility .
the insoluble compounds zones of inhibition therefore can be determined in millimeters relative to the control and used as a rough guide .
since the zone of inhibition of clearance may be affected by other parameters , such as , the nutrient agar depth of the plate and solvent used , the results shown using this method therefore should be used as a guide .
all compounds that showed clearance zones are listed in tables 3 and 4 and were tested in duplicate with the average given .
any zone of inhibition that was noted around the disc was considered sensitive and the zone of clearly was noted .
these results are more useful for compounds that were difficult to dissolve , but equally , these results can indicate resistance if the compound does not diffuse through the agar properly . based on the results obtained in section 2.2.1 ,
based on the results obtained in table 3 , compound 9d has an inhibitor effect on m. smegmatis but in the mic study had no effect .
this could indicate solubility problems with the compound when in solution ; however , this is only speculative ; further studies are required to reveal the cause .
in addition , compound 9d seems to have no inhibitory effect on s. aureus but in the mic studies had a dramatic affect .
this difference in result is unusual but clearly indicates that different methods could reveal different results and therefore it is important to perform both methods prior to further investigation on their inhibitory effects . in the mic studies
, we used 300 g ml as the highest cut - off level .
if a compound has an inhibitory effect on any strain that is greater than this level , then this should be revealed in the disc diffusion assay . however , further investigation is required as some of these compounds are dissolved in dmso and when applied to bacterial cultures can come out of solution .
the disc diffusion assays seem to indicate some sensitivity to fungal cultures despite the fact that they were undetectable in the mic studies . the results in tables 1 and 2 show that the 4-(benzylamino)-2-hydroxybenzoic acid derivative 9d showed the broadest range of activity of the compounds tested , exhibiting activity against the gram - positive and gram - negative bacteria and also m. chlorophenolicum .
furthermore , compound 9d showed to be more active than others against s. aureus with an mic value of 25 g / ml .
compounds 20a , c and 21 ( bis - thiourea products ) were found to be particularly active towards gram - positive b. subtilis at mic values of 12.5 , 25 , and 25 g ml .
in addition , compound 21 also showed activity towards m. smegmatis ( mic 50 g ml ) .
other synthesized compounds which showed an inhibitory effect were 13n which had an inhibitory effect on four bacterial species at 300 g ml and 13d and 13 m which had an inhibitory effect on two of the bacterial species at concentration 200 g ml .
interestingly , e. coli was not inhibited by any of the compounds . in the presence of a compound ,
a zone of clearing was greater than the control which was indicative that the strain was sensitive to the compound , whereas a zone of clearing equal to the control indicated resistance .
the results reveal that none of the compounds had an inhibitory effect on e. coli at concentration 10 g ml ( table 3 ) .
the b. subtilis bacterial species tested showed inhibitory effects to most of the compounds tested , for example , 13d inhibited s. aureus most strongly and compounds 13k , 13f , and 13d inhibited growth of a. baumannii , b. subtilis , and s. agalactiae ( table 3 ) .
some bacterial species that were sensitive to a compound showed similar sized zones of inhibition .
one example was 13k which exhibited activity of 2 mm for a. baumannii and b. subtilis and 5 mm for s. aureus and s. agalactiae .
the same applies to compounds 8d , 9c , and 9d which had shown a 2 mm clearance zone against p. aeruginosa m. smegmatis m. chlorophenolicum . similarly to the broth dilution results , compounds 21 and 9d were found to be active against three fungi species , r. oryzae , a. niger , and a. corymbifera , with clearance zones 25 mm , respectively .
all the compounds tested showed a 24 mm zone of clearing for most of the susceptible species with the exception of compound 13b which had a larger 6 mm zone of inhibition .
this larger zone indicates a hypersensitive effect on the bacterial species ; however , it is specific for the compound and species .
because the mechanism of action of the compound is unknown , it is difficult to explain the reason for the hypersensitive effect .
one possible explanation is that b. subtilis encodes a protein that can transport compound 13b into the cell and this has a more toxic effect than those working from outside the cell .
a similar phenomenon has been shown with bacterial mercury resistance where the presence of a mercury import protein displays a larger zone of clearing in a disc diffusion assay .
the data obtained revealed patterns of inhibition , especially those conducted with the disc diffusion assay .
this suggests that a similar mechanism of action could be involved in the inhibition of growth .
in conclusion , we have prepared seven new compounds of 2-benzylamino - substituted-1,3-benzoxazines , nineteen new n-(benzyl carbamothioyl)-substituted - benzamide and have evaluated some for their activity against bacteria and fungi .
it appears that n-(benzyl carbamothioyl)-substituted - benzamide has shown antibacterial activity such as 20a , 20c , 21 , 13d , 13 m , and 13n .
we are in the process of synthesising new substituted products by replacing the benzyl group of n-(benzyl carbamothioyl ) by 6-aminopenicillanic acid and test their bacteria activity .
h nmr and c nmr spectra were obtained using a bruker ac 200 nmr spectrometer at 200 and 50 mhz , respectively .
all h nmr and c nmr spectral results are recorded as chemical shifts ( ) relative to the internal tms for proton and 77.0 ppm in cdcl3 solvent and 39.4 ppm in dmso - d6 solvent for c nmr .
melting point determinations were carried out using a stuart scientific ( smp3 ) melting point apparatus and all melting points are uncorrected . the stating reagents benzyl amine , sodium hydrogen carbonate , methyl iodide - amino-2-hydroxybenzoic acid , and dry 1,4-dioxane were purchased from aldrich chemical company and were used as received . according to the previously reported method [ 29 , 30 , 37 ] , product 8a was prepared from the reaction of 4-amino-2-hydroxybenzoic acid 7 and acetic anhydride and recrystallised from 1,4-dioxane , 82% yield , mp 221224c ( lit and mp 235c ) .
the physical and spectroscopic data is consistent with the literature values [ 27 , 35 ] .
according to the previously reported method [ 29 , 30 ] , intermediates 8b e were prepared from the appropriate substituted benzaldehyde and 4-amino-2-hydroxy - benzoic acid 7 .
products 8b , c , and e were not identified and used immediately in the synthesis of compound 9a , b , and d.
( z)-4-((3-ethoxy-2-hydroxybenzylidene)amino)-2-hydroxybenzoic acid
8d .
3-ethoxy-2-hydroxybenzaldehyde ( 1.66 g , 0.01 mol ) was allowed to react with 4-amino-2-hydroxybenzoic acid 7 ( 1.53 g , 0.01 mol ) for 1 hour according to the reported procedure [ 29 , 30 ] and gave solid which recrystallised from methanol to give 8d 2.95 g , 98% as red crystals , mp 185188c decomp .
max ( kbr)/cm 1655 ( c = o ) , 1622 , 1600 ( c = n ) : hnmr ( 200 mhz , 300 k , d6-acetone ) 8.93 ( s , 1h , h-8 ) , 8.02 ( d , 1h , jh6,h5 = 8.8 hz , h-6 ) , 7.33 ( dd , 1h , jh15,h14 = 7.8 hz , jh15,h13 = 1.6 hz , h-15 ) , 7.24 ( dd , 1h , jh13,h14 = 8.0 hz , jh13,h15 = 1.6 hz , h-13 ) , 6.917.09 ( m , 3h , h-3,h-5 , and h-14 ) , 4.24 ( q , 2h , jh16,h17 = 7.0 hz , h-16 ) , 1.41 ( t , 3h , jh17,h16 = 7.0 hz , h-17 ) .
general procedure a. in slight modification to a previous reported method , the appropriate 4-substituted ( ( benzylidene ) amino)-hydroxybenzoic acids 8b - c reduced using sodium borohydride ( 2 equiv )
2-hydroxy-4-{[(e)-benzylidene ] amino } benzoic acid 8b ( 2.41 g , 10 mmol ) was allowed to react with sodium borohydride ( 0.76 g , 20 mmol ) according to general procedure a. the resulting solid was recrystallised from methanol / water to give 9a ( 1.76 g , 73% ) , mp 122125c .
max ( kbr)/cm 35003200 br ( oh ) , 3024 , 2569 ( nh ) , 1632(c = o ) ; hnmr ( 200 mhz , 340 k d6-dmso ) 11.34 ( bs , 1h , oh of cooh exchangeable with d2o ) , 7.5 ( d , 1h , jh6,h5 = 8.6 hz , h-6 ) , 7.217.47 ( m , 6h , 5 x ch , 8-nh exchangeable with d2o ) , 6.22 ( dd , 1h , jh5,h6 = 8.6 hz , jh5,h8 = 2.0 hz , h-5 ) , 6.03 ( d , 1h , jh3,h5 = 2.0 hz , h-3 ) , 4.38 ( s , 2h , h-9 ) . 3.32 ( 2-oh under the water envelope ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 171.7 ( c-7 ) , 163.2 ( c-2 ) , 154.8 ( c-4 ) , 139.1 ( c-10 ) , 130.1 ( c-6 ) , 128.1 , 126.9 , 126.6 ( c-12 , c-11 and c-13 ) , 105.3 ( c-5 ) , 100.4 ( c-1 ) , 96.8 ( c-3 ) , 45.8 ( c-9 ) . the resulting product 9a was not stable and was used immediately in the genral procedure b.
2-hydroxy-4-((2-hydroxybenzyl)amino)benzoic acid
9b .
( e)-2-hydroxy-4-((2-hydroxybenzylidene)amino)benzoic acid 8c ( 2.6 g , 10 mmol ) was allowed to react with sodium borohydride ( 0.76 g , 20 mmol ) according to general procedure a. the resulting solid was recrystallised from methanol / water to give 9b as white crystals ( 2.20 g , 85% ) , mp 184186c decomp .
max ( kbr)/cm 35003200 br ( oh ) , 1614 ( c = o ) ; hnmr ( 200 mhz , 300 k d6-dmso ) 11.45 ( bs , 1h , 8-nh or 11-oh exchangeable with d2o ) , 9.61 ( bs , 1h , 11-oh or 8-nh exchangeable with d2o ) 7.44 ( d , 1h , jh6,h5 = 8.8 hz , h-6 ) , 7.16.7 ( m , 6h , h-13 , h-15 , h-14 , h-12 and 11-oh ) , 6.28 ( dd , 1h , jh5,h6 = 8.8 hz , jh5,h3 = 1.1 hz , h-5 ) , 5.92 ( d , 1h , jh3,h5 = 1.1 hz , h-3 ) ; c nmr ( 50 mhz , 350 k , d6-dmso ) 171.9 ( c-7 ) , 163.5 ( c-2 ) , 155.2/155.0 ( c-11/c-4 ) , 131.2 ( c-6 ) , 128.5 ( c-13 ) , 127.9 ( c-14 ) , 125.0 ( c-10 ) , 119.1 ( c-15 ) , 115.3 ( c-12 ) , 105.6 ( c-5 ) , 100.6 ( c-1 ) , 97.0 ( c-3 ) , 41.1 ( c-8 ) .
the resulting product 9b was used immediately in the general procedure b.
4-((3-ethoxy-2-hydroxybenzyl)amino)-2-hydroxybenzoic acid
9c .
( e)-4-((3-ethoxy-2-hydroxybenzylidene)amino)-2-hydroxybenzoic acid 8d ( 10 mmol , 3.0 g ) was allowed to react with sodium borohydride ( 20 mmol , 0.76 g ) according to general procedure a. the resulting solid was collected and recrystallised from methanol / water to give 9d ( 2.66 g , 81% ) as white crystals , mp 159161c decomp .
max ( kbr)/cm 35003200 br ( oh , nh absorption under the oh envelope ) , 1625 ( c = o ) ; hnmr ( 200 mhz , 300 k d6-dmso ) 7.38 ( d , 1h , jh6,h5 = 8.6 hz , h-6 ) , 6.856.65 ( m , 3h , h-13 , h-14 and h-15 ) , 6.35 ( bs , 4h , 3 x oh and nh ) , 6.17 ( dd , 1h , jh5,h6 = 8.6 hz , jh5,h3 = 1.8 hz , h-5 ) , 5.87 ( d , 1h , jh3,h5 = 1.8 hz , h-3 ) , 4.23 ( s , 2h , ch2nh , h-9 ) , 4.05 ( q , 2h , jh16,h17 = 6.8 hz , h-16 ) , 1.35
( t , 3h , jh17,h16 = 6.8 hz , h-17 ) ; c nmr ( 50 mhz , 300 k , d6-dmso ) 172.2 ( c-7 ) , 163.6 ( c-2 ) , 155.3 ( c-4 ) , 146.6 ( c-12 ) , 144.1 ( c-11 ) , 131.4 ( c-6 ) , 125.7 ( c-10 ) , 120.3 ( c-14 ) , 119.2 ( c-15 ) , 111.8 ( c-13 ) , 105.8 ( c-1 ) , 100.2 ( c-5 ) , 96.7 ( c-3 ) , 64.4 ( c-16 ) , 40.8 ( c-9 ) , 15.0 ( c-17 ) ; anal . calcd . for c16h17no5 : c , 63.36 ; h , 4.62 ; n , 5.65 .
found : c , 63.51 ; h , 4.48 ; n , 5.66 . according to the previously reported general procedures [ 2326 ] , the appropriate substituted phenol was used in the synthesis substituted - dihydroxy - di - carboxylic acids 16 and 17 .
general procedure b. the substituted-2-hydroxy benzoic acid was allowed to react with the freshly prepared ph3p(scn)2 according to previously reported general procedure [ 22 , 34 ] .
slightly modified to the previously reported general procedure b [ 22 , 34 ] , 4-(acetyl amino)-2-hydroxybenzoic acid 8a ( 1.56 g , 8 mmol ) was allowed to react with freshly prepared ph3p(scn)2 ( 10 mmol ) at room temperature for 2 hours then under reflux for 16 hours . at the completion of the reaction , the pbbr2 filter cake was washed by acetic acid ( 150 ml ) to extract the desired product .
the acetic acid filtrate was evaporated and minimal toluene was added to dissolve any oil with the product . the crude solid was filtered and recrystallised from ethanol to give 10a ( 1.22 g , 65% ) as light red crystals , mp 285287c decomp .
max ( kbr)/cm 3290 , 3183 ( 9-nh ) , 3072 , 2923 ( 3-nh ) , 1704 ( c = o ) , 1188 ( c = s ) ; hnmr ( 200 mhz , 390 k , d6-dmso ) 13.38 ( bs , 1h , 9-nh ) , 10.56 ( s , 1h , 3-nh ) , 7.86 ( d , 1h , jh5,h6 = 8.6 hz , h-5 ) , 7.77 ( d , 1h , jh8,h6 = 1.8 hz , h-8 ) , 7.44 ( dd , 1h , jh6,h5= 8.6 hz , jh6,h8 = 1.8 hz , h-6 ) , 2.11 ( s , 3h , 11-ch3 ) ; c nmr ( 50 mhz , 330 k , d6-dmso ) 181.9 ( c-2 ) , 169.2 ( c-10 ) , 156.7 ( c-8a ) , 146.0 ( c-7 ) , 127.3 ( c-5 ) , 116.3 ( c-6 ) , 109.6 ( c-4a ) , 104.2 ( c-8 ) , 24.0 ( c-11 ) 155.9 ( c-4 ) ; anal . calcd . for c10h8n2o3s : c , 50.84 ; h , 3.41 ; n , 11.86 c. found : c , 50.69 ; h , 3.53 ; n , 11.86 .
synthesis of 7-amino-2-thioxo-2h - benz[e]oxazin-4(3h)-one
10b . a suspension of ( e)-4-((3-ethoxy-2-hydroxybenzylidene)amino)-2-hydroxybenzoic acid 8d ( 1.2 g , 4 mmol ) in dry dcm ( 20 ml ) was added to a mixture of freshly prepared ph3p(scn)2 ( 10 mmol ) according to the general procedure b. the resulting solid was isolated upon evaporation of the dcm filtrate 10b ( 0.74 g , 91% crude yield ) .
max ( kbr ) cm 3443 , 3328 , ( nh2 ) , 3059 , 2916 ( nh ) , 1749 ( c = o ) , 1679 , 1616
( c = c ) , 1205 ( c = s ) ; hnmr ( 200 mhz , 370 k , d6-dmso ) 13.04 ( s , 1h , 3-nh ) , 7.58 ( d , 1h , jh5,h6 = 8.6 hz , h-5 ) , 6.65 ( s , 2h , 7-nh2 ) , 6.60 ( dd , 1h , jh6,h5 = 8.6 hz , jh6,h8 = 1.8 hz , h-6 ) , 6.37 ( d , 1h , jh8,h6 = 1.8 hz ) ; c nmr ( 50 mhz , 330 k , d6-dmso ) 182.4 ( c-2 ) , 157.3 , 156.9 , 156.5 ( c-4 , c-8a , c-7 ) , 128.1 ( c-5 ) , 112.7 ( c-6 ) , 102.3 ( c-4a ) , 96.8 ( c-8 ) ; anal . calcd . for c8h6n2o2s : c , 49.47 ; h , 3.11 ; n , 14.42 .
in slight modification to previously reported general procedure b , 4-(benzylamino)-2-hydroxybenzoic acid 9a ( 0.94 g , 4 mmol ) was allowed to react with the freshly prepared ph3p(scn)2 ( 10 mmol ) at room temperature for 2 hours then under reflux for 16 hours .
the resulting crude solids ( 0.94 g , 82% ) were filtered , collected , and recrystallised from toluene to give 11a ( 0.88 g , 77% ) as yellow crystals , mp 210212c decomp .
1689 ( c = o ) , 1197 ( c = s ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 12.90 ( bs , 1h , 3-nh exchangeable with d2o ) , 7.61 ( d , 1h , jh5,h6 = 8.8 hz , h-5 ) , 7.377.26 ( m , 6h , ar and 9-nh exchangeable with d2o ) , 6.73 ( dd , 1h , jh6,h5 = 8.8 hz , jh6,h8 = 2.0 hz , h-6 ) , 6.42 ( d , 1h , jh8,h6 = 2.0 hz , h-8 ) , 4.41 ( d , 2h , jh10,h9 = 5.9 hz , h-10 ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 182.1 ( c-2 ) , 157.2 , 156.5 ( c-4 , c-8a ) , 155.2 ( c-7 ) , 138.2 ( c-11 ) , ( 128.2 , 127.3 , 127.0 , 126.8 ) , ( c-13 , c-12 , c-14 and c-5 ) , 111.9 ( c-6 ) , 102.5 ( c-4a ) , 95.1 ( c-8 ) , 45.8 ( c-10 ) ; anal . calcd . for c15h12n2o2s : c , 59.99 ; h , 4.03 ; n , 9.33 .
in slight modification to previously reported general procedure b , 2-hydroxy-4-((2-hydroxybenzyl)amino)benzoic acid 9b ( 1.07 g , 4 mmol ) was allowed to react with the freshly prepared ph3p(scn)2 ( 10 mmol ) at room temperature for 2 hours then under reflux for 16 hours .
the resulting crude solid recrystallised from acetonitrile to give 11b ( 0.55 g , 46% ) as yellow crystals , mp 173175c decomp .
max ( kbr)/cm 35003000 br ( oh ) , 3310 ( 9-nh ) , 2926 ( 3-nh ) , 1684 ( c = o ) , 1192 ( c = s ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 12.86 ( bs , 1h , 3-nh ) , 9.50 ( bs , 1h , 12-oh ) , 7.59 ( d , 1h , jh5,h6 = 8.6 hz , h-5 ) , 7.41 ( bs , 1h , 9-nh ) , 7.057.20 ( m , 2h , h-14 , h-16 ) , 6.696.88 ( m , 3h , h-13 , h-15 and h-6 ) , 6.42 ( d , 1h , jh8,h6 = 2.0 hz , h-8 ) , 4.32 ( s , 2h , h-10 ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 182.2 ( c-2 ) , 157.3 , 156.7 , 155.4 , 154.9 ( c-4 , c-8a , c-12 and c-7 ) , 128.5 , 127.9 , 127.3 ( c-14 , c-16 and c-5 ) 123.9 ( c-11 ) , 118.8 , 115.1 c-15 , c-13 ) , 111.9 ( c-6 ) , 102.2 ( c-4a ) , 94.9 ( c-8 ) , 40.9 ( c-10 ) ; anal . calcd . for c15h12n2o3 : c , 59.99 ; h , 4.03 ; n , 9.33 found : c , 59.83 ; h , 4.14 ; n , 9.45 .
in slight modification to previously reported general procedure [ 22 , 34 ] , 4-((3-ethoxy-2-hydroxybenzyl)amino)-2-hydroxybenzoic acid 9d ( 1.21 g , 4 mmol ) was allowed to react with the freshly prepared ph3p(scn)2 ( 10 mmol ) heated to room temperature for 2 hours then under reflux for 16 hours .
the resulting solids ( 1.94 g ) were recrystallised from acetic acid / water to give 11c ( 0.95 g , 68% ) as yellow crystals , mp 227229c decomp .
max ( kbr)/cm 35003200 br ( oh ) , 3496 ( 7-nh ) , 3301 ( 3-nh ) , 1686 ( c = o ) , 1620 ( c = c ) , 1196 ( c = s ) ; hnmr ( 200 mhz , 390 k , d6-dmso ) 12.86 ( bs , 1h , 3-nh ) , 8.44 ( s , 1h , 12-oh exchangeable with d2o ) , 7.59 ( d , 1h , jh5,h6 = 8.4 hz , h-5 ) , 7.42 ( t , 1h , 9-nh ) 6.896.40 ( m , 4h , ar and h-6 ) , 6.41 ( d , 1h , jh8,h6 = 1.8 hz , h-8 ) , 4.33 ( d , 2h , 10-ch2 ) , 4.07 ( q , 2h , jh17,h18 = 7.0 hz , 17-ch2 ) , 1.36 ( t , 3h , jh18,h17 = 7.0 hz , 18-ch3 ) ; c nmr ( 50 mhz , 390 k , d6-dmso ) 181.8 ( c-2 ) , 156.9 , 156.1 , 155.2 ( c-4 , c-8a , c-7 ) , 146.2 , 144.2 ( c-13 , c-12 ) , 126.9 , 124.4 ( c-15 , c-11 ) , 120.2 , 118.4 ( c-5 , c-16 ) , 112.2 , 111.5 ( c-14 , c-6 ) , 102.1 ( c-4a ) , 94.8 ( c-8 ) , 64.2 ( c-17 ) , 40.7 ( c-10 ) , 14.0 ( c-18 ) ; anal . calcd . for c15h12n2o2s : c , 59.29 ; h , 4.68 . found : c , 59.07 ; h , 4.70 .
2,8-dithioxo-2,3,7,8-tetrahydrobenzo[1,2-e : 5,4-e
] bis(oxazine)-4,6-dione 18a . in slight modification to the general procedure b , 4,6-dihydroxyisophthalic
acid 16a ( 0.79 g , 4 mmol ) was allowed to react with the freshly prepared ph3p(scn)2 ( 10 mmol ) heated to room temperature for 3 hours then under reflux for 5 hours . at the completion of the reaction , the reaction mixture was filtered and the pbbr2 filter cake washed with thf ( 100 ml ) to extract product 18a .
both thf and dcm filtrates were evaporated to dryness and minimal toluene added to remove any oil which may be present .
the crude solid was recrystallised using dioxane / chloroform to give 18a ( 0.63 g , 56% ) as yellow crystals , mp > 300c decomp .
max ( kbr)/cm 3104 , 3031 , 2939 , 2856 ( 3 and 7-nh ) , 1698 ( c = o ) , 1152 ( c = s ) ; hnmr ( 200 mhz , 350 k d6-dmso ) 13.65 ( bs , 2h , 2 x nh ) , 8.27 ( bs , 1h , h-5 ) , 7.69 ( bs , 1h , h-10 ) ; c nmr ( 50 mhz , d6-dmso ) 181.1 ( c-2,8 ) , 159.0 ( c-4,6 ) , 156.1 ( c-9a,10a ) , 126.5 ( c-5 ) , 113.8 ( c-4a,5a ) , 104.1 ( c-10 ) ; anal . calcd . for c10h4n4o4s2 : c , 42.85 ; h , 1.44 ; n , 9.99 . found : c , 42.71 ; h , 1.48 ; n , 10.05 .
10-hydroxy-2,8-dithioxo-2,3,7,8-tetrahydrobenzo[1,2-e : 5,4-e
]
bis ( [ 1,3]oxazine)-4,6-dione 18b . in slight modification to general procedure b , 4,5,6-trihydroxyisophthalic acid 16b ( 0.86 g , 4 mmol ) was allowed to react with the freshly prepared ph3p(scn)2 ( 10 mmol ) at room temperature for 3 hours and then under reflux for 5 hours . at the completion of the reaction ,
the mixture was filtered and the pbbr2 cake washed with 100 ml 1,4-dioxane . both dioxane and dcm filtrates were evaporated to dryness under reduced pressure and minimal toluene was added to remove any oil which may be present .
the resulting solid was recrystallised from 1 , 4 dioxane / chloroform to give 18b ( 0.55 g , 46% ) as yellow crystals , mp 286289c decomp .
max ( kbr)/cm 33003000 br ( oh ) , 3103 , 3047 , 2938 ( nh ) , 1698 ( c = o ) , 1227 ( c = s ) ; h nmr ( 200 mhz , 390 k d6-dmso ) 13.69 ( bs , 2h , 2 x nh ) , 11.64 ( bs , 1h , oh ) , 7.80 ( bs , 1h , h-5 ) ; cnmr ( 50 mhz , 390 k d6-dmso ) 181.0 ( c-2,8 ) , 156.8 ( c-4,6 ) , 148.6 ( c-9a,10a ) , 131.9 ( c-10 ) , 113.8 ( c-5 ) , 113.8 ( c-4a,5a ) ; anal . calcd .
for c10h4n4o4s2 : c , 42.85 ; h , 1.44 ; n , 9.99 . found
: c , 42.71 ; h,1.48 ; n , 10.05 .
10-methyl-2 , 8-dithioxo-2 , 3 , 7 , 8-tetrahydrobenzo[1,2-e : 5,4-e
] bis(oxazine)-4,6-dione 18c . in slight modification to general procedure b , 4,6-dihydroxy-5-methylisophthalic
acid 16c ( 1.7 g , 8 mmol ) was allowed to react with freshly prepared ph3p(scn)2 [ 22 , 34 ] ( 10 mmol ) at room temperature for 3 hours then under reflux for 5 hours . at the completion of the reaction , the reaction mixture was filtered and the pbbr2 cake washed with approx 100 ml thf and filtered .
both thf and dcm filtrates were evaporated to dryness under reduced pressure and minimal toluene was added to remove any oil , which may be present .
the solid which remained was then recrystallised using thf to give product 18c ( 1.18 g , 50% ) .
2,9-dithioxo-2,3,8,9-tetrahydrobenzo[1,2-e:4,3-e
] bis(oxazine)-4,7-dione 19 . in slight modification to the general procedure b [ 22 , 34 ]
, 2,3-dihydroxyterephthalic acid 17 ( 0.86 g , 4 mmol ) was allowed to react with freshly prepared ph3p(scn)2 ( 10 mmol ) at room temperature for 3 hours then under reflux for 5 hours . at the completion of the reaction ,
the reaction mixture was filtered and the pbbr2 cake was washed with thf ( 100 ml ) .
both thf and dcm filtrates were evaporated to dryness under reduced pressure and minimal toluene was added to remove any oil , which may be present .
the remaining solid is recrystallised from ethyl acetate to give 19 ( 0.54 g , 50% ) as yellow crystals , mp > 300c decomp .
max ( kbr)/cm 3079 , 2904 , 2864 ( nh ) , 1718 ( c = o ) , 1252 ( c = s ) ; h nmr ( 200 mhz , 390 k d6-dmso ) 13.90 ( bs , 2h , 2 x nh ) , 7.89 ( s , 2h , h-5 & h-6 ) ; c nmr ( 50 mhz , 390 k ( d6-dmso ) 181.1 ( c-2,9 ) , 157.0 ( c-4,7 ) , 142.7 ( c-10a,10b ) , 122.5 ( c-5,6 ) , 121.3 ( c-4a,6a ) ; anal . calcd . for c10h4n2o4s2 : c , 42.85 ; h , 1.44 ; n , 9.99 . found : c , 42.74 ; h , 1.50 ; n , 9.93 .
general procedure c. the appropriate 2-thio-1,3-benzoxazines ( 1.7 mmole ) 6 , 10a , b , 11c , and 12a m were suspended in a mixture of sodium bicarbonate ( 1 gm ) and water ( 5 ml)/methanol ( 5 ml ) with stirring , then the reaction mixture was warm to 40c for few minutes then benzyl amine ( 4.25 mmol ) was added dropwise , directly from the a pipette , left stirring at room temperature for 4 hours . at the completion of the reaction , the mixture was evaporated to dryness under reduced pressure and the ph was adjusted to 5 - 6 by using conc .
the resulting solid was collected by vacuum filtration and washed with minimal water and recrystallized from an appropriate solvent .
n-(benzyl carbamothioyl)-2-hydroxybenzamide
13a . 2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12a was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from toluene to give 13a ( 75% yield ) , mp 176 - 177c ( lit .
max ( kbr)/cm 3301 ( n h ) , 32002700 ( o h ) , 1661 ( c = o ) , 1605 ( c = s ) ; h nmr ( d6-dmso ) 12.42 ( bs , 1h , h-2 ) , 11.41 ( s , 1h , o
h ) , 11.00 ( t , 1h , j = 5.4 hz , h-4 ) , 7.707.00 ( m , 9h , arh ) , 4.9 ( d , 2h , j = 5.2 hz , h-5 ) ; c nmr ( d6-dmso ) 179.6 ( c-3 ) , 168.3 ( c-1 ) , 160.7 ( c-2 ) , 136.3 ( c-6 ) , 135.9 ( c-4 ) , 129.0 ( c-6 ) , 128.1 ( c-7 ) , 127.9 ( c-8 ) , 127.5 ( c-9 ) , 120.1 ( c-5 ) , 118.8 ( c-3 ) , 113.3 ( c-1 ) , 50.0 ( c-5 ) .
n-(benzyl carbamothioyl)-2-hydroxy-3-methylbenzamide
13b .
8-methyl-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12b was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from toluene to give 13b ( 79% yield ) , mp 220223c .
max ( kbr)/cm 3062 , 2883 ( n h ) , 1696 ( c = o ) , 1610 ( c = s ) ; h nmr ( d6-dmso ) 11.32 ( bs , 1h , h-2 ) , 10.73 ( s , 1h , o
h ) , 8.80 ( t , 1h , j = 5.4 hz , h-4 ) , 7.82 ( d , 1h , j = 7.5 hz , h-6 ) , 7.407.20 ( m , arh , h-4 ) , 6.8 ( t , 1h , j = 7.5 hz , h-5 ) , 4.43 ( d , 2h , j = 6.0 hz , h-5 ) , 2.20 ( s , 3h , 8-ch3 ) ; c nmr ( d6-dmso ) 169.9 ( c-3 ) , 157.7 ( c-1 ) , 152.9 ( c-2 ) , 137.5 ( c-6 ) , 136.0 ( c-4 ) , 128.4 ( c-7 ) , 127.3 ( c-8 ) , 127.0 ( c-9 ) , 126.9 ( c-3 ) , 126.5 ( c-6 ) , 119.0 ( c-5 ) , 115.1 ( c-1 ) , 42.9 ( c-5 ) , 15.9 ( ch3 ) ; anal . calcd . for c16h16n2o3 :
c , 67.59 ; h , 5.67 ; n , 9.85 . found : c , 67.10 ; h , 5.38 ; n , 9.25 .
8-phenyl-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12c was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from toluene to give 13c ( 80% yield ) , mp 250253c .
max ( kbr)/cm 3062 , 2883 ( n h ) , 1686 ( c = o ) , 1595 ( c = s ) ; h nmr ( d6-dmso ) 11.30 ( s , 1h , 2-n
h ) , 10.70 ( s , 1h , o h ) , 8.80 ( t , 1h , j = 5.4 hz , 4-n
h ) , 7.90 ( d , 1h , j = 7.6 hz , h-6 ) , 7.80 ( d , 1h , j = 7.6 hz , h-4 ) , 7.60 ( dd , 1h , jh8,h10 = 1.6 hz , jh8,h9 = 8.0 hz , h-8 ) , 7.50 ( m , 4h , h-5/h-9/h-10 ) , 7.307.20 ( m , arh , h-4 ) , 4.90 ( d , 2h , j = 6.0 hz , h-5 ) ; c nmr ( d6-dmso ) 169.4 ( c-3 ) , 157.7 ( c-1 ) , 155.3 ( c-2 ) , 137.2 ( c-6 ) , 134.2 ( c-4 ) , 129.5 ( c-6 ) , 128.4 ( c-7 ) , 127.3 ( c-8 ) , 127.0 ( c-9 ) , 126.3 ( c-3 ) , 120.1 ( c-1 ) , 118.9 ( c-5 ) , 50.1 ( c-5 ) ; anal . calcd . for c15h14n2o2s :
c , 62.92 ; h , 4.93 ; n , 9.78 . found : c , 63.03 ; h , 4.88 ; n , 9.80 .
6-bromo-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12d was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from toluene to give 13d ( 82% yield ) , mp 185c .
max ( kbr)/cm 3296 , 3073 ( n h ) , 1678 ( c = o ) , 1601 ( c = s ) ; h nmr ( d6-dmso ) 12.42 ( bs , 1h , h-2 ) , 11.41 ( s , 1h , o
h ) , 11.00 ( t , 1h , j = 5.4 hz , h-4 ) , 7.90 ( d , 1h , j = 2.2 hz , h-6 ) , 7.60 ( dd , 1h , jh4,h6 = 2.2 hz , jh4,h3 = 7.5 hz , h-4 ) , 7.407.30 ( m , 5h , arh ) , 7.00 ( d , 1h , j = 7.5 hz , h-3 ) , 4.90 ( d , 2h , j = 5.5 hz , h-5 ) ; c nmr ( d6-dmso ) 179.4 ( c-3 ) , 163.3 ( c-1 ) , 155.9 ( c-2 ) , 137.4 ( c-4 ) , 137.3 ( c-6 ) , 132.9 ( c-6 ) , 128.5 ( c-7 ) , 127.6 ( c-8 ) , 127.3 ( c-9 ) , 119.7 ( c-1 ) , 118.8 ( c-3 ) , 111.1 ( c-5 ) , 48.3 ( c-5 ) ; anal . calcd . for c15h13brn2o2s
: c , 49.33 ; h , 3.59 ; n , 7.67 . found : c , 49.39 ; h , 3.68 ; n , 7.93 .
n-(benzyl carbamothioyl)-5-ethoxy-2-hydroxybenzamide
13e . 6-ethoxy-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12e was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from toluene to give 13e ( 87% yield ) , mp 210c .
max ( kbr)/cm 3300 , 3051 ( n h ) , 1665 ( c = o ) , 1630 ( c = s ) ; h nmr ( d6-dmso ) 12.00 ( bs , 1h , h-2 ) , 11.32 ( s , 1h , o
h ) , 11.12 ( t , 1h , j = 5.4 hz , h-4 ) , 7.50 ( s , 1h , h-6 ) , 7.307.10 ( m , 7h , arh / h-3/h-4 ) , 4.80 ( d , 2h , j = 5.5 hz , h-5 ) , 4.104.00 ( q , 2h , j = 6.7 hz , o
ch2 ) , 1.301.20 ( t , 3h , j = 6.7 hz , ch3 ) ; c nmr ( d6-dmso ) 179.8 ( c-3 ) , 164.3 ( c-1 ) , 164.0 ( c-2 ) , 158.3 ( c-5 ) , 137.4 ( c-6 ) , 128.5 ( c-7 ) , 127.6 ( c-8 ) , 127.3 ( c-9 ) , 120.2 ( c-1 ) , 107.9 ( c-3 ) , 107.7 ( c-4 ) , 101.7 ( c-6 ) , 63.6 ( o ch2 ) , 48.2 ( c-5 ) , 14.5 ( ch3 ) ; anal . calcd . for c17h18n2o3s : c , 61.80 ; h , 5.49 ; n , 8.48 . found : c , 61.90 ; h , 5.55 ; n , 8.61 .
n-(benzyl carbamothioyl)-4-ethoxy-2-hydroxybenzamide
13f . 7-ethoxy-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12f was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from ethyl acetate to give 13f ( 73% yield ) , mp 215218c .
max ( kbr)/cm 3307 , 3071 ( n h ) , 1655 ( c = o ) , 1506 ( c = s ) ; h nmr ( d6-dmso ) 11.91 ( bs , 1h , h-2 ) , 11.00 ( s , 1h , o
h ) , 10.90 ( t , 1h , j = 5.6 hz , h-4 ) , 7.80 ( d , 1h , j = 8.9 hz , h-6 ) , 7.307.20 ( m , 5h , arh ) , 6.60 ( d , 1h , j = 8.9 hz , h-5 ) , 6.40 ( sd , 1h , j = 1.5 hz , h-3 ) , 4.80 ( d , 2h , j = 5.5 hz , h-5 ) , 4.104.00 ( q , 2h , j = 6.7 hz , o
ch2 ) , 1.301.20 ( t , 3h , j = 6.7 hz , ch3 ) ; c nmr ( d6-dmso ) 179.8 ( c-3 ) , 164.3 ( c-1 ) , 164.0 ( c-4 ) , 158.3 ( c-2 ) , 137.4 ( c-6 ) , 130.9 ( c-6 ) , 128.5 ( c-7 ) , 127.6 ( c-8 ) , 127.3 ( c-9 ) , 109.2 ( c-5 ) , 107.9 ( c-1 ) , 101.7 ( c-3 ) , 63.6 ( o ch2 ) , 48.2 ( c-5 ) , 14.5 ( ch3 ) ; anal . calcd . for c17h18n2o3s c , 61.80 ; h , 5.49 ; n , 8.48 . found : c , 62.02 ; h , 5.55 ; n , 8.51 .
8-ethoxy-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12 g was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from toluene to give 13 g ( 82% yield ) , mp 188190c .
max ( kbr)/cm 3310 , 3080 ( n h ) , 1656 ( c = o ) , 1525 ( c = s ) ; h nmr ( d6-dmso ) 11.80 ( s , 1h , 2-n
h ) , 11.60 ( s , 1h , o h ) , 11.00 ( t , 1h , j = 5.4 hz , 4-n h ) , 7.307.20 ( m , 6h , arh / h-5 ) , 7.10 ( d , 1h , j = 7.5 hz , h-6 ) , 6.90 ( d , 1h , j = 7.5 hz , h-4 ) , 4.80 ( d , 2h , j = 5.5 hz , h-5 ) , 4.003.90 ( q , 2h , j = 6.7 hz , o
ch2 ) , 1.301.20 ( t , 3h , j = 6.7 hz , ch3 ) ; c nmr ( d6-dmso ) 179.5 ( c-3 ) , 164.3 ( c-1 ) , 151.8 ( c-3 ) , 150.5 ( c-2 ) , 137.3 ( c-6 ) , 128.5 ( c-7 ) , 127.6 ( c-8 ) , 127.3 ( c-9 ) , 123.0 ( c-5 ) , 118.5 ( c-6 ) , 116.5 ( c-1 ) , 114.2 ( c-4 ) , 63.5 ( o ch2 ) , 48.3 ( c-5 ) , 14.6 ( ch3 ) ; anal . calcd . for c17h18n2o3s : c , 61.80 ; h , 5.49 ; n , 8.48 . found : c , 61.96 ; h , 5.61 ; n , 8.54 .
n-(benzyl carbamothioyl)-2-hydroxy-5-methoxybenzamide
13h .
6-methoxy-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12h was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from ethyl acetate to give 13h ( 69% yield ) , mp 140c .
max ( kbr)/cm 3313 , 3081 ( n h ) , 1667 ( c = o ) , 1610 ( c = c ) , 1516 ( c = s ) ; h nmr ( d6-dmso ) 12.40 ( s , 1h , 2-n
h ) , 11.30 ( s , 1h , o h ) , 11.20 ( t , 1h , j = 5.4 hz , 4-n h ) , 7.507.30 ( m , 8h , arh / h-3/h-4/h-6 ) , 3.80 ( s , 3h , o
ch3 ) ; c nmr ( d6-dmso ) 179.9 ( c-3 ) , 165.4 ( c-1 ) , 151.5 ( c-5 ) , 149.0 ( c-2 ) , 137.4 ( c-6 ) , 128.5 ( c-7 ) , 127.6 ( c-8 ) , 127.3 ( c-9 ) , 121.8 ( c-3 ) , 117.9 ( c-4 ) , 116.4 ( c-1 ) , 56.2 ( o ch3 ) , 48.1 ( c-5 ) ; anal . calcd . for c16h16n2o3s : c , 60.74 ; h , 5.10 ; n , 8.85 .
n-(benzyl carbamothioyl)-2-hydroxy-4-methoxybenzamide
13i . 7-methoxy-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12i was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from ethyl acetate to give 13i ( 87% yield ) , mp 210c .
max ( kbr)/cm 3313 , 3081 ( n h ) , 1667 ( c = o ) , 1610 ( c = c ) , 1520 ( c = s ) ; h nmr ( d6-dmso ) 12.40 ( s , 1h , 2-n
h ) , 11.30 ( s , 1h , o h ) , 11.20 ( t , 1h , j = 5.4 hz , 4-n h ) , 7.70 ( d , 1h , j = 7.5 hz , h-6 ) , 7.507.30 ( m , 5h , arh ) , 6.70 ( m , 2h , h-3/h-5 ) , 3.80 ( s , 3h , o
ch3 ) ; c nmr ( d6-dmso ) 179.9 ( c-3 ) , 165.4 ( c-1 ) , 160.5 ( c-4 ) , 159.0 ( c-2 ) , 137.4 ( c-6 ) , 128.5 ( c-7 ) , 127.6 ( c-8 ) , 127.3 ( c-9 ) , 126.8 ( c-6 ) , 117.9 ( c-5 ) , 116.4 ( c-1 ) , 106.0 ( c-3 ) , 56.2 ( o ch3 ) , 48.1 ( c-5 ) ; anal . calcd . for c16h16n2o3s : c , 60.74 ; h , 5.10 ; n , 8.85 .
n-(benzyl carbamothioyl)-2-hydroxy-3-methoxybenzamide
13j . 8-methoxy-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12j was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from toluene to give 13j ( 82% yield ) , mp 190193c .
max ( kbr)/cm 3313 , 3081 ( n h ) , 1667 ( c = o ) , 1610 ( c = c ) , 1518 ( c = s ) ; h nmr ( d6-dmso ) 12.40 ( s , 1h , 2n
h ) , 11.30 ( s , 1h , o h ) , 11.20 ( t , 1h , j = 5.4 hz , 4-n h ) , 7.50 - 7.20 ( m , 8h , arh / h-4/h-5/h-6 ) , 3.80 ( s , 3h , o
ch3 ) ; c nmr ( d6-dmso ) 179.9 ( c-3 ) , 165.4 ( c-1 ) , 149.5 ( c-3 ) , 148.0 ( c-2 ) , 137.4 ( c-6 ) , 128.5 ( c-7 ) , 127.6 ( c-8 ) , 127.3 ( c-9 ) , 121.8 ( c-6 ) , 117.9 ( c-5 ) , 116.4 ( c-1 ) , 116.0 ( c-4 ) , 56.2 ( o ch3 ) , 48.1 ( c-5 ) ; anal . calcd . for c16h16n2o3s : c , 60.74 ; h , 5.10 ; n , 8.85 .
n-(benzyl carbamothioyl)-2-hydroxy-3,5-diiodobenzamide
13k . 6,8-diiodo-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12k was allowed to react with benzylamine according to general procedure c. the solid was collected and recrystallized from toluene to give 13k ( 75% yield ) , mp 173175c .
max ( kbr)/cm 3264 , 3025 ( n h ) , 1633 ( c = o ) ,
h ) , 10.90 ( t , 1h , j = 5.4 hz , 4-n h ) , 8.20 ( d , 1h , j = 2.2 hz , h-6 ) , 8.00 ( d , 1h , j = 2.2 hz , h-4 ) , 7.407.30 ( m , 5h , arh ) , 4.90 ( d , 2h , j = 5.5 hz , h-5 ) , c nmr ( d6-dmso ) 179.8 ( c-3 ) , 165.6 ( c-1 ) , 157.0 ( c-2 ) , 149.7 ( c-4 ) , 137.9 ( c-6 ) , 137.3 ( c-6 ) , 128.5 ( c-7 ) , 127.7 ( c-8 ) , 127.3 ( c-9 ) , 121.1 ( c-1 ) , 91.75 ( c-5 ) , 81.9 ( c-3 ) , 48.2 ( c-5 ) ; anal . calcd . for c15h12i2n2o2s : c , 33.48 ; h , 2.25 ; n , 5.21 . found : c , 33.43 ; h , 2.66 ; n , 5.78 .
n-(benzyl carbamothioyl)-2,4-dihydroxybenzamide
13l . 7-hydroxy-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12l was allowed to react with benzylamine according to general procedure c. the solid was collected and recrystallized from toluene to give 13l ( 87% yield ) , mp190c .
max ( kbr)/cm 32002700 ( o h ) , 3118 , 3033 ( n h ) , 1665 ( c = o ) ,
1626 ( c = c ) , 1554 ( c = s ) ; h nmr ( d6-dmso ) 12.10 ( bs , 1h , o
h ) , 11.20 ( t , 1h , j = 5.4 hz , n
h ) , 10.50 ( o h ) , 7.80 ( d , 1h , j = 7.6 hz , h-6 ) , 7.30 ( m , 5h , arh ) , 6.40 ( m , 2h , h-3/h-5 ) , 4.80 ( d , 2h , j = 5.7 hz , h-5 ) ; c nmr ( d6-dmso ) 180.0 ( c-3 ) , 164.7 ( c-1 ) , 163.9 ( c-4 ) , 158.5 ( c-2 ) , 137.5 ( c-6 ) , 133.3 ( c-6 ) , 128.7 ( c-7 ) , 127.7 ( c-8 ) , 127.5 ( c-9 ) , 109.2 ( c-5 ) , 107.9 ( c-1 ) , 102.9 ( c-3 ) , 48.2 ( c-5 ) ; anal . calcd . for c15h14n2o3s
: c , 59.59 ; h , 4,67 ; n , 9.27 . found : c , 59.88 ; h , 4.68 ; n , 9.59 .
7-hydroxy-8-methyl-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12 m was allowed to react with benzylamine according to general procedure c. the solid was collected and recrystallized from ethanol to give 13 m ( 78% yield ) , mp 193195c .
max ( kbr ) 3381 , 3225 ( o h ) , 30202800 ( n h ) , 1639 ( c = o ) , 1613 ( c = s ) ; h nmr ( d6-dmso ) 11.60 ( bs , 1h , 2-oh ) , 11.10 ( t , 1h , j = 5.6 hz , 4-nh ) , 10.40 ( bs , 1h , 2-nh ) , 7.70 ( d , 1h , jh6,h5 = 8.8 hz ; h-6 ) , 7.407.30 ( m , 6h / h-7/h-8/h-9/4-oh ) , 6.50 ( d , 1h , jh5,h6 = 8.8 hz ; h-5 ) , 4.90 ( d , 2h , j = 5.6 hz , h-5 ) , 2.00 ( s , 3h , 3-ch3 ) ; c nmr ( d6-dmso ) 179.9 ( c-3 ) , 166.1 ( c-1 ) , 161.5 ( c-4 ) , 157.2 ( c-2 ) , 137.3 ( c-6 ) , 129.1 ( c-7 ) , 128.4 ( c-8 ) , 127.5 ( c-9 ) , 127.2 ( c-6 , c-3 ) , 111.8 ( c-1 ) , 108.3 ( c-5 ) , 48.1 ( c-5 ) , 8.6 ( 3-ch3 ) . anal . calcd . for c16h16n2o3s c , 60.74
; h , 5.10 ; n , 8.85 . found : c , 60.60 ; h , 5.08 ; n , 8.80 ; % .
n-(benzyl carbamothioyl)-2,3-dihydroxybenzamide
13n . 8-hydroxy-2-thioxo-2h - benz[e]-1,3-oxazin-4(3h)-one 12n was allowed to react with benzyl amine according to general procedure c. the crude solid was collected and recrystallized from ethanol to give 13n ( 72% yield ) , mp 172c .
max ( kbr)/cm 3411 , 2942 br ( o h ) , 3118 , 3033 ( n h ) , 1661 ( c = o ) ,
1626 ( c = c ) , 1553 ( c = s ) ; h nmr ( d6-dmso ) 11.61 ( s , 1h , 2-n
h ) , 11.22 ( t , 1h , j = 5.6 hz , 4-n h ) , 10.53 ( o h ) , 7.30 ( m , 7h , arh / h-6 ) , 7.00 ( d , 1h , j = 7.6 hz , h-4 ) , 6.70 ( t , 1h , j = 7.9 hz , h-5 ) , 4.80 ( d , 2h , j = 5.7 hz , h-5 ) ; c nmr ( d6-dmso ) 179.7 ( c-3 ) , 165.0 ( c-1 ) , 146.6 ( c-2 ) , 146.3 ( c-3 ) , 137.3 ( c-6 ) , 128.5 ( c-7 ) , 127.6 ( c-8 ) , 127.3 ( c-9 ) , 120.5 ( c-5 ) , 119.4 ( c-4 ) , 119.2 ( c-5 ) , 116.8 ( c-1 ) , 48.2 ( c-5 ) ; anal . calcd . for c15h14n2o3s
: c , 59.59 ; h , 4.67 ; n , 9.27 . found : c , 60.08 ; h , 5.06 ; n , 9.59 .
4-amino - n-(benzyl carbamothioyl)-2-hydroxybenzamide
13o . 7-amino-2-thioxo-2h - benz[e]oxazin-4(3h)-one 10b was allowed to react with benzylamine following general procedure c. the resulting solid was collected and recrystallised from methanol / water to give 13o ( 0.35 g , 78% ) as off white solid , mp 290293c decomp .
max ( kbr)/cm 35003200 ( oh ) , 3442 , 3332 ( nh ) , 1750 , 1677 ( c = o ) , 1388 ( c = s);. hnmr ( 200 mhz , 300 k , d6-dmso ) 11.54 ( s , 1h , 8-nh exchangeable with d2o ) , 11.3311.28 ( bm , 2h , 10-nh , 2-oh exchangeable with d2o ) , 7.60 ( d , 1h , jh6,h5 = 8.6 hz , h-6 ) , 7.337.29 ( m , 7h , ar , h-11 , h-12 , h-13 and 4-nh2 exchangeable with d2o ) , 6.21 ( dd , 1h , jh5,h6&h5h3 = 8.6 hz , jh5,h3 = 1.6 hz , h-3 ) , ( d , 1h , jh3,h5 = 1.6 hz , h-3 ) , 4.84 ( d , 2h , jh11,h10 = 5.7 hz , h-11 ) ; c nmr ( 50 mhz , 300 k , d6-dmso ) 180.3 ( c-9 ) , 165.2 ( c-7 ) , 158.6 ( c-2 ) , 155.1 ( c-4 ) , 137.7 ( c-12 ) , 133.2 ( c-6 ) , 128.9 ( c-14 ) , 127.8 , 127.7 ( c-13 , c-15 ) , 108.2 ( c-5 ) , 104.3 ( c-1 ) , 99.7 ( c-3 ) , 48.4 ( c-11 ) ; anal . calcd . for c15h15n3o2s : c , 59.78 ; h , 5.02 ; n , 13.94 .
n-(4-oxo-2-thioxo-3 , 4-dihydro-2h - benz[e]oxazin-7-yl ) acetamide 10a was allowed to react with benzylamine following general procedure c. the resulting solid was collected and recrystallised from ethanol to give 13p ( 0.27 g , 47% ) as off white crystals .
max ( kbr)/cm 35003200 ( oh ) , 3292 , 3113 , ( nh ) , 1656 ( c = o ) , 1374 ( c = s ) ; hnmr ( 200 mhz , 300 k , d6-dmso ) 12.09 ( s , 1h , 16-nh ) , 11.40 ( s , 1h , 8-nh ) , 11.18 ( t , 1h , jh10,h11 = 4.6 hz , 10-nh ) , 10.26 ( s , 1h , 2-oh ) , 7.84 ( d , 1h , jh6,h5 = 7.8 hz h-6 ) , 7.64 ( s , 1h , h-3 ) , 7.377.30 ( m , 5h , ar , h-13 , h-14 , h-15 ) , 7.02 ( d , 1h , jh5,h6 = 7.8 hz , h-5 ) , 4.85 ( d , 2h , jh11,h10 = 4.8 hz , h-11 ) , 2.07 ( s , 3h , ch3 ) ; c nmr ( 50 mhz , 300 k , d6-dmso ) 179.7 ( c-9 ) , 169.1 ( c-7 ) , 164.2 ( c-17 ) , 157.4 ( c-2 ) , 145.3 ( c-4 ) , 137.3 ( c-12 ) , 131.9 ( c-6 ) , 128.5 , 127.6 , 127.3 ( c-13 , c-14 , c-15 ) , 111.0 , 110.8 ( c-1 , c-5 ) , 105.9 ( c-3 ) , 48.1 ( c-11 ) , 24.2 ( c-18 ) ; anal . calcd . for c17h17n3o3s : c , 59.46 ; h , 4.97 ; n , 12.24 .
3-ethoxy-2-hydroxy-1,3-benzoxazine 11c was allowed to react with benzylamine according to the general procedure c. the resulting solid was collected and recrystallised from acetonitrile to give 13q ( 0.59 g , 77% ) as yellow crystals , mp 201204c .
max ( kbr)/cm 35003200 ( oh ) , 3496 , 3395 , 3256 ( nh ) , 1686 , 1646 ( c = o ) , 1342 ( c = s ) ; hnmr ( 200 mhz , 300 k , d6-dmso ) 11.26 ( 8-nh ) , 8.33 ( m , 1h , 2-oh ) , 7.63 ( d , 1h , jh6,h5 = 9.0 hz , h-6 ) , 7.667.28 ( m , 5h , ar h-13 , h-14 , h-15 ) , 7.1 ( t , 1h , jh16,h17 = 5.1 hz , h-16 ) , 6.926.66 ( m , 5h , h-21 , h-22 , h-25 , 10-nh and 19-oh exchangeable with d2o ) , 6.30 ( dd , 1h , jh5,h6&h5,h3 = 9.0 hz , jh5,h3 = 1.6 hz , h-5 ) , 6.14 ( d , 1h , jh3,h5 = 1.6 hz , h-3 ) , 4.85 ( d , 2h , jh11,h10 = 5.1 hz , h-11 ) , 4.25 ( d , 2h , jh17,h16 = 5.3 hz , h-17 ) , 4.084.01 ( m , 3h , jh24,h25 = 7.0 hz , h-24 and 16-nh ) , 1.45 ( t , 3h , jh25,h24 = 7.0 hz , h-25 ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 180.1 ( c-9 ) , 164.8 ( c-7 ) , 158.2 ( c-2 ) , 154.7 ( c-4 ) , 146.3 , 144.1 ( c-20 , c-19 ) , 137.2 ( c-12 ) , 132.1 ( c-6 ) , 128.2 , 127.3 , 127.0 ( c-14 , c-13 , c-15 ) , 125.1 ( c-18 ) , 119.9 , 118.5 ( c-21 , c-23 ) , 111.8 ( c-22 ) , 106.1 , 103.6 ( c-1 , c-5 ) , 97.2 ( c-3 ) , 64.1 ( c-24 ) , 47.9 ( c-17 ) , 40.8 ( c-11 ) , 14.4 ( c-25 ) ; anal . calcd . for c24h25n3o4sh2o : c , 61.39 ; h , 5.80 ; n , 8.95 . found : c , 61.59 ; h , 5.57 ; n , 9.38 . in slight modification to the general procedure c , 2 , 8-dithioxo-2 , 3 , 7 , 8-tetrahydrobenzo[1,2-e : 5,4-e]bis(oxazine)-4,6-dione 18a ( 1 mmol , 0.28 g ) was allowed to react with benzylamine ( 3 mmol .
0.32 g ) and sodium hydrogen carbonate solution ( 1 g in 12 ml methanol and 2.4 ml water ) for 16 hour .
the resulting solid was collected and recrystallised from ethanol to give 20a ( 0.31 g , 63% ) as off white crystals.mp 276279c decomp .
max ( kbr)/cm 3313 , ( nh ) , 1672 ( c = o ) , 1328 ( c = s ) , hnmr ( 200 mhz , 340 k , d6-dmso ) 11.25 ( bs , 2h , 2 x nh ) , 11.06 ( t , 2h , jh10,h11 = 5.7 hz , h-10,18 ) , 8.60 ( s , 1h , h-6 ) , 7.387.29 ( 10h , 2 x ar ) , 6.63 ( s , 1h , h-3 ) , 4.87 ( d , 4h , jh11,h10 = 5.7 hz , h-11,20 ) , 3.3 ( oh under the water envelope ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 179.5 ( c-9,17 ) , 163.3 ( c-7,15 ) , 161.5 ( c-2,4 ) , 137.0 , 136.6 ( c-11 , 20 and c-6 ) , 128.2 , 127.3 , 127.0 ( c-13 , 22 , c-12 , 21 and c-14 , 25 ) , 110.3 ( c-1,15 ) , 103.7 ( c-3 ) , 48.0 ( c-11,19 ) ; anal . calcd . for c24h22n4o4s2 : c , 58.28 ; h , 4.48 ; n , 11.33 . found : c , 58.37 ; h , 4.67 ; n , 11.22 . in slight modification to general procedure c , 10-methyl-2,8-bis(methylthio)benzo[1,2-e : 5,4-e]bis(oxazine)-4,6-dione 18c ( 1 mmol , 0.32 g ) was allowed to react with benzylamine ( 3 mmol .
0.32 g ) , sodium hydrogen carbonate solution ( 1 g in 12 ml methanol and 2.4 ml water ) for 16 h. the resulting solid was collected and recrystallised from ethanol to give 20c ( 0.25 g , 49% ) as off white crystals .
max ( kbr)/cm 35003200 ( oh ) , 3414 , 3257 ( nh ) , 1651 ( c = o ) , 1321 ( c = s ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 11.51 ( bs , 2h , 2 x nh ) , 10.96 ( s , 2h , jh10-h11 = 5.7 hz , h-10,19 ) , 8.48 ( s , 1h , h-6 ) , 7.397.28 ( m , 10h , 2 x ar ) , 4.90 ( d , 2h , jh9,h8 = 5.7 hz , h-11 , 20 ) , 2.10 ( s , 3h , h-3 ) , 3.3 ( oh under the water envelope ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 179.6 ( c-9 , 18 ) , 166.3 ( c-7,16 ) , 161.3 ( c-2 , 4 ) , 136.9 ( c-12 , 21 ) , 131.2 ( c-4 ) , 128.1 ( c-14 , 23 ) , 127.3 , 127.0 ( c-13 , 22 and c-15 , 24 ) , 113.2 ( c-1,5 ) , 108.9 ( c-3 ) , 48.1 ( c-11 , 20 ) , 8.4 ( c-1 , 5 ) ; anal . calcd . for c25h25n4o4sh2o : c , 57.02 ; h , 4.98 ; n , 10.64 .
found : c , 57.32 ; h , 4.48 ; n , 10.85 . in slight modification to the general procedure c , 2,9-dithioxo-2,3,8,9-tetrahydrobenzo[1,2-e:4,3-e]bis(oxazine)-4,7-dione 19 ( 1 mmol , 0.28 g ) was allowed to react with benzylamine ( 3 mmol .
0.32 g ) , sodium hydrogen carbonate solution ( 1 g in 12 ml methanol and 2.4 ml water ) for 16 hour .
the resulting solid was collected and recrystallised using ethanol to give 21 ( 0.31 g , 63% ) as off white crystals mp 195198c .
max ( kbr)/cm 35003200 ( oh ) , 3401 , 3248 ( nh ) , 1671 , 1649 ( c = o ) , 1338 ( c = s ) ; hnmr ( 200 mhz , 300 k , d6-dmso ) 11.88 ( bs , 2 x nh , h-8,17 ) , 11.07 ( t , 1h , jh10,h11 = 5.9 hz , h-10,19 ) , 8.56 ( bs , 2h , 2,3-oh ) , 7.397.28 ( m , 12h , 2 x ar and 2 x ch , h-13 , h-14 , h-15 and h-5,6 ) , 4.88 ( d , 4h , jh11,h10 = 5.9 hz , h-11,20 ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 179.6 ( c-9,18 ) , 164.9 ( c-7,16 ) , 147.9 ( c-2,3 ) , 136.9 ( c-12 , 21 ) , 128.2 ( c-14,23 ) , 127.3 ( c-13,22 ) , 127.0 ( c-15,24 ) , 120.9 ( c-1,4 ) , 119.2 ( c-5,6 ) ; anal . calcd . for c24h22n4o4s2h2o : c , 56.23 ; h , 4.72 ; n , 11.33 . found : c , 55.79 ; h , 4.78 ; n , 10.93 .
n-(4-oxo-2-thioxo-3,4-dihydro-2h - benz[e]oxazin-7-yl)acetamide 10a ( 0.59 g , 2.5 mmol ) was allowed to react with methyl iodide following the previously reported .
the resulting beige solid 14h ( 0.61 g , 97% ) is collected and used without further purification but can be crystallised from ethanol , mp 268269c max ( kbr)/cm 3278 , 3142 , 3110 ( nh ) , 3062 ( ch ar ) , 2929 ( ch aliphatic ) , 1760 , 1709 , 1671 ( c = o ) , 1615 ( c =
n ) , 1554 ( c = c ) ; hnmr ( 200 mhz , 300 k d6-dmso ) 10.60 ( s , 1h , 9-nh ) , 7.88 ( b , 1h , h-8 ) , 7.87 ( d , 1h , jh5,h6 = 8.6 hz , h-5 ) , 7.45 ( d , 1h , jh6,h5 = 8.6 hz , h-6 ) , 2.58 ( s , 3h , 11-ch3 ) , 2.12 ( s , 3h , 3-ch3 ) ; c nmr ( 50 mhz , 300 k , d6-dmso ) 172.8 ( c-2 ) , 169.6 ( c-10 ) , 162.1 ( c-4 ) , 155.8 ( c-8a ) , 145.2 ( c-7 ) , 128.0 ( c-5 ) , 117.5 ( c-6 ) , 112.1 ( c-4a ) , 104.4 ( c-8 ) ; anal . calcd . for c15h12n2o2s : c , 59.99 ; h , 4.03 ; n , 9.33 .
general procedure d. the appropriate 2-methylthio-1,3-benzoxazine 14a h ( 2.5 mmol ) was suspended in dry 1,4-dioxane ( 10 ml ) in a 50 ml round - bottomed flask .
benzyl amine ( 12.5 mmol ) was then added dropwise , directly from the pipette , with stirring , and then the reaction mixture was heated to reflux for 4 hours . at the completion of the reaction ,
the reaction mixture was evaporated to dryness under reduced pressure and triturated with minimal diethyl ether .
the resulting solid product 14 was collected by vacuum filtration and recrystallized from an appropriate solvent .
general procedure e. n-(benzyl carbamothioyl)-substituted-2-hydroxy - benzamides 13a , b , e , f , and g ( 0.5 mmol ) were suspended in acetic acid ( 3 ml ) in a 25 ml round - bottomed flask .
the reaction mixture was heated to reflux for 2 hours then ; the acetic acid was evaporated off under reduced pressure .
the oily reaction mixture was triturated with minimal diethyl ether and the resulting solid products 15a , b , e , f , and g were collected by vacuum filtration and recrystallized from an appropriate solvent .
products 15a , b , e , f , and g prepared in this procedure gave identical mp , ir , h nmr and c nmr to the analogues prepared from compound 14 with comparable yields ( scheme 3 ) .
2-(benzyl amino)-4h - benz[e]-1,3-oxazin-4-one
15a . 2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14a was allowed to react with benzyl amine according to general procedure d. the crude solid was collected and recrystallized from ethanol to give 15a ( 75% yield ) , mp 210c .
max ( kbr)/cm 3065 , 2872 ( n h ) , 1681 ( c = o ) ,
n ) ; h nmr ( d6-dmso ) 8.70 ( bs , 1h , n
h ) , 7.90 ( d , 1h , j = 7.5 hz , h-5 ) , 7.70 ( t , 1h , j = 7.5 hz , h-6 ) , 7.607.30 ( m , 7h , arh , h-7 , h-8 ) , 4.50 ( s , 2h , h-9 ) ; c nmr ( d6-dmso ) 165.4 ( c-4 ) , 154.9 ( c-2 ) , 151.6 ( c-8a ) , 137.6 ( c-1 ) , 135.2 ( c-7 ) , 127.7 ( c-2 ) , 126.8 ( c-4 ) , 126.4 ( c-3 ) , 124.3 ( c-5 ) , 124.7 ( c-6 ) , 124.5 ( c-8 ) , 117.0 ( c-4a ) , 43.8 ( c-9 ) ; anal . calcd . for c15h12n2o2 : c , 71.42 ; h , 4.79 ; n , 11.10 . found : c , 71.65 ; h , 4.95 ; n , 11.25 .
2-(benzyl amino)-8-methyl-4h - benz[e]-1,3-oxazin-4-one
15b . 8-methyl-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14b was allowed to react with benzyl amine according to general procedure d. the crude solid was collected and recrystallized from ethyl acetate to give 15b ( 72% yield ) , mp 257258c .
max ( kbr)/cm 3062 , 2883 ( n h ) , 1678 ( c = o ) ,
n ) ; h nmr ( d6-dmso ) 8.80 ( bs , 1h , n
h ) , 7.70 ( d , 1h , j = 7.5 hz , h-5 ) , 7.50 ( d , 1h , j = 7.5 hz , h-7 ) , 7.407.20 ( m , arh / h-6 ) , 4.50 ( s , 2h , h-9 ) 2.30 ( s , 3h , 8-ch3 ) ; c nmr ( d6-dmso ) 165.4 ( c-4 ) , 157.9 ( c-2 ) , 151.6 ( c-8a ) , 137.6 ( c-1 ) , 134.2 ( c-7 ) , 127.7 ( c-2 ) , 126.8 ( c-4 ) , 126.4 ( c-3 ) , 124.3 ( c-5 ) , 124.7 ( c-6 ) , 124.5 ( c-8 ) , 117.0 ( c-4a ) , 43.8 ( c-9 ) , 13.5 ( ch3 ) ; anal . calcd . for c16h14n2o2 :
c , 72.16 ; h , 5.30 ; n , 10.52 . found : c , 72.10 ; h , 5.38 ; n , 10.25 .
2-(benzyl amino)-8-phenyl-4h - benz[e]-1,3-oxazin-4-one
15c . 8-phenyl-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14c was allowed to react with benzyl amine according to general procedure d. the crude solid was collected and recrystallized from ethanol to give 15c ( 65% yield ) , mp 215c .
max ( kbr)/cm 3040 , 2860 ( n h ) , 1679 ( c = o ) ,
n ) ; h nmr ( d6-dmso ) 9.20 ( bs , 1h , n
h ) , 7.90 ( d , 1h , j = 7.5 hz , h-5 ) , 7.707.20 ( m , 11h , arh / arh / h-7 ) , 7.90 ( d , 1h , j = 3.0 hz , h-6 ) , 4.50 ( s , 2h , h-9 ) ; c nmr ( d6-dmso ) 165.1 ( c-4 ) , 157.9 ( c-2 ) , 151.2 ( c-8a ) , 137.6 ( c-1 ) , 134.7 ( c-7 ) , 134.1 ( c-5 ) , 129.2125.8 ( c-2 , c-4 c-3 , c-8 c-9 , c-10 c-11 , c-12 ) , 124.8 ( c-6 ) , 117.7 ( c-4a ) , 43.9 ( c-9 ) ; anal . calcd . for c21h16n2o2
0.5h2o : c , 76.81 ; h , 4.91 ; n , 8.53 . found : c , 74.31 ; h , 4.99 ; n , 8.46 .
2-(benzyl amino)-7-methoxy-4h-1,3-benzoxazin-4-one
15d . 7-methoxy-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14d was allowed to react with benzylamine according to general procedure d. the crude solid was collected and recrystallised from toluene to give 15d ( 83% yield ) , mp 234236c .
max ( kbr)/cm 30692891 ( n h ) , 1678 ( c = o ) ,
n ) ; h nmr ( d6-dmso ) 9.00 ( bs , 1h , n
h ) , 7.80 ( d , 1h , j = 8.6 hz , h-5 ) , 7.30 ( m , 5h , arh ) , 6.90 ( dd , 1h , jh6,h8 = 2.4 hz , jh6,h5 = 8.6 hz , h-6 ) , 6.70 ( d , 1h , j = 2.2 hz , h-8 ) , 4.50 ( s , 2h , h-9 ) , 3.90 ( s , 3h , 7-och3 ) ; c nmr ( d6-dmso ) 164.7 ( c-4 ) , 163.3 ( c-7 ) , 157.9 ( c-2 ) , 154.5 ( c-8a ) , 137.6 ( c-1 ) , 127.7 ( c-5 ) , 127.6 ( c-2 ) , 126.7 ( c-4 ) , 126.4 ( c-3 ) , 112.2 ( c-6 ) , 110.6 ( c-4a ) , 99.3 ( c-8 ) , 55.4 ( och3 ) , 43.7 ( c-9 ) ; anal . calcd . for c16h14n2o3
: c , 68.07 ; h , 5.00 ; n , 9.92 . found : c , 67.86 ; h , 4.89 ; n , 10.01 .
2-(benzyl amino)-7-ethoxy-4h-1,3-benzoxazin-4-one
15e . 7-ethoxy-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14e was allowed to react with benzylamine according to general procedure d. the crude solid was collected and recrystallized from toluene to give 15e ( 80% yield ) , mp 214216c .
max ( kbr)/cm 30692827 ( n h ) , 1673 ( c = o ) , 1600 ( c = c ) , 1466 ( c =
n ) ; h nmr ( d6-dmso ) 9.00 ( bs , 1h , n
h ) , 7.80 ( d , 1h , j = 8.6 hz , h-5 ) , 7.30 ( m , 5h , arh ) , 6.90 ( dd , 1h , jh6,h8 = 2.4 hz , jh6,h5 = 8.6 hz , h-6 ) , 6.70 ( d , 1h , j = 2.2 hz , h-8 ) , 4.50 ( s , 2h , h-9 ) , 4.20 ( q , 2h , j = 5.6 , ch2o ) , 3.90 ( s , 3h , 7-och3 ) , 1.30 ( t , 3h , j = 5.6 , ch3 ) ; c nmr ( d6-dmso ) 164.4 ( c-4 ) , 163.1 ( c-7 ) , 157.9 ( c-2 ) , 154.9 ( c-8a ) , 137.8 ( c-1 ) , 127.6 ( c-5 ) , 127.5 ( c-2 ) , 126.7 ( c-4 ) , 126.4 ( c-3 ) , 112.6 ( c-6 ) , 110.3 ( c-4a ) , 99.8 ( c-8 ) , 63.6 ( ch2o ) , 43.7 ( c-9 ) , 13.5 ( ch3 ) ; anal . calcd . for c17h16n2o3 : c , 68.91 ; h , 5.44 ; n , 9.45 .
2-(benzyl amino)-7-hydroxy-4h-1,3-benzoxazin-4-one
15f . 7-hydroxy-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14f was allowed to react with benzyl amine according to general procedure d. the crude solid was collected and recrystallized from ethanol to give 15f ( 77% yield ) , mp decomp 262c .
max ( kbr)/cm 33003058 ( o h ) , 30582851 ( n h ) , 1667s ( c = o ) ,
n ) ; h nmr ( d6-dmso ) 8.70 ( bs , 1h , n
h ) , 7.50 ( d , 1h , j = 8.6 hz , h-5 ) , 7.307.20 ( m , 6h , arh/7-oh ) , 6.50 ( d , 1h , j = 8.6 hz , h-6 ) , 6.30 ( s , 1h , h-8 ) , 4.50 ( s , 2h , h-9 ) ; c nmr ( d6-dmso ) 169.6 ( c-4 ) , 166.1 ( c-7 ) , 158.1 ( c-2 ) , 155.8 ( c-8a ) , 138.5 ( c-1 ) , 128.3 ( c-2 ) , 127.7 ( c-4 ) , 127.2 ( c-3 ) , 127.0 ( c-5 ) , 116.1 ( c-6 ) , 104.9 ( c-4a ) , 100.6 ( c-8 ) , 43.7 ( c-9 ) ; anal . calcd . for c15h12n2o3
: c , 67.16 ; h , 4.51 ; n , 10.44 . found : c , 67.14 ; h , 4.34 ; n , 10.49 .
7-hydroxy-8-methyl-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14 g was allowed to react with benzylamine according to general procedure d. the crude solid was collected and recrystallised from ethanol to give 15 g ( 74% yield ) , mp 245247c .
max ( kbr)/cm 33002860 ( o h ) , 30582851 ( n h ) , 1678 ( c = o ) ,
n ) ; h nmr ( d6-dmso ) 8.70 ( bs , 1h , n
h ) , 7.60 ( d , 1h , j = 8.6 hz , h-5 ) , 7.307.20 ( m , 6h , h-2/h-3/h-4/7-oh ) , 6.80 ( d , 1h , j = 8.6 hz , h-6 ) , 4.50 ( s , 2h , h-9 ) ; c nmr ( d6-dmso ) 165.2 ( c-4 ) , 159.6 ( c-7 ) , 157.8 ( c-2 ) , 152.5 ( c-8a ) , 137.8 ( c-1 ) , 127.6 ( c-2 ) , 126.7 ( c-4 ) , 126.4 ( c-3 ) , 124.2 ( c-5 ) , 112.0 ( c-6 ) , 109.4 ( c-8 ) , 108.1 ( c-4a ) , 43.7 ( c-9 ) , 6.8 ( 8-ch3 ) ; anal . calcd . for c16h14n2o3 : c , 68.07 ; h , 5.00 ; n , 9.92 . found : c , 67.86 ; h , 5.28 ; n , 9.68 .
in modification to the general procedure d , n-(2-(methylthio)-4-oxo-4h - benz[e]oxazin-7-yl)acetamide 14i ( 0.26 g 1 mmol ) was allowed to react with benzylamine ( 0.2 ml , 1 mmol ) for 4 hours .
the resulting solid was collected and recrystallised from acetonitrile to give 15 h ( 0.1 , 47% ) as white crystals , mp 250253c .
max ( kbr)/cm 3284 , 3234 , 3215 ( nh ) , 1669 ( c = o ) ; hnmr ( 200 mhz , 390 k , d6-dmso ) 10.28 ( s , 1h , h-15 ) , 8.86 ( t , 1h , jh9,h10 = 4.7 hz , h-9 ) , 7.817.78 ( m , 2h , h-8 and h-5 ) , 7.377.26 ( m , 6h , ar , h-1214 and h-6 ) , 4.52 ( d , 2h , jh10,h9 = 4.7 hz , h-10 ) , 2.10
( s , 3h , 17-ch3 ) ; c nmr ( 50 mhz , 390 k , d6-dmso ) 169.3 ( c-16 ) , 165.4 ( c-4 ) , 158.4 ( c-8a ) , 154.2 ( c-2 ) , 144.3 ( c-7 ) , 138.2 ( c-11 ) , 128.4 ( c-5 ) , 127.5/127.3/127.2 ( c-12/c-13/c-14 ) , 115.7 ( c-6 ) , 112.1 ( c-4a ) , 104.2 ( c-8 ) , 44.0 ( c-10 ) , 24.3 ( c-17 ) ; anal . calcd . for c17h15n3o3 :
c , 66.01 ; h , 4.89 ; n , 13.58 . found : c , 65.93 ; h , 5.02 ; n , 13.52 .
following the previously reported , 10-methyl-2 , 8-dithioxo-2 , 3 , 7 , 8-tetrahydrobenzo[1,2-e:5,4-e]bis(oxazine)-4,6-dione 18c ( 0.74 g , 2.5 mmol ) was allowed to react with methyl iodide ( 5.4 ml , 86.8 mmol ) and nahco3 ( 3.0 g , 32 mmol ) for 2 hrs .
the resulting yellow solid 24 ( 0.78 g , 98% ) is collected and used without further purification .
n ) ; h nmr ( 200 mhz , 390 k , d6-dmso ) 8.27 ( s , 1h , h-5 ) , 2.64 ( s , 6h , h-3and h-9 ) , 2.33 ( s , 3h , h-10 ) .
c nmr ( 50 mhz , 390 k , d6-dmso ) 173.0 ( c-2,8 ) , 160.6 ( c-4,6 ) , 155.3 ( c-6a , 9a ) , 124.0 ( c-5 ) 123.6 ( c-10 ) , 115.2 ( c-4a , 5a ) , 13.4 ( c-3 , 9 ) , 6.7 ( c-10 ) .
in slight modification to the general procedure d,10-methyl-2,8-bis(methylthio ) benzo[1,2-e:5,4-e]bis(oxazine)-4,6-dione 24 ( 0.29 g , 1 mmol ) was allowed to react with benzyl amine 0.4 ml , ( 2 mmol ) for 16 hours .
the resulting solid was collected and recrystallised from dmso / water to give 26 ( 0.25 g , 56% ) as an off white solid , mp 285288c decomp .
max ( kbr)/cm 3394 , 3202 , 3029 ( nh ) , 1681 , 1635 ( c = o ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 8.90 ( bs , 2h , h-11,17 ) , 8.27 ( s , 1h , h-5 ) , 7.437.24 ( m , 10h 2 x ar , h-1416 and h-2022 ) , 4.58 ( s , 4h , h-12,18 ) , 2.31 ( s , 3h , h-10 ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 164.0 ( c-4 ) , 164.0 ( c-9a,10a ) , 157.6 ( c-2 ) , 137.2 ( c-13,19 ) , 127.7 , 126.9 , 126.6 , 122.5 ( c-15,21 , c-14,20 and c-16,22 ) , 113.9 ( c-10 ) , 115.5 ( c-4a,5a ) , 44.0 ( c-12,17 ) , 6.6 ( c-10 ) ; anal . calcd . for c25h20n4o42h2o : c , 63.02 ; h , 5.08 ; n , 11.76 . found : c , 63.22 ; h , 4.99 ; n , 11.96 .
general procedure f. the appropriate substituted methylthio-1,3-benzoxazine 2.5 mmol was hydrolysed with 10 ml hydrochloric acid ( 10% ) at 80c for 4 hours . at the completion of the reaction
, the reaction mixture was washed with r.o water , filtered , and recrystallised from an appropriate solvent .
products 22a h were used in the synthesis of products 23a h with no further purification .
2h - benz[e]-1,3-oxazin-2,4(3h)-dione
22a . 2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14a was allowed to react with hydrochloric acid ( 10% ) according to general procedure f. the crude solid was collected and recrystallised from ethanol to give 22a ( 75% yield ) , mp 228c .
max ( kbr)/cm 3179 , 2877 ( n h ) , 1771 ( c = o ) , 1690 ( c = o ) , 1610 ( c = c ) ; h nmr ( 200 mhz , d6-dmso ) 7.90 ( d , 1h , j = 7.5 hz , h-5 ) , 7.80 ( t , 1h , j = 7.5 hz , h-6 ) , 7.40 ( m , 2h , h-7/h-8 ) ; c nmr ( 50 mhz , d6-dmso ) 161.6 ( c-4 ) , 153.7 ( c-2 ) , 147.6 ( c-8a ) , 136.2 ( c-7 ) , 126.9 ( c-5 ) , 125.2 ( c-6 ) , 116.5 ( c-8 ) , 114.6 ( c-4a ) .
8-methyl-2h - benz[e]-1,3-oxazin-2,4(3h)-dione
22b .
8-methyl-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14b was allowed to react with hydrochloric acid ( 10% ) according to general procedure f. the crude solid was collected and recrystallised from ethanol to give 22b ( 85% yield ) , mp 210c ( lit .
max ( kbr)/cm 3221 , 2845 ( n h ) , 1746 ( c = o ) , 1717 ( c = o ) ,
1614 ( c = c ) ; h nmr ( 200 mhz , d6-dmso ) 8.80 ( bs , 1h , n
h ) , 7.70 ( d , 1h , j = 7.5 hz , h-5 ) , 7.60 ( d , 1h , j = 7.5 hz , h-7 ) , 7.307.20 ( t , 1h , j = 7.1 hz , h-6 ) , 2.30 ( s , 3h , 8-ch3 ) ; c nmr ( 50 mhz , d6-dmso ) 161.6 ( c-4 ) , 151.9 ( c-2 ) , 147.3 ( c-8a ) , 136.9 ( c-7 ) , 125.5 ( c-8 ) , 124.6 ( c-5 ) , 124.4 ( c-6 ) , 114.4 ( c-4a ) , 13.8 ( ch3 ) .
7-methoxy-2h - benz[e]-1,3-oxazin-2,4(3h)-dione
22c . 7-methoxy-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14d was allowed to react with hydrochloric acid ( 10% ) according to general procedure f. the crude solid was collected and recrystallised from ethyl acetate to give 22c ( 78% yield ) , mp 213c .
max ( kbr)/cm 3203 , 2930 ( n h ) , 1771 ( c = o ) , 1723 ( c = o ) , 1620 ( c = c ) ; h nmr ( 200 mhz , d6-dmso ) 12.10 ( s , 1h , n
h ) , 7.90 ( d , 1h , j = 8.3 hz , 5-h ) , 7.10 ( s , 1h , 8-h ) , 7.00 ( d , 1h , j = 8.3 hz , 6-h ) , 3.0 ( s , 3h , 7-och3 ) ; c nmr ( 50 mhz , d6-dmso ) 160.6 ( c-4 ) , 157.9 ( c-2 ) , 155.4 ( c-7 ) , 151.6 ( c-8a ) , 128.2 ( c-5 ) , 113.2 ( c-6 ) , 107.1 ( c-4a ) , 100.8 ( c-8 ) , 56.5 ( 7-och3 ) ; anal . calcd . for c9h7no4 :
c , 55.96 ; h , 3.65 ; n , 7.25 . found : c , 55.75 ; h , 3.45 ; n , 7.55 .
7-ethoxy-2h - benz[e]-1,3-oxazin-2,4(3h)-dione
22d . 7-ethoxy-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14e was allowed to react with hydrochloric acid ( 10% ) according to general procedure f. the crude solid was collected and recrystallised from ethyl acetate to give 22e ( 75% yield ) , mp 225227c .
max ( kbr)/cm 31572862 ( n h ) , 1771 ( c = o ) , 1698 ( c = o ) , 1620 ( c = c ) ; h nmr ( 200 mhz , d6-dmso ) 11.90 ( bs , 1h , n
h ) , 7.80 ( d , 1h , j = 8.6 hz , h-5 ) , 6.90 ( m , 2h , h-6/h-8 ) , 4.10 ( q , 2h , j = 6.8 hz , o
ch2 ) , 1.30 ( t , 3h , j = 6.8 hz , ch3 ) ; c nmr ( 50 mhz , d6-dmso ) 164.6 ( c-4 ) , 160.9 ( c-2 ) , 155.4 ( c-7 ) , 147.6 ( c-8a ) , 128.2 ( c-5 ) , 113.2 ( c-6 ) , 107.1 ( c-4a ) , 100.8 ( c-8 ) , 64.4 ( ch2o ) , 14.3 ( ch3 ) ; anal . calcd . for c10h9no4 :
c , 57.97 ; h , 4.38 ; n , 6.76 . found : c , 57.86 ; h , 4.45 ; n , 6.55 .
7-hydroxy-2h - benz[e]-1,3-oxazin-2,4(3h)-dione
22e . 7-hydroxy-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14f was allowed to react with hydrochloric acid ( 10% ) according to general procedure f. the crude solid was collected and recrystallised from ethanol to give 22e ( 65% yield ) , mp 245c ( lit 310 ) .
max ( kbr)/cm 32002700 ( o h ) , 3078 , 2929 ( n h ) , 1780 ( c = o ) , 1688 ( c = o ) , 1616 ( c = c ) ; h nmr ( 200 mhz , d6-dmso ) 11.80 ( s , 1h , n
h ) , 7.70 ( d , 1h , j = 8.6 hz , h-5 ) , 6.80 ( d , 1h , j = 8.6 hz , h-6 ) , 6.60 ( s , 1h , h-8 ) ; c nmr ( 50 mhz , d6-dmso ) 164.6 ( c-4 ) , 160.9 ( c-2 ) , 155.4 ( c-7 ) , 147.6 ( c-8a ) , 128.6 ( c-5 ) , 113.8 ( c-6 ) , 105.9 ( c-4a ) , 101.8 ( c-8 ) ; anal . calcd . for c8h5no4
: c , 53.64 ; h , 2.81 ; n , 7.82 . found : c , 53.75 ; h , 2.45 ; n , 7.55 .
7-hydroxy-8-methyl-2h - benz[e]-1,3-oxazin-2,4(3h)-dione
22f . 7-hydroxy-8-methyl-2-(methylthio)-4h - benz[e]-1,3-oxazin-4-one 14 g was allowed to react with hydrochloric acid ( 10% ) according to general procedure f. the crude solid was collected and recrystallised from ethanol to give 22f ( 70% yield ) , mp 250c decomp .
max ( kbr)/cm 3250 , 2900 ( o h ) , 3188 , 2956 ( n h ) , 1771 ( c = o ) , 1713 ( c = o ) ,
1619 ( c = c ) ; h nmr ( 200 mhz , d6-dmso ) 11.70 ( s , 1h , n
h ) , 7.60 ( d , 1h , j = 8.4 hz , h-5 ) , 6.80 ( d , 1h , j = 8.4 hz , h-6 ) , 2.10 ( s , 3h , 8-ch3 ) ; c nmr ( 50 mhz , d6-dmso ) 162.4 ( c-4 ) , 161.5 ( c-2 ) , 153.4 ( c-7 ) , 147.9 ( c-8a ) , 125.4 ( c-5 ) , 112.6 ( c-6 ) , 110.8 ( c-4a ) , 106.0 ( c-8 ) , 8.1 ( 8-ch3 ) ; anal . calcd . for c9h7no4 :
c , 55.96 ; h , 3.65 ; n , 7.25 . found : c , 55.75 ; h , 3.45 ; n , 7.55 .
n-(2,4-dioxo-3,4-dihydro-2h - benz[e]oxazin-7-yl)acetamide
22 g . in slight modification to the general procedure f , n-(2-(methylthio)-4-oxo-4h - benz[e]oxazin-7-yl ) acetamide 14i ( 0.63 g , 2.5 mmol ) was allowed to reflux with water ( 10 ml ) for 2 hours .
the resulting crude solid was filtered , and recrystallised from methanol to give 22 g ( 0.52 g , 95% ) as light grey solid , mp 284287c ( lit .
max ( kbr)/cm 3350 , 3050 ( nh ) , 1758 , 1704 ( c = o ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 11.80 ( bs , 1h , 3-nh ) , 10.57 ( s , 1h , 9-nh ) , 7.83 ( d , 1h , jh5,h6 = 8.6 hz , h-5 ) , 7.72 ( d , 1h , jh8,h6 = 1.6 hz , h-8 ) , 7.45 ( dd , 1h , jh6,h5 = 8.6 hz , jh6,h8 = 1.6 hz , h-6 ) , 2.11 ( s , 3h , 11-ch3 ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 169.0 ( c-10 ) , 160.3 ( c-4 ) , 154.2 ( c-2 ) , 147.1 , 145.7 ( c-8a , c-7 ) , 127.3 ( c-5 ) , 115.3 ( c-6 ) , 108.6 ( c-4a ) , 104.6 ( c-8 ) , 23.5 ( c-11 ) . anal . calcd . for c10h8n2o4 : c , 54.55 ; h , 3.66 ; n , 12.72 . found : c , 54.49 ; h , 3.77 ; n , 12.69 .
in modification to the general procedure f , n-(2-(methylthio)-4-oxo-4h - benzo[e]oxazin-7-yl ) acetamide 14h ( 0.63 g , 2.5 mmol ) was allowed to react in the presence of hydrochloric acid ( 15 ml , 40% ) for 4 hours . at the completion of the reaction ,
the reaction mixture was neutralized by nahco3 , filtered and recrystallised from ethanol to give 22h ( 0.34 g , 79% ) , mp 284287c decomp .
max ( kbr)/cm 3479 , 3372 ( nh ) , 2843 ( nh ) , 1752 , 1708 ( c = o ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 11.49 ( s , 1h , 3-nh ) , 7.54 ( d , 1h , jh5,h6 = 8.4 hz , h-5 ) , 6.52 ( m , 3h , h-6 and 7-nh2 ) , 6.31 ( s , 1h , h-8).c nmr ( 50 mhz , 340 k , d6-dmso ) 160.8 ( c-4 ) , 156.0 , 155.6 ( c-2 , c-7 ) , 147.9 ( c-8a ) , 127.8 ( c-5 ) , 111.4 ( c-6 ) , 101.5 ( c-4a ) , 97.3 ( c-8 ) .
compound 22h was used for the synthesis of compound 23 g with no further purification .
10-methyl-2,8-bis(methylthio)benzo[1,2-e:5,4-e]bis(oxazine)-4,6-dione 24 ( 0.74 g , 2.5 mmol ) was allowed to react with hydrochloric acid ( 10% ) for 4 hours according to the general procedure f. the resulting solid was recrystallised from dmf to give 25 ( 0.55 g , 85% ) as off white crystals .
max ( kbr)/cm 3183 , 3119 , 3049 ( nh ) , 1784 , 1714 ( c = o ) , 1616 ( c = c ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 12.18 ( bs , 2h , 3,7-nh ) , 8.26 ( s , 1h , h-5 ) , 2.31 ( s , 3h , h-10 ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 159.7 ( c-4,6 ) , 155.4 ( c-2,8 ) , 145.9 ( c-9a,10a ) , 123.3 ( c-5 ) , 113.0 ( c-10 ) , 111.3 ( c-4a,5a ) , 7.3 ( c-10 ) ; anal . calcd . for c11h6n2o6 : c , 50.39 ; h , 2.31 ; n , 10.68 . found : c , 50.54 ; h , 2.42 ; n , 10.74 .
general procedure g. the appropriate 2-dione-1,3-benzoxazines 22a b and 22d i ( 2.5 mmol ) were suspended in dry 1,4-dioxane ( 10 ml ) in a 50 ml round - bottomed flask .
benzyl amine ( 12.5 mmol ) was then added dropwise , directly from the pipette , with stirring , and then the reaction mixture was heated to reflux for 4 hours . at the completion of the reaction , it evaporated to dryness under reduced pressure and triturated with minimal diethyl ether .
the resulting solid was collected by vacuum filtration and recrystallised from an appropriate solvent .
n-(benzyl carbamoyl)-2-hydroxybenzamide
23a . 2h - benz[e]-1,3-oxazin-2,4(3h)-dione 22a was allowed to react with benzyl amine according to general procedure f to give 23a which was recrystallised from ethanol ( 70% yield ) , mp211213c .
max ( kbr)/cm 33402940 ( o h ) , 32303161 ( n h ) , 1687 ( c = o ) , 1648 ( c = o ) ; h nmr ( 200 mhz , d6-dmso ) 11.75 ( bs , 1h , h-2 ) , 10.40 ( bs , 1h , 1-oh ) , 9.05 ( t , 1h , j = 5.6 hz , 4-nh ) , 7.90 ( d , 1h , j = 7.7 hz , h-3 ) , 7.50 ( m , 1h , h-5 ) , 7.307.20 ( m , 5h , arh ) , 7.056.95 ( m , 2h , h-4/h-6 ) , 4.40 ( d , 2h , j = 5.2 hz , h-5 ) ; c nmr ( 50 mhz , d6-dmso ) 166.3 ( c-1 ) , 158.7 ( c-2 ) , 153.1 ( c-3 ) , 137.2 ( c-6 ) , 133.7 ( c-4 ) , 130.8 ( c-6 ) , 128.4 ( c-8 ) , 127.3 ( c-7 ) , 127.0 ( c-9 ) , 119.9 ( c-1 ) , 117.2 ( c-5/c-3 ) , 42.8 ( c-5 ) ; anal . calcd . for c15h14n2o3 : c , 66.66 ; h , 5.22 ; n , 10.36 . found : c , 66.74 ; h , 5.36 ; n , 10.29 .
n-(benzyl carbamoyl)-2-hydroxy-3-methylbenzamide
23b . 8-methyl-2h - benz[e]-1,3-oxazin-2,4(3h)-dione 22b was allowed to react with benzyl amine according to general procedure g to give 23b which was recrystallised from ethanol ( 65% yield ) , mp 220223c .
max ( kbr)/cm 33372946 ( o h ) , 3249 ( n h ) , 1696 ( c = o ) , 1642 ( c = o ) ; h nmr ( 200 mhz , d6-dmso ) 11.30 ( bs , 1h , h-2 ) , 10.70 ( bs , 1h , 1-oh ) , 8.90 ( t , 1h , j = 5.6 hz , 4-nh ) , 7.80 ( d , 1h , j = 7.5 hz , h-6 ) , 7.407.20 ( m , 6h , arh and h-4 ) , 6.80 ( t , 1h , j = 7.5 hz , h-5 ) , 7.006.90 ( m , 2h , h-4 and h-6 ) , 4.60 ( d , 2h , j = 5.2 hz , h-5 ) , 2.20 ( s , 3h , ch3 ) ; c nmr ( 50 mhz , d6-dmso ) 167.9 ( c-1 ) , 159.7 ( c-2 ) , 154.9 ( c-3 ) , 137.2 ( c-6 ) , 135.0 ( c-4 ) , 128.4 ( c-8 ) , 127.3 ( c-7 ) , 127.0 ( c-9 ) , 126.3 ( c-3 ) , 125.5 ( c-6 ) , 119.0 ( c-5 ) , 115.1 ( c-1 ) , 42.9 ( c-5 ) , 15.9 ( ch3 ) ; anal . calcd . for c16h16n2o3
: c , 67.59 ; h , 5.67 ; n , 9.85 . found : c , 67.64 ; h , 5.54 ; n , 9.87 .
n-(benzyl carbamoyl)-4-ethoxy-2-hydroxybenzamide
23c . 7-ethoxy-2h - benz[e]-1,3-oxazin-2,4(3h)-dione 22d was allowed to react with benzyl amine according to general procedure f to give 23c which was recrystallised from ethanol ( 70% yield ) , mp 204206c .
max ( kbr)/cm 33332876 ( o h ) , 32253156 ( n h ) , 1692 ( c = o ) , 1650 ( c = o ) ; h nmr ( 200 mhz , d6-dmso ) 11.90
( bs , 1h , h-2 ) , 10.20 ( bs , 1h , 2-oh ) , 8.00 ( t , 1h , j = 5.4 hz , 4-nh ) , 7.90 ( d , 1h , j = 8.9 hz , h-6 ) , 7.30 ( m , 5h , arh ) , 6.60 ( dd , 1h , j
h-5,h-3 = 2.6 hz , j
h-5,h-6 = 8.9 hz , h-5 ) , 6.50 ( d , 1h , j = 2.2 hz , h-3 ) , 4.40 ( d , 2h , j = 6.0 hz , h-5 ) , 4.10 ( q , 2h , j = 6.8 hz , ch2o ) , 1.30 ( t , 3h , j = 6.8 hz , ch3 ) ; c nmr ( 50 mhz , d6-dmso ) 166.1 ( c-1 ) , 160.7 ( c-4 ) , 158.9 ( c-2 ) , 153.2 ( c-3 ) , 137.2 ( c-6 ) , 132.4 ( c-6 ) , 128.4 ( c-8 ) , 127.3 ( c-7 ) , 127.0 ( c-9 ) , 110.5 ( c-5 ) , 109.4 ( c-1 ) , 101.7 ( c-3 ) , 64.6 ( ch2o ) , 42.8 ( c-5 ) , 14.5 ( ch3 ) ; anal . calcd . for c17h18n2o4 :
c , 64.96 ; h , 5.77 ; n , 8.91 . found : c , 65.03 ; h , 5.82 ; n , 8.84 .
n-(benzyl carbamoyl)-2,4-dihydroxybenzamide
23d . 7-hydroxy-2h - benz[e]-1,3-oxazin-2,4(3h)-dione 22e was allowed to react with benzyl amine according to general procedure g and gave 23d which was recrystallised from ethanol ( 75% yield ) , mp 220c .
max ( kbr)/cm 32002700 ( o h ) , 3118 , 3033 ( n h ) , 1692 ( c = o ) , 1665 ( c = o ) ; h nmr ( 200 mhz , d6-dmso ) 9.00 ( t , 1h , j = 5.6 hz , 4-n h ) , 7.70 ( d , 1h , j = 7.6 hz , h-6 ) , 7.30 ( bm , 6h , arh , 2-oh ) , 6.30 ( s , 1h , h-3 ) , 6.2 ( d , 1h , j = 7.6 hz , h-5 ) , 5.60 ( m , 2h , 4-oh and 2-nh ) 4.40 ( d , 2h , j = 6.0 hz , h-5 ) ; c nmr ( 50 mhz , d6-dmso ) 163.5 ( c-1 ) , 161.9 ( c-4 ) , 158.5 ( c-2 ) , 153.2 ( c-3 ) , 138.5 ( c-6 ) , 135.3 ( c-6 ) , 128.7 ( c-8 ) , 127.7 ( c-7 ) , 127.5 ( c-9 ) , 109.2 ( c-1 ) , 102.9 ( c-5 ) , 107.9 ( c-3 ) , 48.2 ( c-5 ) ; anal . calcd . for c15h14n2o4 : c , 59.59 ; h , 4,67 ; n , 9.27 . found : c , 59.88 ; h , 4.68 ; n , 9.59 .
n-(benzyl carbamoyl)-2,4-dihydroxy-3-methylbenzamide
23e . 7-hydroxy-8-methyl-2h - benz[e]-1,3-oxazin-2,4(3h)-dione 22f was allowed to react with benzylamine according to general procedure g to give 23 g which was recrystallised from ethyl acetate ( 65% yield ) , mp 230c .
max ( kbr)/cm 3381 , 3225 ( o h ) , 30202800 ( n h ) , 1687 ( c = o ) , 1639 ( c = o ) ; h nmr ( 200 mhz , d6-dmso ) 11.00 ( bs , 1h , 2-nh ) , 9.80 ( bs , 1h , 2-oh ) , 8.5 ( t , 1h , j = 5.6 hz , 4-nh ) , 7.5 ( d , 1h , j = 8.8 hz ; h-5 ) , 7.4 ( bm , 5h , arh ) , 6.9 ( d , 1h , j = 8.8 hz ; h-6 ) , 4.50 ( d , 2h , j = 6.0 hz , h-5 ) , 2.00 ( s , 3h , ch3 ) ; c nmr ( 50 mhz , d6-dmso ) 165.2 ( c-1 ) , 162.9 ( c-4 ) , 159.3 ( c-2 ) , 157.2 ( c-3 ) , 137.5 ( c-6 ) , 128.7 ( c-8 ) , 127.7 ( c-7 ) , 127.5 ( c-9 ) , 127.3 ( c-6 ) , 112.2 ( c-1 ) , 110.9 ( c-3 ) , 107.9 ( c-5 ) , 44.2 ( c-5 ) , 8.6 ( 3-ch3 ) ; anal . calcd . for c16h16n2o4 : c , 63.99 ; h , 5.37 ; n , 9.33 . found : c , 63.88 ; h , 5.68 ; n , 9.59 .
4-acetamido-2-hydroxy - n-(phenylcarbamoyl)benzamide
23f . n-(2,4-dioxo-3,4-dihydro-2h - benz[e]oxazin-7-yl ) acetamide 22 g was allowed to react with benzyl amine according to general procedure g. the resulting solid was filtered and recrystallised from ethanol to give 23f ( 0.25 g , 76% ) mp 257260c .
max ( kbr)/cm 35003200 ( oh ) , 3281 , 3122 ( nh ) , 1694 , 1664 ( c = o ) , 1613 , 1559
( c = c ) ; h nmr ( 200 mhz , 340 k , d6-dmso ) 11.63 ( bs , 1h , 10-nh ) , 10.24 , 10.06 ( 15-nh , 2-oh ) , ( s , 1h , jh10,h11 = 5.4 hz , 10-nh ) , 7.84 ( d , 1h , jh6,h5 = 8.8 hz , h-6 ) , 7.58 ( d , 1h , jh3,h5 = 1.8 hz , h-3 ) , 7.387.24 ( m , 5h , arh , h-13 , h-14 , h-15 ) , 7.03 ( dd , jh5,h6 = 8.8 hz , jh5,h3 = 1.8 hz , h-3 ) , 4.45 ( d , 2h , jh11,h10 = 5.9 hz , h-11 ) , 2.07 ( s , 3h , h-17 ) ; c nmr ( 50 mhz , 300 k , d6-dmso ) 168.7 ( c-16 ) , 165.7 ( c-7 ) , 157.5 ( c-2 ) , 152.9 ( c-9 ) , 144.6 ( c-4 ) , 138.8 ( c-12 ) , 131.1 ( c-6 ) , 128.1 , 126.9 , 126.7 ( c-14 , c-13 , c-15 ) , 111.2 ( c-1 ) , 110.5 ( c-5 ) , 106.1 ( c-3 ) , 42.6 ( c-11 ) , 23.9 ( c-17 ) ; anal . calcd . for c17h17n3o4 : c , 62.38 ; h , 5.23 ; n , 12.84 .
7-amino-2h - benz[e]oxazine-2,4(3h)-dione 22h was allowed to react with benzylamine for 16 h in slight modification to general procedure g. the resulting solid was filtered and recrystallised from toluene to give 23 g ( 0.15 g , 54% ) as light brown solid , mp 208211c decomp .
max ( kbr ) cm 35003200 ( oh ) , 3489 , 3390 , 3330 ( nh ) , 1686 1645 ( c = o ) ; h nmr ( 200 mhz , 340 k , d6-dmso ) 10.18 ( bs , 1h , 2-oh ) , 9.00 ( t , 1h , jh10,h11 = 5.1 hz 10-nh ) , 7.65 ( d , 1h , jh6,h5 = 8.6 hz , h-6 ) , 7.347.27 ( m , 6h , ar , h-13 , h-14 , h-15 and 8-nh exchangeable with d2o ) , 6.196.11 ( m , 2h , h-5 and h-3 ) , 5.88 ( bs , 2h , 4-nh2 ) , 4.42 ( d , 2h , jh11,h10 = 5.1 hz , h-11 ) ; c nmr ( 50 mhz , 300 k , d6-dmso ) 166.6 ( c-7 ) , 159.4 ( c-2 ) , 154.8 , 153.3 ( c-4,c-9 ) , 139.0 ( c-12 ) , 131.7 ( c-6 ) , 128.0 ( c-14 ) , 126.9 , 126.6 ( c-15 , c-12 ) , 106.6 ( c-5 ) , 105.5 ( c-1 ) , 99.2 ( c-3 ) , 42.5 ( c-11 ) ; anal . calcd . for c15h15n3o3 : c , 63.15 ; h , 5.30 ; n , 14.73 .
minimal inhibitory concentrations ( mics ) were determined on 7 different bacterial and 4 fungi cultures .
the bacteria include escherichia coli ( fb5 ) , acinetobacter baumannii ( atcc19606 ) , pseudomonas aeruginosa ( pao9503 ) , staphylococcus aureus ( fb13 ) , bacillus subtilis ( pao9503 ) , streptococcus agalactiae ( fb31 ) mycobacterium smegmatis ( con 21 ) , and mycobacterium chlorophenolicum ( con 24 ) .
the fungi include aspergillus niger , rhizopus oryzae , absidia corymbifera , and alternaria alternata .
each one of the different strains of bacteria were cultured into 10 ml of nutrient broth ( nb ) or malt extract broth for fungi and incubated overnight at 37c except for mycobacterium smegmatis at 30c for 24 hours and mycobacterium chlorophenolicum for 4 days .
all fungal cultures were grown at 25c for up to seven days except for alternaria alternate , 3 days 35c .
the grown cultures were diluted ( 1/10 in nb or malt extract broth ) and incubated for a further 2 hours ( to reach exponential phase ) and then used in the mic assay .
compound stock solutions of 10 g / ml were made up in dmso ensuring that a maximum of 30 l is used , otherwise inhibitory effects will be shown on some bacterial / fungal cultures ) . in sterile microcentrifuge tubes ,
varying amounts of exponential phase culture ( 0.1 ml ) were added and nb ( 0.9 ml ) to make up a total volume of 1 ml . each culture
control tubes were made using dmso without the addition of compound . at the completion of the incubation
, the microcentrifuge tubes containing culture were vortexed and compared to their respective controls ( without compound ) .
compounds which displayed an absence of turbidity lower than 50 g / ml were subject to further dilutions , while if there is growth ( or turbidity ) at a particular concentration then the value is recorded as the mic .
the dilution series was carried out in factors of 2 as recommended ( i.e. , at 200 , 100 , 50 , 25 , 12.5 , 6.25 , and 3.125 g ml resp . ) .
compounds that were insoluble in dmso or the nb were evaluated for their antimicrobial activity by agar diffusion assays .
the surface of an na or malt agar plate was flood - inoculated with an overnight nb or malt broth culture of a particular culture adjusted to 10 cfu / ml ( 10 colony forming units per millimeter ) .
each disk contained a specific culture and sterile 12.7-mm paper disk ( oxide ) was placed onto the dry surface for each compound .
the insoluble compound was resuspended in dmso and a 20 l aliquot was impregnated onto the surface of a sterile paper disc including 20 l of dmso control .
the diameter of the zone of inhibition was measured after incubation for 18 hr and compared to the control zone ( dmso ) . | new n-(benzyl carbamothioyl)-2-hydroxy substituted benzamides 13 , 20 , and 21 were synthesized using sodium bicarbonate and benzyl amine with 2-thioxo - substituted-1,3-benzoxazines 6 , 10a , b , 11c , and 12a n . the 2-thioxo - substituted-1,3-oxazines 6 , 10a - b , 11d 12a
n , and 26 were converted to the corresponding 2-methylthio - substituted-1,3-oxazines 14a l and 24 which were then converted to 2-benzyl amino - substituted - benzoxazines 15a
i by refluxing with benzylamine .
products 15a , b , e , f , and g were also synthesized by boiling the corresponding n-(benzyl carbamothioyl)-2-hydroxy substituted benzamides 13a , b , f , l , and m in acetic acid .
2-oxo - substituted-1,3-benzoxazines 22 and 25 were prepared by treating the corresponding 2-methylthio - substituted-1,3-oxazines 14 and 24 with dilute hcl .
the n-(benzyl carbamoyl)-2-hydroxy substituted benzamide 23 was synthesized from the reaction of 2-oxo - substituted-1,3-benzoxazine 22 with benzylamine .
the new products were characterized using ir , 1h , and 13c nmr in addition to microanalysis .
selected compounds were tested in vitro for antibacterial and antifungi activity and the most active compounds were found to be the 4-(substituted - benzylamino)-2-hydroxy benzoic acids 9a and d ( m. chlorophenolicum , mic 50 and 25 gm l1 , resp . ) , n1 , n3-bis ( benzyl carbamothioyl)-4,6-dihydroxy - substituted phthalamides 20a and 20c ( b. subtilis mic 12.5 , 50 gm l1 , resp . ) and 21 ( m. chlorophenolicum , mic 50 gm l1 ) . | 1. Introduction
2. Results and Discussion
3. Conclusion
4. Experimental | in this work
, we developed simple general procedures for the synthesize of n-(benzyl carbamothioyl)-2-hydroxybenzamides 13 , 20 , and 21 , n-(benzyl carbamoyl)-2-hydroxy - substituted - benzamides 23 , and 2-(benzyl amino)-substituted-1,3-benzoxazin-4-one 15 and 26 . the 2-thio - substituted-1,3-benzoxazines 6 , 10 , 11 , 12 ( a r = 8-ch3 , b r = 8-ph , c r = 6-br , d r = 6-och2ch3 , e r = 7-och2ch3 , f r = 8-och2ch3 , g r = 6-och3 , h r = 7-och3 , i r = 8-och3 , j r = 6,8-i , k r = 7-oh , l
r = 7-oh,-8-ch3 , and m r = 8-oh ) , 18 , and 19 were synthesized from the reaction of the substituted 2-hydroxy benzoic acid with freshly prepared ph3p(scn)2 ( schemes 2 and 3 ) following the reported conditions [ 22 , 34 ] with some modifications ( see experimental ) . 7-(substituted benzylamino)-2-thioxo-2,3-dihydro-4h-1,3-benzoxazin-4-one 11a c were synthesized from the reaction of 4-(substituted - benzylamino)-2-hydroxybenzoic acids 9a c with the freshly prepared ph3p(scn)2 [ 22 , 32 ] ( scheme 2 ) . 2-methylthio - substituted-1,3-oxazines 14a i ( a r = h , b r = 8-ch3 , c r = 8-ph , d r = 7-och3 , e r = 7-och2ch3 , f r = 7-oh , g r = 7-oh,-8-ch3 , h
r = acnh , and i r = nh2 ) and 24 were prepared by the reaction of ch3i in the presence of nahco3 with substituted-1,3-oxazines 6 , 10a , b , 11c , and 12 ( a r = 8-ch3 , b r = 8-ph , e r = 7-och2ch3 , h
r = 7-och3 , k r = 7-oh , and l r = 7-oh,-8-ch3 ) according to the previously reported procedure . physical properties , ir , h nmr , and c nmr , collected for products 14a i were found to be identical to the reported data . to overcome this low yield and possible mixture formation ,
the reaction procedure was modified in which the benzoxazines 6 , 10a - b , 11d , and 12b m were mixed with nahco3 and suspended in 1 : 1 mixture of methanol , water and the mixture was then heated to 40c for a few minutes . with slight modification to the procedure b , by altering the ratio of benzylamine to starting material ( 3 : 1 ) and the reaction time to 16 hours , the bis - oxazines 18a , c and
19 were found to react in a similar fashion to give substituted bis(benzyl carbamothioyl ) analogues 20a , c ( yield 63 , 49% ) and 21 ( yield 63% ) ( scheme 4 ) . as mentioned earlier the reaction of the 2-thio-1,3-benzoxazine 6 with a primary amine
the reaction of 2-methylthio-(substituted)-1,3-benzoxazines 14 ( a r = h , b r = 8-ch3 , c r = 8-ph , d r = 7-och3 , e r = 7-och2ch3 , f r = 7-oh , g r = 7-oh,-8-ch3 , and h r = acnh- ) and 24 with excess ( 5-fold ) benzyl amine according to the general procedure c gave 2-benzyl amino - substituted-1,3-benzoxazines 15a h and 26 with moderate to high yields 6283% ( schemes 3 and 5 ) . following their successful synthesis , many of the n-(benzyl carbamothioyl)-2-hydroxybenzamides 13a , b , f , and g were then cyclised by refluxing in acetic acid for 2 hours according to the general procedure d ( scheme 3 ) and gave the corresponding 2-benzylamino-1,3-benzoxazines 15a , b , f , and g fair to good yields . the previously analysed h and c nmr spectra of the parent 2-methylthio-1,3-benzoxazines 14 were used to aid in the analysis of the new products 15b
g s. 2-methylthio - substituted - benzoxazines 14a , b , and d g were allowed to react with 10% hcl according to the general procedure e and gave 2-dione-1,3-benzoxazines 22a h with good to excellent yield ( scheme 5 ) ; however , product 22h was produce if 40% hcl was used in the hydrolysis of 14h . the bacterial species investigated were p. aeruginosa , b. subtilis , s. aureus , a. baumannii , e. coli , s. agalactiae m. smegmatis , and m. chlorophenolicum . compounds 20a , c and 21 ( bis - thiourea products ) were found to be particularly active towards gram - positive b. subtilis at mic values of 12.5 , 25 , and 25 g ml . similarly to the broth dilution results , compounds 21 and 9d were found to be active against three fungi species , r. oryzae , a. niger , and a. corymbifera , with clearance zones 25 mm , respectively . products 8b , c , and e were not identified and used immediately in the synthesis of compound 9a , b , and d.
( z)-4-((3-ethoxy-2-hydroxybenzylidene)amino)-2-hydroxybenzoic acid
8d . max ( kbr)/cm 1655 ( c = o ) , 1622 , 1600 ( c = n ) : hnmr ( 200 mhz , 300 k , d6-acetone ) 8.93 ( s , 1h , h-8 ) , 8.02 ( d , 1h , jh6,h5 = 8.8 hz , h-6 ) , 7.33 ( dd , 1h , jh15,h14 = 7.8 hz , jh15,h13 = 1.6 hz , h-15 ) , 7.24 ( dd , 1h , jh13,h14 = 8.0 hz , jh13,h15 = 1.6 hz , h-13 ) , 6.917.09 ( m , 3h , h-3,h-5 , and h-14 ) , 4.24 ( q , 2h , jh16,h17 = 7.0 hz , h-16 ) , 1.41 ( t , 3h , jh17,h16 = 7.0 hz , h-17 ) . general procedure c. the appropriate 2-thio-1,3-benzoxazines ( 1.7 mmole ) 6 , 10a , b , 11c , and 12a m were suspended in a mixture of sodium bicarbonate ( 1 gm ) and water ( 5 ml)/methanol ( 5 ml ) with stirring , then the reaction mixture was warm to 40c for few minutes then benzyl amine ( 4.25 mmol ) was added dropwise , directly from the a pipette , left stirring at room temperature for 4 hours . max ( kbr)/cm 3301 ( n h ) , 32002700 ( o h ) , 1661 ( c = o ) , 1605 ( c = s ) ; h nmr ( d6-dmso ) 12.42 ( bs , 1h , h-2 ) , 11.41 ( s , 1h , o
h ) , 11.00 ( t , 1h , j = 5.4 hz , h-4 ) , 7.707.00 ( m , 9h , arh ) , 4.9 ( d , 2h , j = 5.2 hz , h-5 ) ; c nmr ( d6-dmso ) 179.6 ( c-3 ) , 168.3 ( c-1 ) , 160.7 ( c-2 ) , 136.3 ( c-6 ) , 135.9 ( c-4 ) , 129.0 ( c-6 ) , 128.1 ( c-7 ) , 127.9 ( c-8 ) , 127.5 ( c-9 ) , 120.1 ( c-5 ) , 118.8 ( c-3 ) , 113.3 ( c-1 ) , 50.0 ( c-5 ) . max ( kbr)/cm 3062 , 2883 ( n h ) , 1686 ( c = o ) , 1595 ( c = s ) ; h nmr ( d6-dmso ) 11.30 ( s , 1h , 2-n
h ) , 10.70 ( s , 1h , o h ) , 8.80 ( t , 1h , j = 5.4 hz , 4-n
h ) , 7.90 ( d , 1h , j = 7.6 hz , h-6 ) , 7.80 ( d , 1h , j = 7.6 hz , h-4 ) , 7.60 ( dd , 1h , jh8,h10 = 1.6 hz , jh8,h9 = 8.0 hz , h-8 ) , 7.50 ( m , 4h , h-5/h-9/h-10 ) , 7.307.20 ( m , arh , h-4 ) , 4.90 ( d , 2h , j = 6.0 hz , h-5 ) ; c nmr ( d6-dmso ) 169.4 ( c-3 ) , 157.7 ( c-1 ) , 155.3 ( c-2 ) , 137.2 ( c-6 ) , 134.2 ( c-4 ) , 129.5 ( c-6 ) , 128.4 ( c-7 ) , 127.3 ( c-8 ) , 127.0 ( c-9 ) , 126.3 ( c-3 ) , 120.1 ( c-1 ) , 118.9 ( c-5 ) , 50.1 ( c-5 ) ; anal . max ( kbr)/cm 35003200 ( oh ) , 3292 , 3113 , ( nh ) , 1656 ( c = o ) , 1374 ( c = s ) ; hnmr ( 200 mhz , 300 k , d6-dmso ) 12.09 ( s , 1h , 16-nh ) , 11.40 ( s , 1h , 8-nh ) , 11.18 ( t , 1h , jh10,h11 = 4.6 hz , 10-nh ) , 10.26 ( s , 1h , 2-oh ) , 7.84 ( d , 1h , jh6,h5 = 7.8 hz h-6 ) , 7.64 ( s , 1h , h-3 ) , 7.377.30 ( m , 5h , ar , h-13 , h-14 , h-15 ) , 7.02 ( d , 1h , jh5,h6 = 7.8 hz , h-5 ) , 4.85 ( d , 2h , jh11,h10 = 4.8 hz , h-11 ) , 2.07 ( s , 3h , ch3 ) ; c nmr ( 50 mhz , 300 k , d6-dmso ) 179.7 ( c-9 ) , 169.1 ( c-7 ) , 164.2 ( c-17 ) , 157.4 ( c-2 ) , 145.3 ( c-4 ) , 137.3 ( c-12 ) , 131.9 ( c-6 ) , 128.5 , 127.6 , 127.3 ( c-13 , c-14 , c-15 ) , 111.0 , 110.8 ( c-1 , c-5 ) , 105.9 ( c-3 ) , 48.1 ( c-11 ) , 24.2 ( c-18 ) ; anal . max ( kbr)/cm 35003200 ( oh ) , 3414 , 3257 ( nh ) , 1651 ( c = o ) , 1321 ( c = s ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 11.51 ( bs , 2h , 2 x nh ) , 10.96 ( s , 2h , jh10-h11 = 5.7 hz , h-10,19 ) , 8.48 ( s , 1h , h-6 ) , 7.397.28 ( m , 10h , 2 x ar ) , 4.90 ( d , 2h , jh9,h8 = 5.7 hz , h-11 , 20 ) , 2.10 ( s , 3h , h-3 ) , 3.3 ( oh under the water envelope ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 179.6 ( c-9 , 18 ) , 166.3 ( c-7,16 ) , 161.3 ( c-2 , 4 ) , 136.9 ( c-12 , 21 ) , 131.2 ( c-4 ) , 128.1 ( c-14 , 23 ) , 127.3 , 127.0 ( c-13 , 22 and c-15 , 24 ) , 113.2 ( c-1,5 ) , 108.9 ( c-3 ) , 48.1 ( c-11 , 20 ) , 8.4 ( c-1 , 5 ) ; anal . the resulting beige solid 14h ( 0.61 g , 97% ) is collected and used without further purification but can be crystallised from ethanol , mp 268269c max ( kbr)/cm 3278 , 3142 , 3110 ( nh ) , 3062 ( ch ar ) , 2929 ( ch aliphatic ) , 1760 , 1709 , 1671 ( c = o ) , 1615 ( c =
n ) , 1554 ( c = c ) ; hnmr ( 200 mhz , 300 k d6-dmso ) 10.60 ( s , 1h , 9-nh ) , 7.88 ( b , 1h , h-8 ) , 7.87 ( d , 1h , jh5,h6 = 8.6 hz , h-5 ) , 7.45 ( d , 1h , jh6,h5 = 8.6 hz , h-6 ) , 2.58 ( s , 3h , 11-ch3 ) , 2.12 ( s , 3h , 3-ch3 ) ; c nmr ( 50 mhz , 300 k , d6-dmso ) 172.8 ( c-2 ) , 169.6 ( c-10 ) , 162.1 ( c-4 ) , 155.8 ( c-8a ) , 145.2 ( c-7 ) , 128.0 ( c-5 ) , 117.5 ( c-6 ) , 112.1 ( c-4a ) , 104.4 ( c-8 ) ; anal . benzyl amine ( 12.5 mmol ) was then added dropwise , directly from the pipette , with stirring , and then the reaction mixture was heated to reflux for 4 hours . general procedure e. n-(benzyl carbamothioyl)-substituted-2-hydroxy - benzamides 13a , b , e , f , and g ( 0.5 mmol ) were suspended in acetic acid ( 3 ml ) in a 25 ml round - bottomed flask . the oily reaction mixture was triturated with minimal diethyl ether and the resulting solid products 15a , b , e , f , and g were collected by vacuum filtration and recrystallized from an appropriate solvent . products 15a , b , e , f , and g prepared in this procedure gave identical mp , ir , h nmr and c nmr to the analogues prepared from compound 14 with comparable yields ( scheme 3 ) . max ( kbr)/cm 30692827 ( n h ) , 1673 ( c = o ) , 1600 ( c = c ) , 1466 ( c =
n ) ; h nmr ( d6-dmso ) 9.00 ( bs , 1h , n
h ) , 7.80 ( d , 1h , j = 8.6 hz , h-5 ) , 7.30 ( m , 5h , arh ) , 6.90 ( dd , 1h , jh6,h8 = 2.4 hz , jh6,h5 = 8.6 hz , h-6 ) , 6.70 ( d , 1h , j = 2.2 hz , h-8 ) , 4.50 ( s , 2h , h-9 ) , 4.20 ( q , 2h , j = 5.6 , ch2o ) , 3.90 ( s , 3h , 7-och3 ) , 1.30 ( t , 3h , j = 5.6 , ch3 ) ; c nmr ( d6-dmso ) 164.4 ( c-4 ) , 163.1 ( c-7 ) , 157.9 ( c-2 ) , 154.9 ( c-8a ) , 137.8 ( c-1 ) , 127.6 ( c-5 ) , 127.5 ( c-2 ) , 126.7 ( c-4 ) , 126.4 ( c-3 ) , 112.6 ( c-6 ) , 110.3 ( c-4a ) , 99.8 ( c-8 ) , 63.6 ( ch2o ) , 43.7 ( c-9 ) , 13.5 ( ch3 ) ; anal . max ( kbr)/cm 3394 , 3202 , 3029 ( nh ) , 1681 , 1635 ( c = o ) ; hnmr ( 200 mhz , 340 k , d6-dmso ) 8.90 ( bs , 2h , h-11,17 ) , 8.27 ( s , 1h , h-5 ) , 7.437.24 ( m , 10h 2 x ar , h-1416 and h-2022 ) , 4.58 ( s , 4h , h-12,18 ) , 2.31 ( s , 3h , h-10 ) ; c nmr ( 50 mhz , 340 k , d6-dmso ) 164.0 ( c-4 ) , 164.0 ( c-9a,10a ) , 157.6 ( c-2 ) , 137.2 ( c-13,19 ) , 127.7 , 126.9 , 126.6 , 122.5 ( c-15,21 , c-14,20 and c-16,22 ) , 113.9 ( c-10 ) , 115.5 ( c-4a,5a ) , 44.0 ( c-12,17 ) , 6.6 ( c-10 ) ; anal . benzyl amine ( 12.5 mmol ) was then added dropwise , directly from the pipette , with stirring , and then the reaction mixture was heated to reflux for 4 hours . | [
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more specifically , acute administration of ethanol damages epithelial cells of the mucosa in the small intestine , increases intestinal permeability , and induces the translocation of endotoxins .
furthermore , it also impairs active transport mechanisms and inhibits the absorption of various nutrients , including vitamins . despite ongoing research ,
the exact pathological mechanisms underlying damage caused by ethanol exposure in the small intestine remain unclear.1,2 several reports have indicated that the structural and functional abnormalities in the small intestine following exposure to alcohol may be related to oxidative stress which was suggested as a possible mechanism for the observed ethanol - induced damage to increase intestinal permeability.3 on the other hand , oxidative stress may induce the synthesis of heat shock proteins ( hsps ) , cellular structural proteins known to play a central role in the elimination and modification of denatured cellular proteins and the prevention of aggregation of those proteins .
they function as molecular chaperones that mediate protein synthesis , folding , transport , translocation , and processing.4 based on their molecular weight , they have been classified into the hsp60 , hsp70 , and hsp90 families , as well as the small hsp family .
several reports have addressed the roles of hsps in intestinal inflammation , however , few investigations regarding the effects of hsps in ethanol - induced damage to the small intestine have been conducted.5 in addition to increased intestinal permeability , bacterial flora may be related to intestinal damage . bacterial flora present in the gastric intestinal tract
are present at a level of 10 colony - forming units ( cfu)/ml or less in the stomach but can be increased up to 10 - 10 bacteria / ml , 10 bacteria / ml , and 10 bacteria / ml in the proximal jejunum , distal ileum , and colon , respectively.6 furthermore , the composition of bacterial flora changes gradually from gram - positive to gram - negative from the proximal to the distal end . as the composition and density of bacterial flora may vary along the length of the small intestine , one might expect that the degree and mechanisms underlying their pathological damage may be different as well .
therefore , the current study was conducted to examine pathological changes in different regions of the small intestine induced by ethanol exposure in order to investigate underlying mechanisms .
furthermore , the association between oxidative stress and small intestine damage was analyzed by assessing antioxidative levels and inflammatory factors .
lastly , changes in the expression of hsps , which are regulatory factors for cellular oxidative damage , were also examined .
in the current study , specific - pathogen - free ( spf ) 8-week - old male icr mice were obtained from dae han biolink co. ( eumsung , korea ) and housed in specific spf chambers at the school of medicine in kangwon national university . the spf chambers in the animal room
were maintained at 231 and 557% relative humidity and mice were acclimated for 3 days prior to the experiments .
animals had free access to water and diets and were housed on a 12:12 hour light - dark cycle .
all experiments were approved by the animal experiment ethics committee of kangwon national university in advance of the study ( approval number : kiacuc-11 - 0012 ) . in the experiment ,
mice were randomly allocated into 4 groups : a control group ( phosphate buffered saline [ pbs ] treated ) , and 3 ethanol groups ( 2.75 g / kg , 5.5 g / kg , and 8.25 g / kg ) .
ethanol ( merck , damstadt , germany ) was diluted with saline ( pbs ) and then administered via intraperitoneal injection .
icr mice were administered either pbs ( control group ) or pbs diluted anhydrous ethanol ( 2.75 g / kg , 5.5 g / kg , or 8.25 g / kg ) via intraperitoneal injection . subsequently , alcohol - induced damage to the small intestines
the small intestine was divided into 3 parts : the proximal jejunum , the middle of the small intestine , and the distal ileum .
each tissue segment with the size of 3 cm was collected and fixed with formalin solution and then histological changes were observed via pathological examinations using optical microscopy ( olympus bx43 ; olympus co. , tokyo , japan ) .
one - hour after the intraperitoneal injection of ethanol , small intestines were harvested from icr mice and total genomic rna was extracted using the easy - spin total rna extraction kit ( intron , seongnam , korea ) according to the manufacturer 's instructions .
quantitative and qualitative analyses of the extracted rna were conducted using the nanodrop nd-1000 ( nanodrop technologies , wilmington , de , usa ) .
the cdna was synthesized using extracted total rna ( 2 g ) , 2.5 mm oligo ( dt ) , and an omniscript rt kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions in order to monitor changes in mrna expression of tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and cyclooxygenase-2 ( cox-2 ) ( geneamp pcr system 2700 ; applied biosystems , foster city , ca , usa ) .
( daejeon , korea ) and base sequences of primers are summarized in table 1 . the respective primer pair for each gene
was added to a mixture ( 20 l ) of synthesized cdna , 1 rox reference dye ( takara bio , otsu , shiga , japan ) , and 1 stbr premix ex tag ( takara bio , otsu , shiga , japan ) .
real - time pcr reactions were performed using the applied biosystems 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) .
changes in the expression level of each gene were applied into the -2 equation using a housekeeping gene ( i.e. , glyceraldehyde-3-phosphate dehydrogenase [ gapdh ] ) and then corrected .
corrected values were compared to those of the pbs - treated control group ( ct = gene of interest ct - gapdh ct , -ct=ct treated-ct control ) .
one - hour after the intraperitoneal injection of ethanol , small intestines were harvested from icr mice and protein was extracted using the pro - prep kit ( intron , seongnam , korea ) per the manufacturer 's instructions.7 protein quantification was conducted using the bca ( sigma , st .
protein samples were separated via sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and then transferred to polyvinylidene fluoride ( pvdf ) membranes .
once transferred , polyvinylidene fluoride membranes were blocked with 5% blocking buffer ( i.e. , non - fat dry milk ) for 30 minutes and then incubated with primary antibody diluted into blocking buffer for 16 hours at 4 ( hsp40 , hsp70 , and hsp90 antibody , 1:1,000 , stressgen , ann arbor , mi , usa ; hsp32 [ heme oxygenase-1 , ho-1 ] antibody , 1:1,000 , abcam , cambridge , ma , usa ; glutathione - s - transferase [ gst ] antibody , 1:1,000 , sigma , st .
the membrane was washed 3 times for 10 minutes and then incubated with secondary antibody for 2 hours at room temperature ( horseradish peroxidase - conjugated goat anti - rabbit igg , horseradish peroxidase - conjugated goat anti - mouse igg , 1:2,000 , jackson immunoresearch , west grove , pa , usa ) .
once incubated , the membrane was washed with tbst 5 times and then incubated again with ecl ( amersham , pittsburgh , pa , usa ) in advance of exposure against x - ray film ( kodak , rochester , ny , usa ) .
developed protein bands on the film were analyzed using the image quant software ( molecular dynamics , sunnyvale , ca , usa ) and then corrected with a housekeeping gene ( -actin ) .
proteins were extracted from the respective segments of the small intestine and then quantified once .
the statistical significance of the results was analyzed via the non - parametric kruskal - wallis test and mann - whitney u test post - hoc analysis ( spss version 12.0 , spss , inc .
in the current study , specific - pathogen - free ( spf ) 8-week - old male icr mice were obtained from dae han biolink co. ( eumsung , korea ) and housed in specific spf chambers at the school of medicine in kangwon national university . the spf chambers in the animal room
were maintained at 231 and 557% relative humidity and mice were acclimated for 3 days prior to the experiments .
animals had free access to water and diets and were housed on a 12:12 hour light - dark cycle .
all experiments were approved by the animal experiment ethics committee of kangwon national university in advance of the study ( approval number : kiacuc-11 - 0012 ) . in the experiment ,
mice were randomly allocated into 4 groups : a control group ( phosphate buffered saline [ pbs ] treated ) , and 3 ethanol groups ( 2.75 g / kg , 5.5 g / kg , and 8.25 g / kg ) .
ethanol ( merck , damstadt , germany ) was diluted with saline ( pbs ) and then administered via intraperitoneal injection .
icr mice were administered either pbs ( control group ) or pbs diluted anhydrous ethanol ( 2.75 g / kg , 5.5 g / kg , or 8.25 g / kg ) via intraperitoneal injection .
the small intestine was divided into 3 parts : the proximal jejunum , the middle of the small intestine , and the distal ileum .
each tissue segment with the size of 3 cm was collected and fixed with formalin solution and then histological changes were observed via pathological examinations using optical microscopy ( olympus bx43 ; olympus co. , tokyo , japan ) .
one - hour after the intraperitoneal injection of ethanol , small intestines were harvested from icr mice and total genomic rna was extracted using the easy - spin total rna extraction kit ( intron , seongnam , korea ) according to the manufacturer 's instructions .
quantitative and qualitative analyses of the extracted rna were conducted using the nanodrop nd-1000 ( nanodrop technologies , wilmington , de , usa ) .
rna samples were stored at -70 until further analysis . the cdna was synthesized using extracted total rna ( 2 g ) , 2.5 mm oligo ( dt ) , and an omniscript rt kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions in order to monitor changes in mrna expression of tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and cyclooxygenase-2 ( cox-2 ) ( geneamp pcr system 2700 ; applied biosystems , foster city , ca , usa ) .
( daejeon , korea ) and base sequences of primers are summarized in table 1 . the respective primer pair for each gene
was added to a mixture ( 20 l ) of synthesized cdna , 1 rox reference dye ( takara bio , otsu , shiga , japan ) , and 1 stbr premix ex tag ( takara bio , otsu , shiga , japan ) .
real - time pcr reactions were performed using the applied biosystems 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) .
changes in the expression level of each gene were applied into the -2 equation using a housekeeping gene ( i.e. , glyceraldehyde-3-phosphate dehydrogenase [ gapdh ] ) and then corrected .
corrected values were compared to those of the pbs - treated control group ( ct = gene of interest ct - gapdh ct , -ct=ct treated-ct control ) .
one - hour after the intraperitoneal injection of ethanol , small intestines were harvested from icr mice and total genomic rna was extracted using the easy - spin total rna extraction kit ( intron , seongnam , korea ) according to the manufacturer 's instructions .
quantitative and qualitative analyses of the extracted rna were conducted using the nanodrop nd-1000 ( nanodrop technologies , wilmington , de , usa ) .
the cdna was synthesized using extracted total rna ( 2 g ) , 2.5 mm oligo ( dt ) , and an omniscript rt kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions in order to monitor changes in mrna expression of tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and cyclooxygenase-2 ( cox-2 ) ( geneamp pcr system 2700 ; applied biosystems , foster city , ca , usa ) .
( daejeon , korea ) and base sequences of primers are summarized in table 1 . the respective primer pair for each gene
was added to a mixture ( 20 l ) of synthesized cdna , 1 rox reference dye ( takara bio , otsu , shiga , japan ) , and 1 stbr premix ex tag ( takara bio , otsu , shiga , japan ) .
real - time pcr reactions were performed using the applied biosystems 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) .
changes in the expression level of each gene were applied into the -2 equation using a housekeeping gene ( i.e. , glyceraldehyde-3-phosphate dehydrogenase [ gapdh ] ) and then corrected .
corrected values were compared to those of the pbs - treated control group ( ct = gene of interest ct - gapdh ct , -ct=ct treated-ct control ) .
one - hour after the intraperitoneal injection of ethanol , small intestines were harvested from icr mice and protein was extracted using the pro - prep kit ( intron , seongnam , korea ) per the manufacturer 's instructions.7 protein quantification was conducted using the bca ( sigma , st .
protein samples were separated via sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and then transferred to polyvinylidene fluoride ( pvdf ) membranes .
i.e. , non - fat dry milk ) for 30 minutes and then incubated with primary antibody diluted into blocking buffer for 16 hours at 4 ( hsp40 , hsp70 , and hsp90 antibody , 1:1,000 , stressgen , ann arbor , mi , usa ; hsp32 [ heme oxygenase-1 , ho-1 ] antibody , 1:1,000 , abcam , cambridge , ma , usa ; glutathione - s - transferase [ gst ] antibody , 1:1,000 , sigma , st .
the membrane was washed 3 times for 10 minutes and then incubated with secondary antibody for 2 hours at room temperature ( horseradish peroxidase - conjugated goat anti - rabbit igg , horseradish peroxidase - conjugated goat anti - mouse igg , 1:2,000 , jackson immunoresearch , west grove , pa , usa ) .
once incubated , the membrane was washed with tbst 5 times and then incubated again with ecl ( amersham , pittsburgh , pa , usa ) in advance of exposure against x - ray film ( kodak , rochester , ny , usa ) .
developed protein bands on the film were analyzed using the image quant software ( molecular dynamics , sunnyvale , ca , usa ) and then corrected with a housekeeping gene ( -actin ) .
proteins were extracted from the respective segments of the small intestine and then quantified once .
the statistical significance of the results was analyzed via the non - parametric kruskal - wallis test and mann - whitney u test post - hoc analysis ( spss version 12.0 , spss , inc . ,
chicago , il , usa ) . a p - value<0.05 was considered as statistically significant .
in the current study , the small intestines were dissected following 1 hour of ethanol administration .
this was based on the results of a preliminary study which tested various time points ( i.e. , 30 minutes , 1 hour , 3 hours , and 5 hours ) in order to determine the optimal timing for harvesting tissue .
the preliminary study results showed that mice treated with high concentrations of ethanol died even before they reached several of the specific time points , which lead us to harvest tissues after 1 hour of ethanol administration , as all animals survived at least that long regardless of the ethanol dose .
pathological analyses were conducted to explore differences among the regions of the small intestine according to the concentration of ethanol administered .
regarding damage on mucosal membranes , samples from pbs - treated mice displayed well maintained villi and normal lymphoid follicles without infiltration of inflammatory cells . in contrast , mice treated with 2.75 g / kg of ethanol showed partial falling of the villi , slight edema of the glandular layers , damage to the villi in the proximal segments and more pronounced damage to the villi in distal segments . in mice treated with 5.5 g / kg , falling of the villi
was observed more frequently , as were destroyed glandular layers , erosions , and ulcers . in particular , severe erosions and ulcers were detected in distal segments where lymphoid follicles were penetrated by inflammatory cells .
mice treated with 8.25 g / kg of ethanol showed an overall loss of glandular layers , extremely severe ulcers , and damage to the serosa .
furthermore , irregular expansion of the lumen was observed , possibly due to ischemic changes in the gastrointestinal tract ( fig .
collectively , all ethanol - treated groups displayed more serious damage in distal segments as compared to proximal segments based on histological observations of the small intestines .
a variety of changes in the expression of hsps were found in the small intestines in response to ethanol administration .
such effects were more pronounced in the distal segments of the small intestines ( fig .
hsp40 was significantly increased in the control and low dose ( 2.75 g / kg ) ethanol groups from the distal segment as compared to the proximal and middle segments .
in contrast , hsp40 was significantly decreased in the distal segments of the small intestines from the 5.5 g / kg and 8.25 g / kg ethanol - treated groups ( fig .
similar to hsp40 , hsp90 was significantly elevated in the control group and decreased as the ethanol dosage increased in the distal segment of the small intestines ( fig .
in addition , hsp90 was significantly higher in the proximal and middle segments of the small intestines of mice exposed to ethanol at 5.5 g / kg and was elevated in the middle segment in mice treated with 8.25 g / kg of ethanol , indicating site - specific differences and significant changes according to the concentration of ethanol are not observed in hsp90 expression . in the control group , hsp32 was significantly increased in the middle and distal segments of the small intestines but decreased in the proximal segment by comparison . although hsp32 was decreased in the distal segment in a dose dependent manner , its expression pattern was irregular in the proximal segment ( fig .
no difference in gst , an anti - oxidative enzyme , was noted between the control and ethanol - treated groups .
additionally , no changes were found in the different segments of the small intestines ( fig .
although expression of the cytokine tnf- increased in a dose dependent manner , there was no difference among segments of the small intestines ( fig .
similarly , il-1 expression increased with increasing ethanol dosages but did not demonstrated site - specific differences ( fig .
the inflammatory mediator cox-2 was elevated without site - specific changes as well ( fig .
in the current study , the small intestines were dissected following 1 hour of ethanol administration .
this was based on the results of a preliminary study which tested various time points ( i.e. , 30 minutes , 1 hour , 3 hours , and 5 hours ) in order to determine the optimal timing for harvesting tissue .
the preliminary study results showed that mice treated with high concentrations of ethanol died even before they reached several of the specific time points , which lead us to harvest tissues after 1 hour of ethanol administration , as all animals survived at least that long regardless of the ethanol dose .
pathological analyses were conducted to explore differences among the regions of the small intestine according to the concentration of ethanol administered .
regarding damage on mucosal membranes , samples from pbs - treated mice displayed well maintained villi and normal lymphoid follicles without infiltration of inflammatory cells .
in contrast , mice treated with 2.75 g / kg of ethanol showed partial falling of the villi , slight edema of the glandular layers , damage to the villi in the proximal segments and more pronounced damage to the villi in distal segments . in mice treated with 5.5 g / kg , falling of the villi
was observed more frequently , as were destroyed glandular layers , erosions , and ulcers . in particular , severe erosions and ulcers were detected in distal segments where lymphoid follicles were penetrated by inflammatory cells .
mice treated with 8.25 g / kg of ethanol showed an overall loss of glandular layers , extremely severe ulcers , and damage to the serosa .
furthermore , irregular expansion of the lumen was observed , possibly due to ischemic changes in the gastrointestinal tract ( fig .
collectively , all ethanol - treated groups displayed more serious damage in distal segments as compared to proximal segments based on histological observations of the small intestines .
a variety of changes in the expression of hsps were found in the small intestines in response to ethanol administration .
such effects were more pronounced in the distal segments of the small intestines ( fig .
hsp40 was significantly increased in the control and low dose ( 2.75 g / kg ) ethanol groups from the distal segment as compared to the proximal and middle segments . in contrast , hsp40 was significantly decreased in the distal segments of the small intestines from the 5.5 g / kg and 8.25 g / kg ethanol - treated groups ( fig .
similar to hsp40 , hsp90 was significantly elevated in the control group and decreased as the ethanol dosage increased in the distal segment of the small intestines ( fig .
in addition , hsp90 was significantly higher in the proximal and middle segments of the small intestines of mice exposed to ethanol at 5.5 g / kg and was elevated in the middle segment in mice treated with 8.25 g / kg of ethanol , indicating site - specific differences and significant changes according to the concentration of ethanol are not observed in hsp90 expression . in the control group , hsp32 was significantly increased in the middle and distal segments of the small intestines but decreased in the proximal segment by comparison . although hsp32 was decreased in the distal segment in a dose dependent manner , its expression pattern was irregular in the proximal segment ( fig .
no difference in gst , an anti - oxidative enzyme , was noted between the control and ethanol - treated groups .
additionally , no changes were found in the different segments of the small intestines ( fig .
although expression of the cytokine tnf- increased in a dose dependent manner , there was no difference among segments of the small intestines ( fig .
3b ) . similarly , il-1 expression increased with increasing ethanol dosages but did not demonstrated site - specific differences ( fig .
the inflammatory mediator cox-2 was elevated without site - specific changes as well ( fig .
acute exposure to ethanol is known to cause damage to the epithelial cells of the intestinal mucosa .
possible mechanisms underlying this phenomenon include increased intestinal permeability and the translocation of endotoxins by bacterial flora .
as there are regional differences in intestinal bacteria , it is likely that structural and functional changes in the small intestines may differ from one to another region . in the current study
, small intestinal damage was induced via acute administration of ethanol in order to compare various biochemical changes in the different regions and elucidate putative mechanisms underlying the damage .
ethanol - induced damage to the small intestines has been associated with oxidative stress , which results in increased carbonylation and nitrotyrosination of cellular proteins including actin and the microtubule cytoskeleton .
these changes are known to increase membrane permeability and induce the loss of integrity of tight junctions.3 similar results were also demonstrated in a recent study that investigated ethanol - induced permeability of the epithelial cells of the lungs.8 collectively , the results of the aforementioned studies suggest that oxidative stress may play a critical role in modulating intestinal permeability .
hsps , molecular chaperones , are critical to cell survival and detrimental to protect cells against the harmful effects of stress - related stimuli .
hsps are synthesized in various cells when exposed not only to ethanol , but also to other environmental stressors .
although most hsps mediate similar protective effects in response to stress , differences are apparent in their expression patterns and the biological roles depending on their class .
for instance , hsp70 is known to be strongly induced by stress , whereas hsp90 is essentially expressed regardless of stress.9 an additional study reported that hsp70 was increased in non - steroidal anti - inflammatory drug - induced damage , while geranylgeranylacetone , a known hsp inducer used clinically as anti - ulcer drug , increased hsp70 in tissues of the small intestine and had a protective effect against damage to the intestinal mucosa.5 furthermore , a protective effect of hsp70 has been demonstrated in intestinal inflammation , generating interest in potential novel roles of hsps in the treatment of intestinal diseases.10 in rats , hsp70 and hsp32 were induced when ischemia - reperfusion injuries were generated.11 however , the roles of hsps in the small intestine have not yet been fully elucidated .
therefore , the current study was conducted with the aim of investigating differences in the expression of hsps in the small intestine and their potential roles in ethanol - induced intestinal damage .
there are multiple hsps with varying functions . in the current study , which was intended to examine the effects of hsps on the different regions of the small intestine ( i.e. , proximal , middle , and distal parts ) damaged by ethanol , a review of relevant literature led to the investigation of hsp70 , hsp90 , hsp40 , and hsp32 .
the results of the current study demonstrated distinct differences in damage to the small intestines of icr mice depending on the concentration of ethanol and that damage was more pronounced in the distal region as compared to the rest of small intestines .
when pbs was administered to controls , hsp40 and hsp90 were higher in the distal region while hsp70 was elevated regardless of the region examined .
conversely , hsp32 was lower in the proximal region in controls . in the ethanol - treated mice , it was difficult to find any specific correlation or site - specific associations between ethanol exposure and expression of hsps , although differences in the expression levels of hsps were detected and dependent on the region examined .
hsp70 decreased in a dose - dependent manner in response to ethanol , particularly in the distal region .
similar findings were observed for hsp32 , hsp40 and hsp90 , however , displayed a site - specific pattern similar to that of hsp70 at a dose of 5.5 g / kg only , with different expression patterns found in the 2.75 g / kg and 8.5 g / kg ethanol - treated groups . when comparing the site - specific expression patterns of hsps in this model , varying expression patterns and degrees of histological damage were found , in keeping with the known associations between the expression of hsps and degrees of histological damage . in the small intestine of icr mice ,
hsp70 generally showed the highest levels of expression and these levels were inversely correlated with the concentration of ethanol .
it is important to note that the drastic change in hsp70 observed in distal regions was associated with severe tissue damage .
in addition , the expression pattern of hsp32 was very similar to that of hsp70 , suggesting that both hsps may be critical in damage to the small intestine . however , it is difficult to elucidate the exact roles of these hsps based on the results of the current study . upon the administration of ethanol , cytokines ( i.e. , tnf- , and il-1 ) and inflammatory mediator ( i.e. , cox-2 ) were not showing any noticeable change among different regions of the small intestine , as found in macroscopic observations .
furthermore , no differences in the expression of gst , an anti - oxidative enzyme known to play an important role in protecting cells against oxidative stress , were detected between the control and ethanol groups , or among the regions of the small intestines in response to intestinal damage .
however , it is difficult to exclude the possibility of an association between gst and intestinal damage , as the potential for differences in enzyme activity may be more significant than differences in protein expression levels .
additional studies examining putative associations between gst and intestinal damage may be warranted . in the current study , varying magnitudes of histological damage and hsp expression in different regions of the small intestines were observed .
one can expect that such site - specific changes are because we injected ethanol on lower right abdominal side .
we decided to inject ethanol on the lower right side of the abdomen where there was a minimal risk of tissue damages due to a less dense distribution of blood vessels .
however , the injection site itself may have influenced the results ( i.e. , a site specific effect of ethanol ) .
this would seem unlikely , as hsp40 and hsp90 were exclusively increased in distal regions and hsp70 expression was elevated in a non - site - specific manner in the control group .
furthermore , hsp32 and hsp70 demonstrated similar expression patterns . therefore , different patterns of hsp expression were likely due to their inherent features rather than the injection site . in order to provide further clarification regarding the effects of ethanol exposure on hsp expression patterns in the small intestines
it may be possible for intestinal bacteria to impact patterns of hsp expression given the differences in distribution and classes of bacteria among regions of the small intestines .
physiological and environmental stress ( e.g. , microbial infection ) may induce the synthesis of cellular protective hsps on the intestinal epithelial cells . according to the results of previous studies ,
hsps were modulated in response to multiple stimuli on the intestinal epithelial cells and such modulations were contingent on the exposure to intestinal probiotic bacteria.12 therefore , the regional differences in hsp expression in the small intestine are likely attributable , at least in part , to the intestinal bacteria that differ in type and distribution throughout the small intestine . as opposed to the proximal region of the small intestine which contains few bacteria , more bacteria and their metabolites
are present in the distal region and may result in more pronounced effects on hsp expression .
some hsps are known to be expressed under stress - free environments to maintain cellular integrity . in a previous study that examined metronidazole administration in a small intestinal model ,
an intestinal bacterial flora demonstrated the ability to modulate physiological expression of the cellular protective hsps 25 and 72.13 although not examined in the current study , the administration of antibiotics may be useful in further elucidating associations between hsps and intestinal bacteria in a mouse model .
for two different reasons , ethanol was administrated via intraperitoneal injection instead of oral exposure in the current study .
first , the original goal of the study was to investigate differences among the regions of the small intestines in response to ethanol exposure .
although oral administration is the route of exposure for ethanol in humans , it may result in exposure to higher concentrations of ethanol in the proximal region compared to the distal regions and thereby make it difficult to analyze effects on the respective regions in a quantitative manner .
second , the bioavailability of ethanol in the small intestines differs due to varying degrees of gastric emptying .
as such , exposure to ethanol via oral administration might be more suitable to examine chronic effects but is not recommended to determine acute effects .
therefore , ethanol was administered via intraperitoneal injection primarily to provide uniform concentrations over the small intestines in order to examine its impact on different regions . in previous studies
, ethanol has been administered through intraperitoneal injection to achieve similar research objectives.14 the current study had another limitation .
more extensive and accurate results may have been generated if the time course of the damaging effects of ethanol on the small intestines at the concentrations of ethanol high enough to make such changes ( i.e. , 5.5 g / kg or higher ) was monitored .
however , the initial study objective was to determine damage to varying regions of the small intestines .
to better understand the mechanisms underlying ethanol - induced damage , further studies regarding the time course of effects as well as the magnitudes of intestinal damage dependent on exposure time should be conducted .
collectively , results from the current study demonstrated that the expression patterns of the hsps assessed varied in general and also in a region - specific manner .
specifically , more pronounced histological damage was found in the distal region and hsp70 and hsp32 were decreased in particular when ethanol was administered intraperitoneally .
the endotoxins produced by bacterial flora may have influenced the expression of hsps in ethanol - induced damage to the small intestine and future studies should address this possibility . | background / aimsethanol administration causes intestinal epithelial cell damage by increasing intestinal permeability and the translocation of endotoxins from intestinal bacterial flora .
heat shock proteins ( hsps ) are associated with recovery and protection from cell damage .
the aim of the current study was to investigate differences in the expression of hsps in the small intestine and the biochemical changes attributable to ethanol - induced intestinal damage.methodsethanol ( 20% ) was injected intraperitoneally ( 2.75 g / kg , 5.5 g / kg , 8.25 g / kg ) in icr mice and the same volume of saline was administered to controls .
after 1 hour , the proximal , middle , and distal segments were taken from the small intestine and the degree of damage was analyzed . in each segment , the expression of hsps was analyzed by western blotting .
the expression of inflammatory mediators including interleukin-1 ( il-1 ) , tumor necrosis factor- ( tnf- ) , cyclooxygenase-2 ( cox-2 ) , and antioxidant enzyme such as glutathione - s - transferase were compared using real - time polymerase chain reaction assays.resultsin the control group , hsp70 increased in all segments of small intestine .
additionally , increases in the expression of hsp40 and hsp90 in the distal regions and an increase in hsp32 in the middle regions were observed .
after ethanol treatment , greater histological damage was observed in the distal small intestine and significant decreases in hsps were observed generally .
increased expression of il-1 , tnf- , and cox-2 was observed in small intestinal tissues exposed to ethanol - induced damage .
however , there was no significant difference in the expression of an antioxidant enzyme.conclusionssignificant differences in the expression of hsps in different intestinal regions were observed .
these differences may have been attributable to the distribution of intestinal bacteria . | INTRODUCTION
METHODS
1. Experimental Animals and Ethanol Injection
2. Macroscopic Assessment and Histological Analyses
3. RNA Analysis
1) RNA Extraction
2) The Synthesis of cDNA and Real-time PCR Analysis
4. Protein Analysis
5. Statistical Analysis
RESULTS
1. Determination of the Time Point for Harvesting Small Intestines Following the Administration of Ethanol
2. Pathological Analysis of Damage to the Small Intestine Induced by Ethanol Administration
3. Changes in HSPs
4. Changes in an Anti-oxidative Enzyme and Cytokines
DISCUSSION | more specifically , acute administration of ethanol damages epithelial cells of the mucosa in the small intestine , increases intestinal permeability , and induces the translocation of endotoxins . despite ongoing research ,
the exact pathological mechanisms underlying damage caused by ethanol exposure in the small intestine remain unclear.1,2 several reports have indicated that the structural and functional abnormalities in the small intestine following exposure to alcohol may be related to oxidative stress which was suggested as a possible mechanism for the observed ethanol - induced damage to increase intestinal permeability.3 on the other hand , oxidative stress may induce the synthesis of heat shock proteins ( hsps ) , cellular structural proteins known to play a central role in the elimination and modification of denatured cellular proteins and the prevention of aggregation of those proteins . they function as molecular chaperones that mediate protein synthesis , folding , transport , translocation , and processing.4 based on their molecular weight , they have been classified into the hsp60 , hsp70 , and hsp90 families , as well as the small hsp family . several reports have addressed the roles of hsps in intestinal inflammation , however , few investigations regarding the effects of hsps in ethanol - induced damage to the small intestine have been conducted.5 in addition to increased intestinal permeability , bacterial flora may be related to intestinal damage . bacterial flora present in the gastric intestinal tract
are present at a level of 10 colony - forming units ( cfu)/ml or less in the stomach but can be increased up to 10 - 10 bacteria / ml , 10 bacteria / ml , and 10 bacteria / ml in the proximal jejunum , distal ileum , and colon , respectively.6 furthermore , the composition of bacterial flora changes gradually from gram - positive to gram - negative from the proximal to the distal end . as the composition and density of bacterial flora may vary along the length of the small intestine , one might expect that the degree and mechanisms underlying their pathological damage may be different as well . therefore , the current study was conducted to examine pathological changes in different regions of the small intestine induced by ethanol exposure in order to investigate underlying mechanisms . furthermore , the association between oxidative stress and small intestine damage was analyzed by assessing antioxidative levels and inflammatory factors . lastly , changes in the expression of hsps , which are regulatory factors for cellular oxidative damage , were also examined . in the current study , specific - pathogen - free ( spf ) 8-week - old male icr mice were obtained from dae han biolink co. ( eumsung , korea ) and housed in specific spf chambers at the school of medicine in kangwon national university . in the experiment ,
mice were randomly allocated into 4 groups : a control group ( phosphate buffered saline [ pbs ] treated ) , and 3 ethanol groups ( 2.75 g / kg , 5.5 g / kg , and 8.25 g / kg ) . icr mice were administered either pbs ( control group ) or pbs diluted anhydrous ethanol ( 2.75 g / kg , 5.5 g / kg , or 8.25 g / kg ) via intraperitoneal injection . subsequently , alcohol - induced damage to the small intestines
the small intestine was divided into 3 parts : the proximal jejunum , the middle of the small intestine , and the distal ileum . the cdna was synthesized using extracted total rna ( 2 g ) , 2.5 mm oligo ( dt ) , and an omniscript rt kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions in order to monitor changes in mrna expression of tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and cyclooxygenase-2 ( cox-2 ) ( geneamp pcr system 2700 ; applied biosystems , foster city , ca , usa ) . , non - fat dry milk ) for 30 minutes and then incubated with primary antibody diluted into blocking buffer for 16 hours at 4 ( hsp40 , hsp70 , and hsp90 antibody , 1:1,000 , stressgen , ann arbor , mi , usa ; hsp32 [ heme oxygenase-1 , ho-1 ] antibody , 1:1,000 , abcam , cambridge , ma , usa ; glutathione - s - transferase [ gst ] antibody , 1:1,000 , sigma , st . proteins were extracted from the respective segments of the small intestine and then quantified once . in the current study , specific - pathogen - free ( spf ) 8-week - old male icr mice were obtained from dae han biolink co. ( eumsung , korea ) and housed in specific spf chambers at the school of medicine in kangwon national university . in the experiment ,
mice were randomly allocated into 4 groups : a control group ( phosphate buffered saline [ pbs ] treated ) , and 3 ethanol groups ( 2.75 g / kg , 5.5 g / kg , and 8.25 g / kg ) . icr mice were administered either pbs ( control group ) or pbs diluted anhydrous ethanol ( 2.75 g / kg , 5.5 g / kg , or 8.25 g / kg ) via intraperitoneal injection . the small intestine was divided into 3 parts : the proximal jejunum , the middle of the small intestine , and the distal ileum . the cdna was synthesized using extracted total rna ( 2 g ) , 2.5 mm oligo ( dt ) , and an omniscript rt kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions in order to monitor changes in mrna expression of tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and cyclooxygenase-2 ( cox-2 ) ( geneamp pcr system 2700 ; applied biosystems , foster city , ca , usa ) . the cdna was synthesized using extracted total rna ( 2 g ) , 2.5 mm oligo ( dt ) , and an omniscript rt kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions in order to monitor changes in mrna expression of tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and cyclooxygenase-2 ( cox-2 ) ( geneamp pcr system 2700 ; applied biosystems , foster city , ca , usa ) . , non - fat dry milk ) for 30 minutes and then incubated with primary antibody diluted into blocking buffer for 16 hours at 4 ( hsp40 , hsp70 , and hsp90 antibody , 1:1,000 , stressgen , ann arbor , mi , usa ; hsp32 [ heme oxygenase-1 , ho-1 ] antibody , 1:1,000 , abcam , cambridge , ma , usa ; glutathione - s - transferase [ gst ] antibody , 1:1,000 , sigma , st . proteins were extracted from the respective segments of the small intestine and then quantified once . in the current study , the small intestines were dissected following 1 hour of ethanol administration . , 30 minutes , 1 hour , 3 hours , and 5 hours ) in order to determine the optimal timing for harvesting tissue . pathological analyses were conducted to explore differences among the regions of the small intestine according to the concentration of ethanol administered . in contrast , mice treated with 2.75 g / kg of ethanol showed partial falling of the villi , slight edema of the glandular layers , damage to the villi in the proximal segments and more pronounced damage to the villi in distal segments . in mice treated with 5.5 g / kg , falling of the villi
was observed more frequently , as were destroyed glandular layers , erosions , and ulcers . mice treated with 8.25 g / kg of ethanol showed an overall loss of glandular layers , extremely severe ulcers , and damage to the serosa . a variety of changes in the expression of hsps were found in the small intestines in response to ethanol administration . such effects were more pronounced in the distal segments of the small intestines ( fig . hsp40 was significantly increased in the control and low dose ( 2.75 g / kg ) ethanol groups from the distal segment as compared to the proximal and middle segments . in contrast , hsp40 was significantly decreased in the distal segments of the small intestines from the 5.5 g / kg and 8.25 g / kg ethanol - treated groups ( fig . similar to hsp40 , hsp90 was significantly elevated in the control group and decreased as the ethanol dosage increased in the distal segment of the small intestines ( fig . in addition , hsp90 was significantly higher in the proximal and middle segments of the small intestines of mice exposed to ethanol at 5.5 g / kg and was elevated in the middle segment in mice treated with 8.25 g / kg of ethanol , indicating site - specific differences and significant changes according to the concentration of ethanol are not observed in hsp90 expression . in the control group , hsp32 was significantly increased in the middle and distal segments of the small intestines but decreased in the proximal segment by comparison . additionally , no changes were found in the different segments of the small intestines ( fig . although expression of the cytokine tnf- increased in a dose dependent manner , there was no difference among segments of the small intestines ( fig . in the current study , the small intestines were dissected following 1 hour of ethanol administration . , 30 minutes , 1 hour , 3 hours , and 5 hours ) in order to determine the optimal timing for harvesting tissue . pathological analyses were conducted to explore differences among the regions of the small intestine according to the concentration of ethanol administered . in contrast , mice treated with 2.75 g / kg of ethanol showed partial falling of the villi , slight edema of the glandular layers , damage to the villi in the proximal segments and more pronounced damage to the villi in distal segments . in mice treated with 5.5 g / kg , falling of the villi
was observed more frequently , as were destroyed glandular layers , erosions , and ulcers . mice treated with 8.25 g / kg of ethanol showed an overall loss of glandular layers , extremely severe ulcers , and damage to the serosa . a variety of changes in the expression of hsps were found in the small intestines in response to ethanol administration . such effects were more pronounced in the distal segments of the small intestines ( fig . hsp40 was significantly increased in the control and low dose ( 2.75 g / kg ) ethanol groups from the distal segment as compared to the proximal and middle segments . in contrast , hsp40 was significantly decreased in the distal segments of the small intestines from the 5.5 g / kg and 8.25 g / kg ethanol - treated groups ( fig . similar to hsp40 , hsp90 was significantly elevated in the control group and decreased as the ethanol dosage increased in the distal segment of the small intestines ( fig . in addition , hsp90 was significantly higher in the proximal and middle segments of the small intestines of mice exposed to ethanol at 5.5 g / kg and was elevated in the middle segment in mice treated with 8.25 g / kg of ethanol , indicating site - specific differences and significant changes according to the concentration of ethanol are not observed in hsp90 expression . in the control group , hsp32 was significantly increased in the middle and distal segments of the small intestines but decreased in the proximal segment by comparison . additionally , no changes were found in the different segments of the small intestines ( fig . although expression of the cytokine tnf- increased in a dose dependent manner , there was no difference among segments of the small intestines ( fig . possible mechanisms underlying this phenomenon include increased intestinal permeability and the translocation of endotoxins by bacterial flora . in the current study
, small intestinal damage was induced via acute administration of ethanol in order to compare various biochemical changes in the different regions and elucidate putative mechanisms underlying the damage . ethanol - induced damage to the small intestines has been associated with oxidative stress , which results in increased carbonylation and nitrotyrosination of cellular proteins including actin and the microtubule cytoskeleton . these changes are known to increase membrane permeability and induce the loss of integrity of tight junctions.3 similar results were also demonstrated in a recent study that investigated ethanol - induced permeability of the epithelial cells of the lungs.8 collectively , the results of the aforementioned studies suggest that oxidative stress may play a critical role in modulating intestinal permeability . for instance , hsp70 is known to be strongly induced by stress , whereas hsp90 is essentially expressed regardless of stress.9 an additional study reported that hsp70 was increased in non - steroidal anti - inflammatory drug - induced damage , while geranylgeranylacetone , a known hsp inducer used clinically as anti - ulcer drug , increased hsp70 in tissues of the small intestine and had a protective effect against damage to the intestinal mucosa.5 furthermore , a protective effect of hsp70 has been demonstrated in intestinal inflammation , generating interest in potential novel roles of hsps in the treatment of intestinal diseases.10 in rats , hsp70 and hsp32 were induced when ischemia - reperfusion injuries were generated.11 however , the roles of hsps in the small intestine have not yet been fully elucidated . therefore , the current study was conducted with the aim of investigating differences in the expression of hsps in the small intestine and their potential roles in ethanol - induced intestinal damage . in the current study , which was intended to examine the effects of hsps on the different regions of the small intestine ( i.e. , proximal , middle , and distal parts ) damaged by ethanol , a review of relevant literature led to the investigation of hsp70 , hsp90 , hsp40 , and hsp32 . the results of the current study demonstrated distinct differences in damage to the small intestines of icr mice depending on the concentration of ethanol and that damage was more pronounced in the distal region as compared to the rest of small intestines . when pbs was administered to controls , hsp40 and hsp90 were higher in the distal region while hsp70 was elevated regardless of the region examined . in the ethanol - treated mice , it was difficult to find any specific correlation or site - specific associations between ethanol exposure and expression of hsps , although differences in the expression levels of hsps were detected and dependent on the region examined . similar findings were observed for hsp32 , hsp40 and hsp90 , however , displayed a site - specific pattern similar to that of hsp70 at a dose of 5.5 g / kg only , with different expression patterns found in the 2.75 g / kg and 8.5 g / kg ethanol - treated groups . when comparing the site - specific expression patterns of hsps in this model , varying expression patterns and degrees of histological damage were found , in keeping with the known associations between the expression of hsps and degrees of histological damage . in the small intestine of icr mice ,
hsp70 generally showed the highest levels of expression and these levels were inversely correlated with the concentration of ethanol . in addition , the expression pattern of hsp32 was very similar to that of hsp70 , suggesting that both hsps may be critical in damage to the small intestine . however , it is difficult to elucidate the exact roles of these hsps based on the results of the current study . , tnf- , and il-1 ) and inflammatory mediator ( i.e. , cox-2 ) were not showing any noticeable change among different regions of the small intestine , as found in macroscopic observations . furthermore , no differences in the expression of gst , an anti - oxidative enzyme known to play an important role in protecting cells against oxidative stress , were detected between the control and ethanol groups , or among the regions of the small intestines in response to intestinal damage . however , it is difficult to exclude the possibility of an association between gst and intestinal damage , as the potential for differences in enzyme activity may be more significant than differences in protein expression levels . in the current study , varying magnitudes of histological damage and hsp expression in different regions of the small intestines were observed . this would seem unlikely , as hsp40 and hsp90 were exclusively increased in distal regions and hsp70 expression was elevated in a non - site - specific manner in the control group . in order to provide further clarification regarding the effects of ethanol exposure on hsp expression patterns in the small intestines
it may be possible for intestinal bacteria to impact patterns of hsp expression given the differences in distribution and classes of bacteria among regions of the small intestines . according to the results of previous studies ,
hsps were modulated in response to multiple stimuli on the intestinal epithelial cells and such modulations were contingent on the exposure to intestinal probiotic bacteria.12 therefore , the regional differences in hsp expression in the small intestine are likely attributable , at least in part , to the intestinal bacteria that differ in type and distribution throughout the small intestine . as opposed to the proximal region of the small intestine which contains few bacteria , more bacteria and their metabolites
are present in the distal region and may result in more pronounced effects on hsp expression . in a previous study that examined metronidazole administration in a small intestinal model ,
an intestinal bacterial flora demonstrated the ability to modulate physiological expression of the cellular protective hsps 25 and 72.13 although not examined in the current study , the administration of antibiotics may be useful in further elucidating associations between hsps and intestinal bacteria in a mouse model . first , the original goal of the study was to investigate differences among the regions of the small intestines in response to ethanol exposure . although oral administration is the route of exposure for ethanol in humans , it may result in exposure to higher concentrations of ethanol in the proximal region compared to the distal regions and thereby make it difficult to analyze effects on the respective regions in a quantitative manner . more extensive and accurate results may have been generated if the time course of the damaging effects of ethanol on the small intestines at the concentrations of ethanol high enough to make such changes ( i.e. , 5.5 g / kg or higher ) was monitored . however , the initial study objective was to determine damage to varying regions of the small intestines . to better understand the mechanisms underlying ethanol - induced damage , further studies regarding the time course of effects as well as the magnitudes of intestinal damage dependent on exposure time should be conducted . collectively , results from the current study demonstrated that the expression patterns of the hsps assessed varied in general and also in a region - specific manner . specifically , more pronounced histological damage was found in the distal region and hsp70 and hsp32 were decreased in particular when ethanol was administered intraperitoneally . the endotoxins produced by bacterial flora may have influenced the expression of hsps in ethanol - induced damage to the small intestine and future studies should address this possibility . | [
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] |
bipolar disorder ( bd ) is associated with poor health outcomes and is responsible for the highest rate of suicide among all mental disorders .
cannabis is the most common illicit substance used among individuals with bipolar disorder and up to 38% of the individuals with bipolar disorder misuse it .
cannabis abuse has particularly been reported to be high among young bipolar patients , and chronic cannabis use is associated with higher severity of illness and greater noncompliance to treatment among individuals with bipolar disorder . in bipolar disorder , there is evidence that , with many patients , substance abuse precedes the onset of bd , and it has been suggested that affective deregulation may increase the risk of bipolar disorder due to substance abuse . the relationship between substance abuse and age at onset of bipolar disorder is not really well understood . among individuals with cooccurring cud , age at onset of bipolar disorder
was 6 years lower and the mean number of manic , hypomanic , and depressive episodes per year was greater compared to individuals without cud .
lev - ran et al . also reported that cannabis use may decrease the age at onset in both schizophrenia and bd .
this is consistent with the view that cannabis use may unmask a preexisting genetic liability that is partly shared between patients with schizophrenia and bipolar disorder .
. found a significant association indicating a dose - response relationship between cannabis use and age at onset , which remained statistically significant after controlling for possible confounders ( gender , bipolar subtype , family history of severe mental illness , and alcohol or other substance use disorders ) .
the mean difference in age at onset between the groups with and without cannabis use disorder in that study was 5 years .
it is important to review the definitions of age at onset , since these definitions are controversial in the psychiatric literature .
patients displaying bipolar disorder at a younger age at onset have higher prevalence rates of psychotic symptoms , substance abuse comorbidity , learning disabilities , and episodes of rapid cycling of mood [ 10 , 11 ] .
moreover , there is an increased risk factor for suicide attempts . among common mental disorders ,
. the risk of suicide in bipolar patients is 2030 times higher than that of the general population .
the risk is greater among those who have been admitted to inpatient care due to bipolar disorder and especially high in bipolar patients admitted to inpatient care after suicide attempts .
the main aims of this study are to review ( 1 ) the definition of the age at onset of bd , ( 2 ) the association of cannabis use as a risk factor for early onset of bd , and ( 3 ) the relationship of cannabis use and suicidal behavior in bd .
between june and september 2014 , pubmed database was searched for articles using combinations of the following key words : bipolar disorder , suicide , suicide attempts , cannabis , marijuana , early age at onset , and early onset .
duplicates and repetitive reviews were excluded . in the cases of similar studies performed by the same group , the study with bigger sample size was included in this review .
function of the pubmed database , the reference list of selected articles , conference abstracts , and google scholar were also used to identify additional articles .
the entry criteria of the articles included in this review should meet at least one of the following requirements : ( 1 ) the selected study should provide data that allowed evaluating the age at onset of bipolar disorder ; ( 2 ) the paper should present the proportion of bipolar patients who used cannabis ; ( 3 ) patients included in these studies should have had an early age at onset of bd or suicide attempts .
initially , for the keywords early onset , bipolar disorder , 1,017 articles were found ; for cannabis and bipolar disorder , 143 articles ; for suicide attempts and bipolar disorder , 1,069 ; for suicide and bipolar disorder , 2,289 ; for marijuana and bipolar disorder , 190 ; and for early age at onset and bipolar disorder , 626 .
, 77 papers ( all of them published ) fulfilled the entry criteria and were included , as shown in the table 1 .
they were published between 1994 and 2014 , 40 of them ( 51% ) in the last 5 years .
the age at which the first bipolar episode occurs is relevant because an early age is associated with poor prognosis .
if an early diagnosis of bd is not established , the treatment starts late , compromising its good outcome .
subgroups defined as early onset and very early onset were associated with greater rates of comorbid anxiety disorders and substance abuse , violent behavior , rapid cycling , and shorter periods of euthymia compared to the late onset subgroup .
the dsm - iv suggests the age of 21 , supported by genetic studies , as the maximum age to be included as the early age at onset of bd .
an epidemiological study with 61,392 community adults in 11 countries found that the mean age at onset of bd type i was 18.4 ( 0.7 ) years , bd type ii was 20.0 ( 0.6 ) years , and subthreshold bd was 21.9 ( 0.4 ) years .
other studies described different definitions of early age at onset , being < 21 years the most common age defined as early onset , as shown in table 2 .
table 2 shows chronological studies with different definitions of early and late ages at onset .
the following variables are described : year of publication ; sample size ; study design ; and ages at very early , early , intermediate , and late onset .
the definition of early age ranged 17.421 , while late age ranged 1840 . in a study comparing a sample of early ( n = 58 ) and late onset ( n = 39 ) bipolar patients , the early onset group had a more severe form of the disorder with more psychotic features , mixed episodes , comorbid panic disorder , and poorer response to lithium .
the early onset group was defined as younger than 18 and late onset as older than 40 .
another paper comprised 52 ( 50 bd type i , 2 bd type ii ) patients who had an early onset and 38 ( 30 bd type i , 8 bd type ii ) a late onset .
it was observed that the early onset group is characterized by a higher frequency of psychotic symptoms as compared to patients with late onset .
a sample of 320 individuals diagnosed with bd i or ii was stratified into subjects with early age ( 18 years ) and late ( > 18 years ) age at onset of bd .
a significant earlier age at onset in subjects with anxiety disorders and rapid cycling course was found . when clinical characteristics between earlier and later onset of bd were compared , subjects with early aao had more frequent suicidal ideation / attempts , axis i comorbidity , substance use disorders , and rapid cycling course .
the odds ratios associated with these variables were 1.4 ( suicide ideation ) , 1.6 ( axis i comorbidity ) , 1.4 ( substance abuse ) , and 2.0 ( rapid cycling course ) .
a study with 368 patients investigated the cut - off in the age at onset in three subgroups ( early , intermediate , and late ) .
the mean age in each group was estimated to be 17.4 ( sd = 2.3 ) , 25.1 ( sd = 6.2 ) , and 40.4 years ( sd = 11.3 )
. a sample of 1,000 adults with bipolar disorder was divided into three groups : very early ( < 13 years ) , early ( 1318 years ) , and late ( > 18 years ) onset of mood symptoms and it resulted in 983 patients whose age at onset could be determined : 272 ( 27.7% ) had very early onset and 370 ( 37.6% ) experienced early onset .
early onset again was associated with higher rates of comorbid anxiety disorders and substance abuse , more recurrences , shorter periods of euthymia , greater likelihood of suicide attempts , and violence , suggesting a more severe course of disease in terms of chronicity and comorbidity .
an article reported the age at onset of 211 families with bd type i probands .
they divided the analysis into two variables : early - onset subgroup as 21 years and late onset
clinical features , such as comorbid substance abuse , rapid cycling , suicidality , and increased episode frequency , were correlated with the early onset group .
history of high depressive recurrence ( without history of mania / hypomania ) has been proposed as a mood subtype close to bipolar disorders . a study with 224 outpatients diagnosed with major depressive disorder and with 336 outpatients with bd type ii
was conducted on such putative bipolar validators ( early age at onset , high recurrence , mixed depression , and bipolar family history ) as early age at onset of first major depressive episode ( before 21 years ) .
early onset was the only variable which identified a major depressive disorder subgroup significantly associated with all bipolar validators .
this major depressive disorder subgroup was similar to bd type ii in age at onset and bipolar family history and had a high frequency of mixed depression .
a sample of 1,369 bd type i patients was divided into three subgroups : early onset as < 22 years ; intermediate onset as 2537 years ; and late onset as > 40 years .
the early onset subgroup had more rapid cycling , family history for affective disorders , and episodes of depression and mania when compared to other subgroups .
a study with two independent samples from france ( n = 480 ) and from the united states ( n = 714 ) was carried out with bd type i patients .
a correlation of early age at onset , substance use , and suicidal behavior was found .
there is no consensus about the criteria of frequency or quantity of cannabis use in bd to establish the definition of abuse , as shown in table 3 .
criteria range from the number of joints per day in the last 12 months to the qualification of heavy consumers
. the frequency of use was adopted as a parameter in two studies , but with different approaches . while henquet et al . stratified the cannabis use into daily , weekly , and monthly frequencies , tijssen et al .
classified , in their sample , all patients who had made the use of cannabis five times or more in their lifetime .
table 3 shows the criteria adopted in several studies to establish the frequency of cannabis use .
the following variables are described : year of publication , study design , follow - up period , measures , comparisons , and frequency of cannabis use .
sample sizes ranged from 151 to 4,815 . a study with a sample of 4,815 individuals between 18 and 64 years old was examined , using the composite international diagnostic interview , and it was found that cannabis use can trigger manic symptoms independently of age , ethnicity , neuroticism , sex , educational level , marital status , use of other drugs or alcohol .
patients who used it 3 - 4 days per week were more likely to manifest manic symptoms than those who used it less frequently .
a study in a prospective cohort of 705 adolescents followed up during 8 years analyzed the association between risk factors , such as cannabis use , and the manifestation of manic or depressive symptoms in bipolar patients and found that cannabis use was involved with manic manifestation .
a sample with 766 patients between 16 and 65 years old , 676 of them diagnosed with schizophrenia and 90 with bd , was interviewed and cannabis use was linked to early age at onset of schizophrenia and bd .
another study investigated 151 patients with bd types i and ii who were in psychiatric treatment and it was observed that excessive cannabis use was related to early onset of bd and that it had been used at an earlier age than the excessive use of alcohol .
data from the national epidemiological survey of alcohol and related conditions ( nesarc wave 1 , 2001 - 2002 ) were analyzed and they showed that 1,905 patients were diagnosed with bipolar disorder and 7.2% of these were identified with cannabis use disorder ( cud ) .
it was observed that the cooccurrence of bd and cud was a risk factor for alcohol , nicotine , and other drug dependence . in a study involving approximately 2,000 individuals ( 471 bipolar individuals and 1,761 controls ) ,
almost 30% of the bd group that had made use of cannabis at least once fulfilled the criteria for cud of dsm - iv .
individuals with bd and cud were 1.8 times more likely to have disability due to the disorder than those with the diagnosis of bd that had no history of cud , even after controlling sociodemographic variables , substance use , and psychiatric covariates .
individuals with bd and cud had more mixed episodes and a higher probability of suicide attempts than those with bd but without cud . in a study selecting adult patients from france and the united states that met dsm
iv criteria for bd type i , the age at onset was classified into two subgroups : early age at onset ( < 21 years ) and late age at onset ( 21 years ) .
they analyzed the association of clinical and demographic variables with age at onset and polarity .
the relationships between age at onset , alcohol / drug misuse , and suicidal behavior are the most important in both samples : early age at onset was associated with suicidal behavior ( france : or = 2.16 95% ci ( 1.483.15 ) sensibility = 35% , specificity = 46% ; usa : or = 2.05 95% ci ( 1.442.92 ) sensibility = 27% , specificity = 54% ) and lifetime cannabis misuse ( france : or = 2.60 95% ci ( 1.514.48 ) sensibility = 9% , specificity = 79% ; usa : or = 1.75 95% ci ( 1.023.01 ) sensibility = 30% , specificity = 59% ) .
a cross - sectional study was conducted on a population - based national representative sample , the national epidemiological survey of alcohol and related conditions ( nesarc ) .
individuals with lifetime prevalence of bd ( n = 1 , 905 ) were analyzed regarding sociodemographic characteristics and prevalence of comorbid psychiatric disorder among bd patients with and without cud ( in the last 12 months ) .
they found among bd patients with cud ( n = 119 ) an earlier age at onset of the first manic or hypomanic episode ( mean age = 19.5 years ) and of the first depressive episode ( mean age = 18.5 years ) compared to patients without cud ( n = 1 , 786 ) , whose mean ages were 25.1 years and 24.4 years , respectively ( p < 0.0001 ) . in this study ,
cud was associated with earlier onset of bipolar disorder and greater number of depressive and hypomanic / manic episodes . a secondary analysis of data collected by the netherlands mental health survey and incidence study ( nemesis)a longitudinal study with three measurements at 1996 , 1997 , and 1999 ( follow - up)of the dutch adult population ( 1864 years ) was conducted .
at baseline , 7,076 individuals were interviewed , and , in the last follow - up , 4,848 participants were interviewed . to investigate
if cannabis use predicted the first episodes of mood and anxiety disorders , the analyses were made with 3,881 people who had had no lifetime mood disorder and with 3,854 people who had had no lifetime anxiety disorder .
it was found that cannabis was a predictor of subsequent mood episodes , especially bipolar disorder , and that cannabis use was associated with increasing the risk of bd ( or = 7.6 ; p < 0.001 ) .
an earlier onset was found in patients that used cannabis ( 676 schizophrenia patients and 90 bd patients ) .
the reduction was 9 years in the age at onset in the bipolar group . in a sample of 151 bd types i and ii
, cannabis use was associated with an earlier onset of bd independently of the history of psychosis or polarity of the first episode , not only manic or psychotic , as described by henquet et al . and ngr et al . .
( 2009 ) reported that comorbid lifetime cannabis , but not alcohol , abuse / dependence was associated with a statistically significant 3-year - earlier age at onset of psychosis in schizophrenia ( n = 80 ) , schizoaffective disorder ( n = 61 ) , and bipolar disorder with psychotic features ( n = 92 ) .
patients fulfilled the criteria for cannabis abuse / dependence an additional 3 years before psychosis . in a study with 324 bd types i and ii patients
, an independent dose - response was shown between cannabis use and age at onset of bd : there was a statistically significant decrease in age at onset with increasing levels of lifetime cannabis use , from 23.2 years ( 9.7 ) for patients who never used cannabis or used cannabis < 10 times during one month lifetime , 20.5 years ( 7.3 ) for patients who used cannabis > 10 times during one month lifetime , and 18.6 years ( 5.0 ) for patients with a lifetime cannabis use disorder ( abuse or dependence ) . according to the world health organization ( who ) , about 3,000 people commit suicide every day worldwide one every 40 seconds . for each suicide ,
the annual number of suicides is currently around one million and represents about half of all violent deaths recorded in the world . among psychiatric disorders , bd has the highest risk of suicide , reaching rates 2030 times higher than in the general population .
approximately 56% of patients with bd who committed suicide had attempted suicide at least once in their life ; and 1 in 15 bipolar patients are victims of suicide .
the rate that expresses suicide attempts at least once in their life in bipolar patients varies between 25% and 50% ; however , 8% to 19% will commit suicide .
the number of suicide attempts in bipolar types i and ii is a subject of controversy .
another study shows not statistically significant rates in suicide risk among bipolar patients either with type i or type ii in a follow - up of bipolar patients up to 18 months and in a study in barcelona that analyzes 290 bipolar patients .
more suicide attempts are committed by bipolar females than bipolar males ( 3419% ) . however ,
bd associated with substance use in males doubles the number of suicidal behaviors . the early age at onset of bd and gender , prior suicide attempts , suicide in different mood episodes of bipolar disorder ,
episode polarity and polarity of first affective episode , and drug and alcohol abuse disorders show a significantly increased risk of suicide attempts .
the early onset of this disorder increases the risk of suicide and that early onset is around age 20 [ 42 , 43 ] .
based on a cohort of 6,086 ( 60% females ) patients followed up annually 20052012 , it was found that early onset is a significant predictor of suicide attempts only in females .
early age at onset was also associated with suicide attempts in samples from france and the united states .
another study with a population - based longitudinal cohort sample included 1,542 patients with bipolar depression attending a registry of 388,624 inhabitants of keelung city , taiwan , from 1999 to 2004 .
this cohort was followed until the end of 2008 and data were from the national health insurance dataset ( nhid ) and the national mortality registry ( nmr ) .
the risk of suicide in these patients was doubled in comparison with other depressive patients .
the following variables are described : year of publication ; number of individuals ; suicide attempts ; suicide attempts and substance use ; suicide attempts ; and cannabis use .
the following percentages in bipolar patients were found : suicide attempts 3.642% ; suicide attempts and substance use ranging from 5 to 60% ; and suicide attempts and cannabis use ranging from 15 to 42% .
it is important to notice that higher percentages of suicide attempts were found in the studies with smaller number of participants , which is statistically understandable , but the real number of patients that attempted suicide was significant in all studies taking in credit that all suicide attempts can potentially lead to death .
a sample of 1,556 bipolar patients was examined along 2 years of follow - up .
they were participants in the systematic treatment enhancement program for bipolar disorder ( step - bd ) , a multicenter study which evaluates longitudinal outcomes in patients with bd .
this study analyzed the association between baseline clinical and demographic variables and subsequent suicide attempts and completions .
suicidal ideation , percent of anxiety , depressed and irritable days in past year , history of suicide attempts , age at onset , marital and smoking status , age , and gender were considered for comparison .
the sample with complete baseline data was categorized by whether or not participants experienced a suicide attempt or completion over the 2-year follow - up period . a rate of 3.66% ( 57 patients ) attempted or completed suicide . after analysis ,
only history of suicide ( or = 4.52 ) and percent of depressed days in the past year ( or = 1.16 ) were significant .
the age at onset of bd ( < 13 years ) presented or = 1.37 .
a prospective study was conducted in the jorvi bipolar study ( jobs ) screening 1,630 patients , of whom 546 obtained a positive mdq screen or suspicion of bd .
490 patients of this sample could be interviewed ; however , 201 had the diagnosis of bd confirmed by the structured clinical interview for dsm - iv disorders ( scid - i ) and 10 patients refused to participate .
this research aimed to investigate the risk for suicide attempts in psychiatric inpatients and outpatients with bd , and it found that during the 18-month follow - up , 20% of patients ( 35/176 ) attempted suicide . in a cox regression model ,
baseline previous suicide attempts , hopelessness , depressive phase at index episode , and younger age at intake were independent risk factors for suicide attempts during follow - up , whereas factors such as bipolar i or ii or comorbidity did not reach statistical significance .
recent affective episodes predicted attempted suicide during follow - up in male ( or = 3.63 ) and in female ( or = 2.81 ) patients as well as previous suicide attempts ( male : or = 3.93 ; female : or = 4.24 ) and recent psychiatric inpatient care ( male : or = 3.57 ; female : or = 2.68 ) .
comorbid substance use disorder was a predictor in male , while many lifetime mixed episodes , early onset of mental disorder , personality disorder , and social problems related to the primary group were predictors in women .
a study was conducted with a group of 1,369 bipolar patients , divided into three subgroups : the first was represented by patients who had had an early age at onset , the second an intermediate age at onset of bd , and the third a late onset , producing a sample of 1,225 individuals ( 144 with borderline values were excluded ) .
results showed that 44.3% of the first group attempted suicide while in the second and third groups , respectively , the rate was 33.7 and 28.7% .
the early onset group in comparison with the others had a greater frequency of suicide attempts . in a prospective study with a sample of 2,219 bipolar patients who provided data about their lifetime history , 663 ( 29.9% )
factors that were associated with a history of suicide attempts included the following variables : female gender , a history of alcohol abuse , a history of substance abuse , early onset , longer disorder duration , greater depressive symptom severity , current benzodiazepine use , higher overall symptom severity , and poor compliance .
of the 663 patients with suicide attempts , 17.3% of them had lifetime cannabis abuse . of the sample without suicidal behavior ( 1,556 ) ,
10.7% had lifetime cannabis abuse . a study conducted with a bipolar cohort of 87 males and 70 females aimed to detect factors that may be predictive for suicide attempt . among 157 patients , 59 of them had a history of at least one suicide attempt .
white race , family history of completed suicide , and history of cocaine abuse / dependence were predictive of suicide attempt histories .
clinical and dimensional characteristics in bipolar patients were analyzed through diagnostic interview and questionnaires . in a sample of 652 patients , 280 ( 42.9% )
some variables were associated with a lifetime history of suicidal behavior like being a woman , a history of head injury , tobacco misuse , early age at onset , high number of depressive episodes , positive history of rapid cycling , alcohol misuse , and social phobia .
data was analyzed comparing two different groups : bipolar patients with and without suicidal behavior ( bd + sb and bd sb ) and an earlier age at onset was associated with suicidal behavior ( 23.1 9.1 in bp + sb versus 27.0 11.1 in bd sd ) .
cannabis misuse was indicated in 41 ( 15.1% ) of 280 ( 42.9% ) bipolar patients that made a suicide attempt . in a cohort of 3,083 bipolar patients , 140 ( 4.6% )
the strongest predictor of a suicide event was a history of suicide attempt , in line with prior literature .
additional predictors were younger age , a high total score on the personality disorder questionnaire , and a high percentage of days spent depressed in the year prior to study entry . a meta - analysis was conducted in order to relate comorbidities and suicide attempts in patients with bd .
twenty - nine of 222 studies assessed for eligibility met the inclusion criteria , comprising a total of 31,294 individuals with bd , of whom 6,308 ( 20.1% ) had documented suicide attempts .
as compared to controls , subjects with bd and comorbid alcohol and other substance use disorder were more likely to attempt suicide .
the association between suicide and substance abuse in bipolar patients can be demonstrated by substantial evidence of suicidal behavior in patients with comorbidities for substance use disorders .
this relationship can be sustained on the basis of data stating that cannabis use affects negatively the course of bipolar disorder .
the effects of substance use on suicidal behavior were registered by a large epidemiological study in 1,643 bipolar patients among 43,093 general - population respondents who were interviewed in the 2001 - 2002 by the national epidemiologic survey on alcohol and related conditions ( nesarc ) .
the use of cannabis reduces the age at onset of bd ; it may also increase manic episodes and their durations and may increase the risk of suicide in patients with bipolar depression . a study involving 101 patients concluded that the first exposure to cannabis represented strong evidence of an earlier age at onset of first depressive episode .
cannabis use in patients with bipolar disorder increases the incidence of psychotic symptoms and suicide attempts and still decreases the response to treatment by lithium compared to patients who do not use this substance .
the abusive use of cannabis may act as a predictor of early onset of bipolar disorder and both conditions can interfere with increasing the risk of suicide in patients with bd .
greater suicidal behavior in bipolar patients who experienced a reduction in the age at onset in the context of cannabis use was also demonstrated .
a group of 125 bipolar patients with psychotic features was analyzed and it was demonstrated that bipolar disorder occurred earlier in a subgroup that had substance abuse associated with other comorbidities from axis i .
a meta - analysis of 29 studies evaluated suicide attempts and substance use ( including cannabis ) specifically in bd population .
it comprised 31,294 individuals with bd of which 6,308 ( 20.1% ) had suicide attempts .
cannabis use disorders were evaluated in 4 studies comprising 3,439 individuals with specific information on bd , suicide attempts , and cud ( 559 with bd and lifetime cannabis use disorders ) .
individuals misusing cannabis showed an increased risk of suicide ( or = 1.44 ) when compared with their noncomorbid counterparts .
the definition of early age at onset of bd is not clear ; however , it is associated with poor prognosis of bd .
if an early diagnosis of bd is not established , the treatment starts late , compromising a good outcome .
nine studies between 2000 and 2012 were evaluated and a wide variation in the meaning of early age at onset was found ( 622 years ) , as shown in table 2 .
the definition of the age at onset of bipolar disorder is important because there is robust evidence suggesting that the age at onset distribution of individuals affected with bipolar disorder is composed of at least two age distributions ( early and late onset ) [ 10 , 17 , 18 , 22 , 43 ] , three divided into early , intermediate , and late [ 19 , 21 , 23 ] and only one into very early , early , and late onset .
the three normal distributions ( early , intermediate , and late onset ) may reflect genetic heterogeneity within bipolar i disorder .
results may vary in the literature relating to age at onset because different criteria were used to establish the definition of age at onset .
hamshere et al . incorporated the duration of the disorder at the time of the interview and gender as covariates , while benazzi and akiskal used age at assessment - time , gender , bipolar type ii , and mania / hypomania family history .
perlis et al . also included the duration of the disorder and finally lin et al
the definition of very early age at onset is even more troublesome because the first symptoms of mood variation that could be diagnosed as the first bipolar episode are difficult to diagnose by mini and other instruments .
the relationship between early age at onset and the severity of bipolar disorder is well established .
early age at onset is associated with a greater number of rapid cycling [ 18 , 21 , 23 ] , mixed episodes , and psychotic episodes [ 10 , 17 ] and panic disorder , anxiety disorder [ 18 , 20 ] , substance use disorder [ 3 , 18 , 20 , 21 ] , and major depression , even worse response to lithium and suicidal behavior [ 3 , 18 , 20 , 21 ] .
cannabis is the most common illicit substance used among individuals with bd ( 7.2% in bd versus 1.2% in general population ) .
the use of marijuana seems to be a predictor of mood episodes in bd patients and it reduces the age at onset in 69 years [ 8 , 9 ] .
cannabis use disorder triggers earlier episodes of bd when compared with alcohol use disorder . especially in cases of cannabis abuse , the relationship between abuse and age at onset is not well understood .
it happens because substance use disorder can be a prodromal symptom of mood disorder and can be both a consequence and a cause of bd .
the interaction of childhood stressor events and cannabis use appears to be the potential factor for a vicious cycle [ 51 , 52 ] .
another interesting point was that patients with lifetime cannabis and alcohol abuse / dependence had a later onset compared with those who had only cannabis abuse / dependence .
there is an association between early age at onset of bd and suicide attempts . among psychiatric disorders , bd has the highest risk of suicide .
early age at onset is associated with suicide attempts and is considered a predictor , especially in females .
it is still necessary to elucidate whether early age at onset in bd is an independent risk factor for suicide attempts or whether it depends on other factors , such as disorder severity , rapid cycling , more psychiatric comorbidity , abuse in childhood , family history of mood disorder , differences of gender , and bd subtypes [ 53 , 54 ] .
but when gender was compared , females showed higher rate of suicide attempts , while males presented higher rates of completed suicide .
bd type ii was associated with higher rates of suicide attempts , while other studies have shown no significant differences between bd types i and ii .
the findings of an association between cannabis use , early age at onset , and suicide attempts may be taken as a support of the view that cannabis negatively affects the course of the disorder .
recent research confirms the importance of the genetics of the bipolar disorder , although the involvement of no specific chromosome region or gene has been specifically confirmed [ 5558 ] .
the pathophysiology of this interaction is not completely elucidated and thus points to the importance of researching common biomarkers of both disorders for a better understanding of the course of the disorder .
it may be hypothesized that cannabis use and bd may share a common genetic background .
the neuropathological bases of comorbidity between bipolar disorder and cannabis use disorder are a challenge because there are few studies addressing the pathophysiology of these two disorders .
areas of medial temporal cortex and prefrontal and subcortical regions with structural and functional abnormalities are associated with emotional and motivational processing in adolescents with bd and substance use disorder .
thus , neurophysiological dysfunctions may overlap in some individuals predisposed to the development of both disorders : bd and substance use disorder .
only one study addressed the relationship between bipolar patients and the expression of cb1-r and did not find a direct relation to neither increase nor decrease the density in bipolar patients .
however , it was observed that bipolar patients who were taking first - generation antipsychotics had their levels of cb1-r immunoreactive glial cells reduced .
type 1 cannabinoid receptor , cb1-r , is a g protein - coupled receptor and located mainly in the central and peripheral nervous system .
this is due to influence on the mesolimbic pathway , especially in the nucleus accumbens region .
another research found that polymorphism of akt1 gene may be related to the use of cannabis and to the developing of psychosis , but not specifically for bipolar patients .
bipolar adolescents with cooccurring cannabis use disorder had structural differences in frontal and temporal cortical regions and the right caudate nucleus , which is extended and is related to emotional and motivational regulation .
in addition , patients who use cannabis and tobacco had lower activation of the right hippocampus when compared with controls . in patients with cooccurrence of bd and cannabis use disorder
an increase in gray matter volume in the right caudate and precentral gyrus and increased gray matter density in the occipital , middle right fusiform , and cerebellar vermis were observed , while there was a reduction in gray matter volume in the left fusiform .
abnormalities in the subcortical region have been linked to dysfunction in the reward system in patients with substance use disorder .
the activity of the caudate is related to desire and the fusiform gyrus , which is altered in bipolar patients with cannabis use disorder , is associated with craving and drug seeking behavior .
the results of studies attempting to correlate structural and functional changes in the brains of patients with bd and cannabis use disorder found some limitations because they were not able to differentiate preexisting structural brain abnormalities from the consequences of repeated exposure to abused substances .
furthermore , a preliminary study with a small sample of patients ( n = 10 ) found no significant changes in the brain of patients who were frequently using cannabis .
several studies have demonstrated that bd is more prevalent in individuals who have experienced early - life events stressors .
neuroendocrine , autonomic , immune , and oxidative responses are triggered , once a state of chronic inflammation is elicited , altering cellular mediators of plasticity and energy metabolism , besides the deleterious programing of the hypothalamic - pituitary - adrenal ( hpa ) axis .
van leeuwen et al . proposed opposite effects on the hypothalamic - pituitary - adrenal ( hpa ) axis when childhood abuse and cannabis use cooccur .
that is why cannabis seems to decrease the programming of hpa while childhood trauma is linked as a stressor to hpa , triggering a factor for long - term hyperactivation of hpa .
the catechol - o - methyltransferase has been studied because it is involved in the metabolism of catecholamines ( dopamine , adrenaline , and noradrenaline ) .
massat et al . observed the influence of catechol - o - methyltransferase variants on major depression and bd , particularly in early onset subjects .
experimental studies support a theory which shows an interaction between cannabis and catechol - o - methyltransferase as a mechanism for cognitive abnormalities .
the polymorphism of the gene responsible for enzyme sat1 is related to dysfunction in the catabolism of polyamines .
bipolar patients who had been hospitalized with or without suicidal ideation had a reduced amount of sat1 when compared with those who had not been hospitalized .
there is sparse literature on the issue involving cannabis use , early age at onset of bd , and suicide attempts .
the definition of early age at onset is not consensual ; therefore studies may not be entirely comparable .
some studies report data on the bipolar group as a whole , not specifying the relationship of cannabis use , early age at onset , and suicide attempts .
other studies report data involving the early age at onset in bipolar and schizophrenia making it harder to distinguish to which group of patients the study is referring .
a definition of what constitutes a prodromal phase or an index episode in many studies is lacking .
information on whether there is only cannabis use / abuse or other psychiatric comorbidities in terms of drug use / abuse is missing in many studies . the definition of cannabis use / abuse is subject of controversy .
finally , some studies published data only on bd type i and others on bd type ii and both ( bd i and ii ) and , in some of them , it is not specified whether the sample refers to bd and schizophrenia [ 7275 ] . however , in this review , as in other studies , the relationship between abuse / dependence and age at onset differentiated on the basis of psychiatric diagnosis or gender aspects has not been investigated , although some of these associations are potentially plausible .
in conclusion , the age at onset of bipolar disorder was reviewed due to its prognostic value , making it clear that the first episode should have an early diagnosis .
the evidence of cannabis use as a risk factor to early onset of bipolar disorder highlights the necessity to be attentive to the first bipolar episode so that treatment can be started promptly .
the use of cannabis is an important factor that may trigger early onset of bd and , by itself , is associated with higher rates of suicidal behavior in bd .
however , it is not clear whether the effect of cannabis at the age at onset and suicide attempts are independent of each other or not . |
introduction . bipolar disorder ( bd ) implies risk of suicide .
the age at onset ( aao ) of bd carries prognostic significance .
substance abuse may precede the onset of bd and cannabis is the most common illicit drug used .
the main goal of this study is to review the association of cannabis use as a risk factor for early onset of bd and for suicide attempts .
materials and methods .
pubmed database was searched for articles using key words bipolar disorder , suicide attempts ,
cannabis , marijuana , early age at onset , and early onset .
results .
the following percentages in bipolar patients were found : suicide attempts 3.642% ; suicide attempts and substance use 560% ; suicide attempts and cannabis use 1542% .
an early aao was associated with cannabis misuse .
the mean age of the first manic episode in individuals with and without bd and cannabis use disorder ( cud ) was 19.5 and 25.1 years , respectively .
the first depressive episode was at 18.5 and 24.4 years , respectively .
individuals misusing cannabis showed increased risk of suicide .
discussion .
cannabis use is associated with increased risk of suicide attempts and with early aao .
however , the effect of cannabis at the aao and suicide attempts is not clear . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion | bipolar disorder ( bd ) is associated with poor health outcomes and is responsible for the highest rate of suicide among all mental disorders . cannabis is the most common illicit substance used among individuals with bipolar disorder and up to 38% of the individuals with bipolar disorder misuse it . cannabis abuse has particularly been reported to be high among young bipolar patients , and chronic cannabis use is associated with higher severity of illness and greater noncompliance to treatment among individuals with bipolar disorder . in bipolar disorder , there is evidence that , with many patients , substance abuse precedes the onset of bd , and it has been suggested that affective deregulation may increase the risk of bipolar disorder due to substance abuse . the relationship between substance abuse and age at onset of bipolar disorder is not really well understood . among individuals with cooccurring cud , age at onset of bipolar disorder
was 6 years lower and the mean number of manic , hypomanic , and depressive episodes per year was greater compared to individuals without cud . found a significant association indicating a dose - response relationship between cannabis use and age at onset , which remained statistically significant after controlling for possible confounders ( gender , bipolar subtype , family history of severe mental illness , and alcohol or other substance use disorders ) . the mean difference in age at onset between the groups with and without cannabis use disorder in that study was 5 years . it is important to review the definitions of age at onset , since these definitions are controversial in the psychiatric literature . patients displaying bipolar disorder at a younger age at onset have higher prevalence rates of psychotic symptoms , substance abuse comorbidity , learning disabilities , and episodes of rapid cycling of mood [ 10 , 11 ] . moreover , there is an increased risk factor for suicide attempts . the risk of suicide in bipolar patients is 2030 times higher than that of the general population . the main aims of this study are to review ( 1 ) the definition of the age at onset of bd , ( 2 ) the association of cannabis use as a risk factor for early onset of bd , and ( 3 ) the relationship of cannabis use and suicidal behavior in bd . between june and september 2014 , pubmed database was searched for articles using combinations of the following key words : bipolar disorder , suicide , suicide attempts , cannabis , marijuana , early age at onset , and early onset . the entry criteria of the articles included in this review should meet at least one of the following requirements : ( 1 ) the selected study should provide data that allowed evaluating the age at onset of bipolar disorder ; ( 2 ) the paper should present the proportion of bipolar patients who used cannabis ; ( 3 ) patients included in these studies should have had an early age at onset of bd or suicide attempts . initially , for the keywords early onset , bipolar disorder , 1,017 articles were found ; for cannabis and bipolar disorder , 143 articles ; for suicide attempts and bipolar disorder , 1,069 ; for suicide and bipolar disorder , 2,289 ; for marijuana and bipolar disorder , 190 ; and for early age at onset and bipolar disorder , 626 . the age at which the first bipolar episode occurs is relevant because an early age is associated with poor prognosis . subgroups defined as early onset and very early onset were associated with greater rates of comorbid anxiety disorders and substance abuse , violent behavior , rapid cycling , and shorter periods of euthymia compared to the late onset subgroup . the dsm - iv suggests the age of 21 , supported by genetic studies , as the maximum age to be included as the early age at onset of bd . an epidemiological study with 61,392 community adults in 11 countries found that the mean age at onset of bd type i was 18.4 ( 0.7 ) years , bd type ii was 20.0 ( 0.6 ) years , and subthreshold bd was 21.9 ( 0.4 ) years . other studies described different definitions of early age at onset , being < 21 years the most common age defined as early onset , as shown in table 2 . in a study comparing a sample of early ( n = 58 ) and late onset ( n = 39 ) bipolar patients , the early onset group had a more severe form of the disorder with more psychotic features , mixed episodes , comorbid panic disorder , and poorer response to lithium . a sample of 320 individuals diagnosed with bd i or ii was stratified into subjects with early age ( 18 years ) and late ( > 18 years ) age at onset of bd . when clinical characteristics between earlier and later onset of bd were compared , subjects with early aao had more frequent suicidal ideation / attempts , axis i comorbidity , substance use disorders , and rapid cycling course . the mean age in each group was estimated to be 17.4 ( sd = 2.3 ) , 25.1 ( sd = 6.2 ) , and 40.4 years ( sd = 11.3 )
. a sample of 1,000 adults with bipolar disorder was divided into three groups : very early ( < 13 years ) , early ( 1318 years ) , and late ( > 18 years ) onset of mood symptoms and it resulted in 983 patients whose age at onset could be determined : 272 ( 27.7% ) had very early onset and 370 ( 37.6% ) experienced early onset . early onset again was associated with higher rates of comorbid anxiety disorders and substance abuse , more recurrences , shorter periods of euthymia , greater likelihood of suicide attempts , and violence , suggesting a more severe course of disease in terms of chronicity and comorbidity . a study with 224 outpatients diagnosed with major depressive disorder and with 336 outpatients with bd type ii
was conducted on such putative bipolar validators ( early age at onset , high recurrence , mixed depression , and bipolar family history ) as early age at onset of first major depressive episode ( before 21 years ) . a correlation of early age at onset , substance use , and suicidal behavior was found . a study in a prospective cohort of 705 adolescents followed up during 8 years analyzed the association between risk factors , such as cannabis use , and the manifestation of manic or depressive symptoms in bipolar patients and found that cannabis use was involved with manic manifestation . a sample with 766 patients between 16 and 65 years old , 676 of them diagnosed with schizophrenia and 90 with bd , was interviewed and cannabis use was linked to early age at onset of schizophrenia and bd . another study investigated 151 patients with bd types i and ii who were in psychiatric treatment and it was observed that excessive cannabis use was related to early onset of bd and that it had been used at an earlier age than the excessive use of alcohol . data from the national epidemiological survey of alcohol and related conditions ( nesarc wave 1 , 2001 - 2002 ) were analyzed and they showed that 1,905 patients were diagnosed with bipolar disorder and 7.2% of these were identified with cannabis use disorder ( cud ) . it was observed that the cooccurrence of bd and cud was a risk factor for alcohol , nicotine , and other drug dependence . individuals with bd and cud were 1.8 times more likely to have disability due to the disorder than those with the diagnosis of bd that had no history of cud , even after controlling sociodemographic variables , substance use , and psychiatric covariates . individuals with bd and cud had more mixed episodes and a higher probability of suicide attempts than those with bd but without cud . in a study selecting adult patients from france and the united states that met dsm
iv criteria for bd type i , the age at onset was classified into two subgroups : early age at onset ( < 21 years ) and late age at onset ( 21 years ) . they analyzed the association of clinical and demographic variables with age at onset and polarity . the relationships between age at onset , alcohol / drug misuse , and suicidal behavior are the most important in both samples : early age at onset was associated with suicidal behavior ( france : or = 2.16 95% ci ( 1.483.15 ) sensibility = 35% , specificity = 46% ; usa : or = 2.05 95% ci ( 1.442.92 ) sensibility = 27% , specificity = 54% ) and lifetime cannabis misuse ( france : or = 2.60 95% ci ( 1.514.48 ) sensibility = 9% , specificity = 79% ; usa : or = 1.75 95% ci ( 1.023.01 ) sensibility = 30% , specificity = 59% ) . they found among bd patients with cud ( n = 119 ) an earlier age at onset of the first manic or hypomanic episode ( mean age = 19.5 years ) and of the first depressive episode ( mean age = 18.5 years ) compared to patients without cud ( n = 1 , 786 ) , whose mean ages were 25.1 years and 24.4 years , respectively ( p < 0.0001 ) . in this study ,
cud was associated with earlier onset of bipolar disorder and greater number of depressive and hypomanic / manic episodes . it was found that cannabis was a predictor of subsequent mood episodes , especially bipolar disorder , and that cannabis use was associated with increasing the risk of bd ( or = 7.6 ; p < 0.001 ) . in a sample of 151 bd types i and ii
, cannabis use was associated with an earlier onset of bd independently of the history of psychosis or polarity of the first episode , not only manic or psychotic , as described by henquet et al . ( 2009 ) reported that comorbid lifetime cannabis , but not alcohol , abuse / dependence was associated with a statistically significant 3-year - earlier age at onset of psychosis in schizophrenia ( n = 80 ) , schizoaffective disorder ( n = 61 ) , and bipolar disorder with psychotic features ( n = 92 ) . in a study with 324 bd types i and ii patients
, an independent dose - response was shown between cannabis use and age at onset of bd : there was a statistically significant decrease in age at onset with increasing levels of lifetime cannabis use , from 23.2 years ( 9.7 ) for patients who never used cannabis or used cannabis < 10 times during one month lifetime , 20.5 years ( 7.3 ) for patients who used cannabis > 10 times during one month lifetime , and 18.6 years ( 5.0 ) for patients with a lifetime cannabis use disorder ( abuse or dependence ) . the early age at onset of bd and gender , prior suicide attempts , suicide in different mood episodes of bipolar disorder ,
episode polarity and polarity of first affective episode , and drug and alcohol abuse disorders show a significantly increased risk of suicide attempts . the early onset of this disorder increases the risk of suicide and that early onset is around age 20 [ 42 , 43 ] . early age at onset was also associated with suicide attempts in samples from france and the united states . the following variables are described : year of publication ; number of individuals ; suicide attempts ; suicide attempts and substance use ; suicide attempts ; and cannabis use . the following percentages in bipolar patients were found : suicide attempts 3.642% ; suicide attempts and substance use ranging from 5 to 60% ; and suicide attempts and cannabis use ranging from 15 to 42% . suicidal ideation , percent of anxiety , depressed and irritable days in past year , history of suicide attempts , age at onset , marital and smoking status , age , and gender were considered for comparison . the age at onset of bd ( < 13 years ) presented or = 1.37 . in a cox regression model ,
baseline previous suicide attempts , hopelessness , depressive phase at index episode , and younger age at intake were independent risk factors for suicide attempts during follow - up , whereas factors such as bipolar i or ii or comorbidity did not reach statistical significance . comorbid substance use disorder was a predictor in male , while many lifetime mixed episodes , early onset of mental disorder , personality disorder , and social problems related to the primary group were predictors in women . a study was conducted with a group of 1,369 bipolar patients , divided into three subgroups : the first was represented by patients who had had an early age at onset , the second an intermediate age at onset of bd , and the third a late onset , producing a sample of 1,225 individuals ( 144 with borderline values were excluded ) . in a prospective study with a sample of 2,219 bipolar patients who provided data about their lifetime history , 663 ( 29.9% )
factors that were associated with a history of suicide attempts included the following variables : female gender , a history of alcohol abuse , a history of substance abuse , early onset , longer disorder duration , greater depressive symptom severity , current benzodiazepine use , higher overall symptom severity , and poor compliance . in a sample of 652 patients , 280 ( 42.9% )
some variables were associated with a lifetime history of suicidal behavior like being a woman , a history of head injury , tobacco misuse , early age at onset , high number of depressive episodes , positive history of rapid cycling , alcohol misuse , and social phobia . data was analyzed comparing two different groups : bipolar patients with and without suicidal behavior ( bd + sb and bd sb ) and an earlier age at onset was associated with suicidal behavior ( 23.1 9.1 in bp + sb versus 27.0 11.1 in bd sd ) . the association between suicide and substance abuse in bipolar patients can be demonstrated by substantial evidence of suicidal behavior in patients with comorbidities for substance use disorders . the use of cannabis reduces the age at onset of bd ; it may also increase manic episodes and their durations and may increase the risk of suicide in patients with bipolar depression . a study involving 101 patients concluded that the first exposure to cannabis represented strong evidence of an earlier age at onset of first depressive episode . cannabis use in patients with bipolar disorder increases the incidence of psychotic symptoms and suicide attempts and still decreases the response to treatment by lithium compared to patients who do not use this substance . the abusive use of cannabis may act as a predictor of early onset of bipolar disorder and both conditions can interfere with increasing the risk of suicide in patients with bd . greater suicidal behavior in bipolar patients who experienced a reduction in the age at onset in the context of cannabis use was also demonstrated . a meta - analysis of 29 studies evaluated suicide attempts and substance use ( including cannabis ) specifically in bd population . cannabis use disorders were evaluated in 4 studies comprising 3,439 individuals with specific information on bd , suicide attempts , and cud ( 559 with bd and lifetime cannabis use disorders ) . individuals misusing cannabis showed an increased risk of suicide ( or = 1.44 ) when compared with their noncomorbid counterparts . the definition of early age at onset of bd is not clear ; however , it is associated with poor prognosis of bd . if an early diagnosis of bd is not established , the treatment starts late , compromising a good outcome . the definition of the age at onset of bipolar disorder is important because there is robust evidence suggesting that the age at onset distribution of individuals affected with bipolar disorder is composed of at least two age distributions ( early and late onset ) [ 10 , 17 , 18 , 22 , 43 ] , three divided into early , intermediate , and late [ 19 , 21 , 23 ] and only one into very early , early , and late onset . also included the duration of the disorder and finally lin et al
the definition of very early age at onset is even more troublesome because the first symptoms of mood variation that could be diagnosed as the first bipolar episode are difficult to diagnose by mini and other instruments . early age at onset is associated with a greater number of rapid cycling [ 18 , 21 , 23 ] , mixed episodes , and psychotic episodes [ 10 , 17 ] and panic disorder , anxiety disorder [ 18 , 20 ] , substance use disorder [ 3 , 18 , 20 , 21 ] , and major depression , even worse response to lithium and suicidal behavior [ 3 , 18 , 20 , 21 ] . cannabis is the most common illicit substance used among individuals with bd ( 7.2% in bd versus 1.2% in general population ) . especially in cases of cannabis abuse , the relationship between abuse and age at onset is not well understood . there is an association between early age at onset of bd and suicide attempts . early age at onset is associated with suicide attempts and is considered a predictor , especially in females . it is still necessary to elucidate whether early age at onset in bd is an independent risk factor for suicide attempts or whether it depends on other factors , such as disorder severity , rapid cycling , more psychiatric comorbidity , abuse in childhood , family history of mood disorder , differences of gender , and bd subtypes [ 53 , 54 ] . bd type ii was associated with higher rates of suicide attempts , while other studies have shown no significant differences between bd types i and ii . the findings of an association between cannabis use , early age at onset , and suicide attempts may be taken as a support of the view that cannabis negatively affects the course of the disorder . the neuropathological bases of comorbidity between bipolar disorder and cannabis use disorder are a challenge because there are few studies addressing the pathophysiology of these two disorders . areas of medial temporal cortex and prefrontal and subcortical regions with structural and functional abnormalities are associated with emotional and motivational processing in adolescents with bd and substance use disorder . in patients with cooccurrence of bd and cannabis use disorder
an increase in gray matter volume in the right caudate and precentral gyrus and increased gray matter density in the occipital , middle right fusiform , and cerebellar vermis were observed , while there was a reduction in gray matter volume in the left fusiform . the activity of the caudate is related to desire and the fusiform gyrus , which is altered in bipolar patients with cannabis use disorder , is associated with craving and drug seeking behavior . the results of studies attempting to correlate structural and functional changes in the brains of patients with bd and cannabis use disorder found some limitations because they were not able to differentiate preexisting structural brain abnormalities from the consequences of repeated exposure to abused substances . there is sparse literature on the issue involving cannabis use , early age at onset of bd , and suicide attempts . some studies report data on the bipolar group as a whole , not specifying the relationship of cannabis use , early age at onset , and suicide attempts . other studies report data involving the early age at onset in bipolar and schizophrenia making it harder to distinguish to which group of patients the study is referring . however , in this review , as in other studies , the relationship between abuse / dependence and age at onset differentiated on the basis of psychiatric diagnosis or gender aspects has not been investigated , although some of these associations are potentially plausible . in conclusion , the age at onset of bipolar disorder was reviewed due to its prognostic value , making it clear that the first episode should have an early diagnosis . the evidence of cannabis use as a risk factor to early onset of bipolar disorder highlights the necessity to be attentive to the first bipolar episode so that treatment can be started promptly . the use of cannabis is an important factor that may trigger early onset of bd and , by itself , is associated with higher rates of suicidal behavior in bd . however , it is not clear whether the effect of cannabis at the age at onset and suicide attempts are independent of each other or not . | [
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the ne separates the nucleoplasm from the cytoplasm in eukaryotic cells and generates the spatial and functional compartmentalization essential for the maintenance and processing of genetic information .
the cellular role of the ne , however , extends far beyond its function as a physical barrier . as an integral element of cellular architecture , the ne impacts on cytoskeleletal structure and chromatin organization , and ne components regulate gene expression , signal transduction and cell cycle progression .
the physiological role of the ne becomes evident in a number of genetic diseases , referred to as nuclear envelopathies or laminopathies , which are caused by mutations in genes of ne proteins .
nuclear envelopathies commonly manifest in dystrophic , degenerative or premature aging syndromes . in most cases ,
pathogenesis is still poorly understood , but it likely involves both structural and regulatory dysfunction of the ne .
the inm and the onm enclose the perinuclear space ( pns ) , an evenly - spaced luminal compartment continuous with the lumen of the endoplasmic reticulum ( er ) .
the ne is bridged by two types of elaborate , multi - subunit protein complexes , namely nuclear pore complexes ( npcs ) and linc complexes , both of which are highly conserved throughout evolution .
npcs decorate the sites of fusion between the inm and the onm and generate aqueous pores in the ne , which guide the selective exchange of molecules between the nucleoplasm and the cytoplasm .
they are built from sun and kash domain proteins , tm proteins of the inm and the onm , respectively . in the nucleoplasm ,
sun domain proteins engage in a network of inm proteins , chromatin and the nuclear lamina , an intermediate filament mesh that lines and supports the inm in metazoan cells . in the cytoplasm , kash domain proteins bind to cytoskeletal elements , like actin , intermediate filaments and microtubule motors .
the hallmark of the linc complex , however , lies in the lumen of the ne , where the conserved sun and kash domains interact .
thereby , the complexes bridge the ne and connect the structural elements of the nucleus and the cytoskeleton .
sun - kash complexes are thought to serve as handles in the ne , which bear or transmit both cytoskeleton and chromatin - derived forces .
consistently , functions of linc complexes involve mechanical action and crosstalk between the two sides of the ne , such as during nuclear anchorage and migration , or in meiotic chromosome movements .
based on their central position in cellular architecture and function , sun and kash domain proteins are essential for development , differentiation and reproduction .
this idea was based on the prediction of coiled coil elements preceding the c - terminal sun domain in the luminal part of all typical sun homologs ( figs . 1 and 2 ) ) , as well as on the oligomerization of the coiled coil region of human sun1 in vitro .
their n - terminal portions localize in the nucleo / cytoplasm , while their c - terminal portions , consisting of a coiled coil region ( blue ) and the conserved sun domain ( orange ) , are exposed to the lumen of the ne / er . tm regions , coiled coils and sun domains are indicated based on bioinformatic predictions ( tmhmm2 , parcoil2 , prosite ) , literature and visual comparison of homologs .
note that additional hydrophobic stretches or potential tm regions are present in most sun domain proteins , but have been omitted from the scheme for clarity .
further , although only short coiled coil elements are predicted ( solid blue ) , it is conceivable that the entire luminal region preceding the sun domain forms one continuous coiled coil ( blue shadow , length indicated ) .
atypical sun domain proteins . besides the typical sun domain proteins , an unusual second subfamily exists .
this subfamily is also highly conserved throughout evolution and includes , for instance , human suco / c1orf9 , saccharomyces cerevisiae slp1 ( sun - like protein 1 ) and several plant homologs like zea mays sun4 .
interestingly , zmsun4 has recently been shown to concentrate in the nuclear periphery in maize .
the domain structure of this type of sun domain proteins is strikingly different to the conventional one .
the sun domain is located in the middle of the protein instead of at the c terminus and is followed and not as usual preceded by a putative coiled coil region .
further , the proteins are potentially membrane - anchored at both their n and c termini and might thus form ne bridges independently of binding partners .
presently , it is unclear if these atypical sun domain proteins interact with kash - like peptides and whether they function in force transduction , ne spacing or membrane fusion .
most likely , however , their distinct domain structure , i.e. , the inverse order of sun domain , coiled coils and membrane anchor , might reflect a unique role of these proteins in the ne .
tm regions , coiled coils and sun domains were predicted as in figure 1 . a novel type of sun domain proteins in the nuclear envelope .
the first crystal structures of a sun domain protein , human sun2 , therefore bore some surprise : sun2 is a trimer ( fig . 3 ) .
the shape of the complex resembles a lollipop , with the three sun domains forming a globular head , from which the n - terminal extensions of the domains emanate as a helical stalk .
a conserved salt bridge between the stalk and the sun domains holds the two elements in a rigid orientation and may serve to organize the spatial arrangement of the globular head .
in fact , the trimeric organization of sun2 is the essential prerequisite for the assembly of linc complexes .
isolated sun domains lacking their helical extensions are monomeric in solution and are deficient in recruiting kash domain proteins both in vitro and in vivo .
three kash peptides ( yellow , orange , green ) bind to the subunit interfaces of a sun domain trimer ( shades of blue ) , where they are tightly anchored by the binding pocket and the kash lid of the sun domain .
the significance of sun trimerization is immediately evident in the mode of sun - kash interaction .
kash domain proteins are tail - anchored proteins that contain a short luminal portion of 2030 residues , the kash peptide , which directly contacts sun . crystal structures of sun2 in complex with the kash peptides of either nesprin-1 or -2 have revealed the molecular details of their interaction ( fig .
importantly , the kash peptides are accommodated at the interfaces of adjacent sun domain subunits , clearly illustrating how their binding sites can be shaped only by the trimeric arrangement of sun .
note that this principle mode of sun - kash binding , as first reported by sosa et al .
, has recently been confirmed by wang et al . the kash peptides act as the molecular glue of the linc complex ( fig . 3 ) .
each peptide is buried in an extended binding groove at the interface of two subunits and engages in multiple contacts with both neighboring sun domains .
the c terminus of the peptide is anchored in a tailor - made binding pocket on the sun domain with its terminal carboxyl group forming hydrogen bonds to conserved binding pocket side chains .
a stretch of three conserved proline residues in the terminal pppx motif of the peptide helps to orient the carboxyl into the binding pocket .
sun - kash interactions do not tolerate any interference with this terminal anchorage mechanism as evident from both in vitro and in vivo binding studies .
the central part of the kash peptide is clamped between the kash lid , a -hairpin extension of the sun domain and the core of the neighboring sun subunit ( fig .
the peptide backbone and the ' kash lid ' form a joint -sheet , and hydrophobic peptide side chains in this region are buried in deep binding clefts formed between the sun domain subunits . besides the terminal binding pocket ,
the kash lid adds the second essential and conserved contribution to sun - kash interaction .
in addition , many metazoan linc complexes seem to contain a customized safety lock . complexes of human sun1 or sun2 and nesprins are covalently linked via intermolecular disulfide bonds .
the bonds are formed between a cysteine residue in the n - terminal , membrane - facing part of the kash peptide and a cysteine on the sun domain . at both positions ,
cysteines are conserved throughout a range of metazoan homologs suggesting that the covalent linkage has been preserved during evolution .
however , the disulfide bond does not seem to be required for the association of sun and kash domain proteins per se .
rather , it may represent a backup mechanism to prevent the disruption of complexes under peak force loads .
alternatively , the covalent linkage of sun and kash may serve to regulate the dynamics of sun - kash association or the turnover of the proteins .
the extensive network of non - covalent and covalent contacts between sun and kash domains , in combination with the binding avidity gained by the association of three kash peptides with each sun trimer , suggests a highly stable complex . the hexameric assembly appears , in fact , ideally suited to bear and transmit forces on the ne . during processes like nuclear migration or chromosome movement , however , linc complexes likely do not act as individual units , but organize into higher - order structures .
the most striking description of a regular , higher - order assembly of linc components stems from fibroblasts studied in wounded cell monolayers . in preparation for cell migration
, fibroblasts polarize by rearward movement of their nucleus , which is dependent on retrograde actin flow and its coupling to the ne via linc complexes . during this nuclear movement , sun2 and
nesprin-2normally distributed homogenously throughout the ne were shown to organize into linear arrays aligned with actin cables , which were termed tm actin - associated nuclear ( tan ) lines .
another deviation from the usual homogenous ne distribution of mammalian linc components is observed in meiotic cells .
a complex between sun1 and the germ cell - specific kash domain protein kash5 seems to be dedicated to a role during the prophase of meiosis .
the linc complex serves to tether the telomeres of the meiotic chromosomes to the ne , which is thought to facilitate meiotic prophase - specific chromosome movements and to assist the pairing and recombination of the homologs . during this process
, sun1 and kash5 were shown to cluster at the sites of telomere attachment to the ne .
nevertheless , sun2 is expressed in meiotic cells and has been detected at telomere attachment sites , indicating that it may participate in the formation of meiosis - specific higher - order linc assemblies .
it is well conceivable that linc complexes form permanent or transient higher - order structures also in other cell types , which could so far not be resolved by conventional imaging techniques .
their organization may be established by oligomerization of kash domain proteins , or may be mediated via nucleo- or cytoskeletal interaction partners , such as actin filaments or the nuclear lamina .
higher - order structures of linc complexes may be flexible and adapted to dedicated functions , as suggested for tan line formation .
importantly , networks of sun and kash domain proteins in the ne could potentiate the force load , which the complexes can withstand .
linc complexes in the ne might thus function in nucleo - cytoskeletal force transduction analogous to integrins in the plasma membrane , which assemble in focal adhesions to transmit forces between the extracellular matrix and the cytoskeleton .
the mechanical coupling between the nucleus and the cytoplasm can directly be assessed by analyzing changes in nuclear structure and shape upon the application of forces to either the cytoplasm or to the cell surface .
a role of linc complexes in the mechanical coupling of cellular compartments has been demonstrated in several experimental setups , supporting the idea that nucleo - cytoskeletal force transduction is a general principle of linc complex function elucidating the contributions of the characteristic molecular features of the sun - kash assembly in dedicated assays may in future provide insight into the mechanisms of intracellular force transduction and the basis of fundamental cellular processes .
the physiological role of the linc complex , however , may go far beyond these relatively well - characterized tasks .
the molecular architecture of the complex , together with its integral position in the ne , suggests important functions in membrane shaping and organization . by spanning the double membrane of the ne
, assemblies of sun and kash domain proteins may be critical to generate and/or maintain the characteristic shape of the nuclear membrane system .
indeed , observations by electron microscopy indicate that the even spacing of the nuclear membranes is lost upon the co - depletion of sun1 and sun2 from hela cells , giving rise to dilations of the onm and irregular expansions of the pns .
linc complexes may in fact serve as the molecular rulers , which set the distance between the inm and the onm . when the luminal region of sun2 preceding the sun domain is extrapolated as a continuous and extended , trimeric coiled coil , it reaches a length of ~45 nm .
together with the globular sun domain assembly , the entire luminal portion of sun2 would thus span a distance of ~48 nm , fitting strikingly well the observed spacing of the nuclear membranes in mammalian cells ( fig .
importantly , domain organization and size of the luminal parts are conserved between sun1 and sun2 ( fig . 1 ) .
although it can not be excluded that they simply represent adaptions to a given membrane distance , the existing experimental evidence clearly points toward an active and collaborative role of linc complexes in shaping the mammalian ne .
sun and kash domain proteins ( blue and yellow ) are tm proteins of the inm and the onm , respectively .
sun - kash complexes may serve as nuclear membrane spacers and determine the regular shape of the ne .
observed inm - onm distances in mammalian cells fit well the luminal sizes of both sun1 and sun2 given that their coiled coil regions form continuous and extended structures .
analogously , other linc complexes might determine the spacing of the nuclear membranes in different cell types or species . but is this function limited to sun1 and sun2 ? or may sun domain homologs in other cell types and organisms fulfill a similar task ?
besides sun1 and sun2 , the mammalian genome encodes at least three further sun domain proteins ( fig . 1 ) . in sharp contrast to the ubiquitous expression of the former , sun35 are highly tissue - specific and almost exclusively found in male germ cells
sun3 was shown to localize to the ne of developing spermatids , where it accumulates in specific regions in the posterior part of the nucleus .
its localization corresponds to the contact region between the ne and the manchette , a spermatid - specific , calyx - like microtubule structure that is thought to assist the elongation and compaction of the nucleus during sperm head formation .
sun3-containing linc complexes were suggested to tether the manchette microtubules to the ne , potentially required for the remodeling of the sperm nucleus . besides sun3 ,
the fourth mammalian sun domain family member , known as sun4 or spag4 , has also been detected in spermatid - specific microtubule structures .
its d. melanogaster homolog was shown to focus in microtubule- and centriole - associated regions of the ne during spermatogenesis and was required for the attachment of the spermatid 's centriole to the nucleus .
interestingly , early electron microscopic studies on both rodent and fly spermatids have described dense material , or even rod - like structures , between microtubules and the ne , which might from our current point of view well be interpreted as linc complexes . although the overall domain architecture is shared between all typical sun domain homologs , the sperm - specific family members sun35 differ from sun1 and 2 in some critical aspects .
they contain sequence variations in the kash lid , and the cysteine involved in disulfide - bonding to kash is not conserved .
most strikingly , however , the coiled coil regions in the luminal portions of sun35 are significantly shorter than the ones of their larger brothers80130 amino acids ( aa ) compared with ~300 aa in both sun1 and sun2 ( fig . 1 ) .
might a close proximity between the inm and the onm in the sperm nucleus be intended ?
in electron micrographs of the manchette region , the ne indeed seems narrow with a width of ~25 nm as estimated from comparison with neighboring microtubules .
close membrane coupling might in fact provide particular stability and rigidity to the ne at the posterior pole of the sperm nucleus .
it might assist nuclear remodeling during sperm development , or might enable the nucleus of mature spermatozoa to withstand potentially immense cytoskeletal forces generated during sperm movement .
the potential function of linc complexes in ne organization and membrane spacing may be conserved throughout the evolution of eukaryotes . c. elegans and
d. melanogaster possess sun domain homologs with similar molecular properties and sub - cellular localizations as mammalian sun1/2 ( fig . 1 ) , which would thus be well suited to determine ne structure in these organisms .
in contrast to the homogenous distribution of most metazoan sun and kash domain proteins in the ne , linc components in both s. cerevisiae and s. pombe cluster at the spindle pole bodies ( spbs ) during vegetative growth . nevertheless , a minor pool of both sun and kash domain proteins appears to be dispersed throughout the ne in both budding and fission yeast , which may well be sufficient to tether the inm and the onm together and to generate the homogenous shape of the nuclear membrane system .
interestingly , the lengths of the luminal coiled coil regions differ significantly not only between mammalian sun domain homologs , but also between family members from metazoans and yeast ( fig . 1 ) .
while the coiled coils of s. cerevisiae mps3 are with ~250 aa only slightly shorter than the ones of sun1 and sun2 , s. pombe sad1 contains only ~130 aa between its tm helix and the c - terminal sun domain . to our knowledge ,
we therefore attempted to estimate values from electron micrographs acquired under conditions optimized for the preservation of nuclear membrane structure , which obtained inm - onm distances of ~20 nm for both s. cerevisiae and s. pombe .
this would fit well with the predicted length of the sad1 coiled coil region , but be too narrow to accommodate an extended coiled coil for mps3 .
this discrepancy sheds some doubt on the suggested role of linc complexes in ne spacing in yeasts .
yet , it is presently unclear whether the entire luminal region preceding the sun domain adopts a continuous coiled coil structure in all sun domain proteins .
taken together , sun domain proteins may serve as molecular rulers to set the distance between the inm and onm in a wide range of eukaryotes .
moreover , the composition of linc complexes might be adapted in certain cell types , or even within specific regions of the ne , to modulate membrane spacing for dedicated functions .
clearly , more systematic analyses of ne structure in different cell types and organisms , and directed experimental approaches will be required to verify a role of linc complexes in nuclear membrane spacing .
expression and localization patterns of sun domain proteins and correlations between the lengths of their coiled coil regions and inm - onm distances may uncover new facets of intra - cellular membrane shaping .
fundamental cellular processes may rely on the proper structural organization of the nuclear membrane system .
the coupling of the nuclear membranes may be particularly important for membrane remodeling and fusion events , such as the breakdown and reformation of the ne during open mitosis in metazoan cells , or the assembly of new npcs during interphase .
interphase npc biogenesis occurs by insertion of new npcs into the intact double membrane of the ne .
a fusion event between the inm and the onm is thus required to form the aqueous pore .
membrane fusion , in general , necessitates a very close proximity of two membranes which at the sites of npc insertion into the ne might be assisted by linc complexes that sew inm and onm together . in line with this
, human sun1 was found to locally cluster around npcs and to be required for the equal distribution of npcs in the ne .
moreover , it has been proposed that sun1 assists npc biogenesis specifically during interphase . in vitro studies revealed that the formation of aqueous pores in the ne was prohibited in the absence of sun1 , suggesting a function in the membrane fusion step of npc biogenesis .
consistently , the assembly of npcs during ne reformation at the end of mitosis , which could in principle mechanistically differ from interphase assembly and must not necessarily require membrane fusion , was unaffected by sun1 depletion .
transient interactions between the luminal portions of sun1 and the tm nucleoporin pom121 were suggested to facilitate the fusion between the inm and the onm , but such mechanism remains to be validated experimentally .
similar to npcs , the spbs of yeast are gigantic , multi - subunit protein complexes inserted into the double membrane of the ne .
spbs are the main microtubule - organizing centers of yeast cells , and their integration into the ne is critical for the formation of the intra - nuclear spindle during mitosis . in s.
cerevisiae , spbs are inserted into the ne as a late step of their duplication in g1 and remain embedded throughout the cell cycle , whereas spbs of s. pombe become integrated into the ne only temporarily during mitosis .
also the insertion of spbs thus necessitates the fusion between the inm and the onm and likely poses strict requirements on nuclear membrane structure .
sun and kash domain proteins are integral components of spbs in both budding and fission yeast , and their importance for spb structure and function is well established .
the s. cerevisiae sun domain protein mps3 has been implicated specifically in the membrane insertion step of spb duplication .
mps3 was found to influence the lipid composition of the nuclear membranes , potentially by recruiting lipid - modifying enzymes to the ne , which was proposed to modulate physical membrane properties required for spb insertion . for a detailed discussion on the connections between npc and spb insertion in yeast , and on potential roles of s. cerevisiae mps3 in these processes
. how might fusion of inm and onm for insertion of npcs and spbs depend on linc complexes mechanistically ? as an underlying principle , linc complexes may simply generate and/or maintain ne structure , in particular the even spacing of the nuclear membranes . their molecular architecture the extended coiled coil region in combination with the robustly coupled sun - kash assembly renders the complexes ideally suited for this task . or might linc complexes even play a more direct role ?
although purely speculative at this point , it is conceivable that sun - kash complexes actively mediate membrane fusion .
both linc and viral fusion complexes are trimeric protein assemblies , which at least at certain steps during the fusion process
influenza hemagglutinin and linc complexes further share their composition of extended , trimeric coiled coils and globular head domains . similar to viral fusion complexes , sun - kash assemblies might thus employ conformational changes , potentially coupled to association / dissociation cycles , to induce nuclear membrane fusion .
however , it is currently unclear whether the extended luminal coiled coil regions of sun domain proteins are indeed capable of undergoing such changes .
clearly , much remains to be discovered with respect to structure , function and dynamics of these versatile lincs in the ne . | linker of nucleoskeleton and cytoskeleton ( linc ) complexes span the double membrane of the nuclear envelope ( ne ) and physically connect nuclear structures to cytoskeletal elements .
linc complexes are envisioned as force transducers in the ne , which facilitate processes like nuclear anchorage and migration , or chromosome movements .
the complexes are built from members of two evolutionary conserved families of transmembrane ( tm ) proteins , the sun ( sad1/unc-84 ) domain proteins in the inner nuclear membrane ( inm ) and the kash ( klarsicht / anc-1/syne homology ) domain proteins in the outer nuclear membrane ( onm ) . in the lumen of the ne ,
the sun and kash domains engage in an intimate assembly to jointly form a ne bridge .
detailed insights into the molecular architecture and atomic structure of linc complexes have recently revealed the molecular basis of nucleo - cytoskeletal coupling .
they bear important implications for linc complex function and suggest new potential and as yet unexplored roles , which the complexes may play in the cell . | The LINC Between the Nucleus and the Cytoskeleton
The Molecular Architecture of the LINC Complex
The LINC Complex and Force Transduction
The LINC Complex and Nuclear Membrane Shape
The LINC Complex and Membrane Fusion | the ne separates the nucleoplasm from the cytoplasm in eukaryotic cells and generates the spatial and functional compartmentalization essential for the maintenance and processing of genetic information . the cellular role of the ne , however , extends far beyond its function as a physical barrier . as an integral element of cellular architecture , the ne impacts on cytoskeleletal structure and chromatin organization , and ne components regulate gene expression , signal transduction and cell cycle progression . the physiological role of the ne becomes evident in a number of genetic diseases , referred to as nuclear envelopathies or laminopathies , which are caused by mutations in genes of ne proteins . in most cases ,
pathogenesis is still poorly understood , but it likely involves both structural and regulatory dysfunction of the ne . the inm and the onm enclose the perinuclear space ( pns ) , an evenly - spaced luminal compartment continuous with the lumen of the endoplasmic reticulum ( er ) . the ne is bridged by two types of elaborate , multi - subunit protein complexes , namely nuclear pore complexes ( npcs ) and linc complexes , both of which are highly conserved throughout evolution . npcs decorate the sites of fusion between the inm and the onm and generate aqueous pores in the ne , which guide the selective exchange of molecules between the nucleoplasm and the cytoplasm . they are built from sun and kash domain proteins , tm proteins of the inm and the onm , respectively . in the nucleoplasm ,
sun domain proteins engage in a network of inm proteins , chromatin and the nuclear lamina , an intermediate filament mesh that lines and supports the inm in metazoan cells . in the cytoplasm , kash domain proteins bind to cytoskeletal elements , like actin , intermediate filaments and microtubule motors . the hallmark of the linc complex , however , lies in the lumen of the ne , where the conserved sun and kash domains interact . thereby , the complexes bridge the ne and connect the structural elements of the nucleus and the cytoskeleton . sun - kash complexes are thought to serve as handles in the ne , which bear or transmit both cytoskeleton and chromatin - derived forces . consistently , functions of linc complexes involve mechanical action and crosstalk between the two sides of the ne , such as during nuclear anchorage and migration , or in meiotic chromosome movements . based on their central position in cellular architecture and function , sun and kash domain proteins are essential for development , differentiation and reproduction . their n - terminal portions localize in the nucleo / cytoplasm , while their c - terminal portions , consisting of a coiled coil region ( blue ) and the conserved sun domain ( orange ) , are exposed to the lumen of the ne / er . note that additional hydrophobic stretches or potential tm regions are present in most sun domain proteins , but have been omitted from the scheme for clarity . further , although only short coiled coil elements are predicted ( solid blue ) , it is conceivable that the entire luminal region preceding the sun domain forms one continuous coiled coil ( blue shadow , length indicated ) . atypical sun domain proteins . besides the typical sun domain proteins , an unusual second subfamily exists . this subfamily is also highly conserved throughout evolution and includes , for instance , human suco / c1orf9 , saccharomyces cerevisiae slp1 ( sun - like protein 1 ) and several plant homologs like zea mays sun4 . interestingly , zmsun4 has recently been shown to concentrate in the nuclear periphery in maize . the domain structure of this type of sun domain proteins is strikingly different to the conventional one . the sun domain is located in the middle of the protein instead of at the c terminus and is followed and not as usual preceded by a putative coiled coil region . further , the proteins are potentially membrane - anchored at both their n and c termini and might thus form ne bridges independently of binding partners . presently , it is unclear if these atypical sun domain proteins interact with kash - like peptides and whether they function in force transduction , ne spacing or membrane fusion . , the inverse order of sun domain , coiled coils and membrane anchor , might reflect a unique role of these proteins in the ne . a novel type of sun domain proteins in the nuclear envelope . the shape of the complex resembles a lollipop , with the three sun domains forming a globular head , from which the n - terminal extensions of the domains emanate as a helical stalk . a conserved salt bridge between the stalk and the sun domains holds the two elements in a rigid orientation and may serve to organize the spatial arrangement of the globular head . in fact , the trimeric organization of sun2 is the essential prerequisite for the assembly of linc complexes . isolated sun domains lacking their helical extensions are monomeric in solution and are deficient in recruiting kash domain proteins both in vitro and in vivo . three kash peptides ( yellow , orange , green ) bind to the subunit interfaces of a sun domain trimer ( shades of blue ) , where they are tightly anchored by the binding pocket and the kash lid of the sun domain . the significance of sun trimerization is immediately evident in the mode of sun - kash interaction . kash domain proteins are tail - anchored proteins that contain a short luminal portion of 2030 residues , the kash peptide , which directly contacts sun . crystal structures of sun2 in complex with the kash peptides of either nesprin-1 or -2 have revealed the molecular details of their interaction ( fig . importantly , the kash peptides are accommodated at the interfaces of adjacent sun domain subunits , clearly illustrating how their binding sites can be shaped only by the trimeric arrangement of sun . the kash peptides act as the molecular glue of the linc complex ( fig . each peptide is buried in an extended binding groove at the interface of two subunits and engages in multiple contacts with both neighboring sun domains . the c terminus of the peptide is anchored in a tailor - made binding pocket on the sun domain with its terminal carboxyl group forming hydrogen bonds to conserved binding pocket side chains . a stretch of three conserved proline residues in the terminal pppx motif of the peptide helps to orient the carboxyl into the binding pocket . the central part of the kash peptide is clamped between the kash lid , a -hairpin extension of the sun domain and the core of the neighboring sun subunit ( fig . the peptide backbone and the ' kash lid ' form a joint -sheet , and hydrophobic peptide side chains in this region are buried in deep binding clefts formed between the sun domain subunits . besides the terminal binding pocket ,
the kash lid adds the second essential and conserved contribution to sun - kash interaction . in addition , many metazoan linc complexes seem to contain a customized safety lock . the bonds are formed between a cysteine residue in the n - terminal , membrane - facing part of the kash peptide and a cysteine on the sun domain . however , the disulfide bond does not seem to be required for the association of sun and kash domain proteins per se . alternatively , the covalent linkage of sun and kash may serve to regulate the dynamics of sun - kash association or the turnover of the proteins . the extensive network of non - covalent and covalent contacts between sun and kash domains , in combination with the binding avidity gained by the association of three kash peptides with each sun trimer , suggests a highly stable complex . during processes like nuclear migration or chromosome movement , however , linc complexes likely do not act as individual units , but organize into higher - order structures . in preparation for cell migration
, fibroblasts polarize by rearward movement of their nucleus , which is dependent on retrograde actin flow and its coupling to the ne via linc complexes . during this nuclear movement , sun2 and
nesprin-2normally distributed homogenously throughout the ne were shown to organize into linear arrays aligned with actin cables , which were termed tm actin - associated nuclear ( tan ) lines . a complex between sun1 and the germ cell - specific kash domain protein kash5 seems to be dedicated to a role during the prophase of meiosis . the linc complex serves to tether the telomeres of the meiotic chromosomes to the ne , which is thought to facilitate meiotic prophase - specific chromosome movements and to assist the pairing and recombination of the homologs . nevertheless , sun2 is expressed in meiotic cells and has been detected at telomere attachment sites , indicating that it may participate in the formation of meiosis - specific higher - order linc assemblies . it is well conceivable that linc complexes form permanent or transient higher - order structures also in other cell types , which could so far not be resolved by conventional imaging techniques . their organization may be established by oligomerization of kash domain proteins , or may be mediated via nucleo- or cytoskeletal interaction partners , such as actin filaments or the nuclear lamina . higher - order structures of linc complexes may be flexible and adapted to dedicated functions , as suggested for tan line formation . importantly , networks of sun and kash domain proteins in the ne could potentiate the force load , which the complexes can withstand . linc complexes in the ne might thus function in nucleo - cytoskeletal force transduction analogous to integrins in the plasma membrane , which assemble in focal adhesions to transmit forces between the extracellular matrix and the cytoskeleton . the mechanical coupling between the nucleus and the cytoplasm can directly be assessed by analyzing changes in nuclear structure and shape upon the application of forces to either the cytoplasm or to the cell surface . a role of linc complexes in the mechanical coupling of cellular compartments has been demonstrated in several experimental setups , supporting the idea that nucleo - cytoskeletal force transduction is a general principle of linc complex function elucidating the contributions of the characteristic molecular features of the sun - kash assembly in dedicated assays may in future provide insight into the mechanisms of intracellular force transduction and the basis of fundamental cellular processes . the physiological role of the linc complex , however , may go far beyond these relatively well - characterized tasks . the molecular architecture of the complex , together with its integral position in the ne , suggests important functions in membrane shaping and organization . by spanning the double membrane of the ne
, assemblies of sun and kash domain proteins may be critical to generate and/or maintain the characteristic shape of the nuclear membrane system . indeed , observations by electron microscopy indicate that the even spacing of the nuclear membranes is lost upon the co - depletion of sun1 and sun2 from hela cells , giving rise to dilations of the onm and irregular expansions of the pns . linc complexes may in fact serve as the molecular rulers , which set the distance between the inm and the onm . when the luminal region of sun2 preceding the sun domain is extrapolated as a continuous and extended , trimeric coiled coil , it reaches a length of ~45 nm . together with the globular sun domain assembly , the entire luminal portion of sun2 would thus span a distance of ~48 nm , fitting strikingly well the observed spacing of the nuclear membranes in mammalian cells ( fig . although it can not be excluded that they simply represent adaptions to a given membrane distance , the existing experimental evidence clearly points toward an active and collaborative role of linc complexes in shaping the mammalian ne . sun and kash domain proteins ( blue and yellow ) are tm proteins of the inm and the onm , respectively . sun - kash complexes may serve as nuclear membrane spacers and determine the regular shape of the ne . analogously , other linc complexes might determine the spacing of the nuclear membranes in different cell types or species . besides sun1 and sun2 , the mammalian genome encodes at least three further sun domain proteins ( fig . in sharp contrast to the ubiquitous expression of the former , sun35 are highly tissue - specific and almost exclusively found in male germ cells
sun3 was shown to localize to the ne of developing spermatids , where it accumulates in specific regions in the posterior part of the nucleus . its localization corresponds to the contact region between the ne and the manchette , a spermatid - specific , calyx - like microtubule structure that is thought to assist the elongation and compaction of the nucleus during sperm head formation . sun3-containing linc complexes were suggested to tether the manchette microtubules to the ne , potentially required for the remodeling of the sperm nucleus . besides sun3 ,
the fourth mammalian sun domain family member , known as sun4 or spag4 , has also been detected in spermatid - specific microtubule structures . its d. melanogaster homolog was shown to focus in microtubule- and centriole - associated regions of the ne during spermatogenesis and was required for the attachment of the spermatid 's centriole to the nucleus . interestingly , early electron microscopic studies on both rodent and fly spermatids have described dense material , or even rod - like structures , between microtubules and the ne , which might from our current point of view well be interpreted as linc complexes . they contain sequence variations in the kash lid , and the cysteine involved in disulfide - bonding to kash is not conserved . most strikingly , however , the coiled coil regions in the luminal portions of sun35 are significantly shorter than the ones of their larger brothers80130 amino acids ( aa ) compared with ~300 aa in both sun1 and sun2 ( fig . might a close proximity between the inm and the onm in the sperm nucleus be intended ? in electron micrographs of the manchette region , the ne indeed seems narrow with a width of ~25 nm as estimated from comparison with neighboring microtubules . close membrane coupling might in fact provide particular stability and rigidity to the ne at the posterior pole of the sperm nucleus . the potential function of linc complexes in ne organization and membrane spacing may be conserved throughout the evolution of eukaryotes . in contrast to the homogenous distribution of most metazoan sun and kash domain proteins in the ne , linc components in both s. cerevisiae and s. pombe cluster at the spindle pole bodies ( spbs ) during vegetative growth . nevertheless , a minor pool of both sun and kash domain proteins appears to be dispersed throughout the ne in both budding and fission yeast , which may well be sufficient to tether the inm and the onm together and to generate the homogenous shape of the nuclear membrane system . interestingly , the lengths of the luminal coiled coil regions differ significantly not only between mammalian sun domain homologs , but also between family members from metazoans and yeast ( fig . while the coiled coils of s. cerevisiae mps3 are with ~250 aa only slightly shorter than the ones of sun1 and sun2 , s. pombe sad1 contains only ~130 aa between its tm helix and the c - terminal sun domain . to our knowledge ,
we therefore attempted to estimate values from electron micrographs acquired under conditions optimized for the preservation of nuclear membrane structure , which obtained inm - onm distances of ~20 nm for both s. cerevisiae and s. pombe . this would fit well with the predicted length of the sad1 coiled coil region , but be too narrow to accommodate an extended coiled coil for mps3 . this discrepancy sheds some doubt on the suggested role of linc complexes in ne spacing in yeasts . yet , it is presently unclear whether the entire luminal region preceding the sun domain adopts a continuous coiled coil structure in all sun domain proteins . moreover , the composition of linc complexes might be adapted in certain cell types , or even within specific regions of the ne , to modulate membrane spacing for dedicated functions . clearly , more systematic analyses of ne structure in different cell types and organisms , and directed experimental approaches will be required to verify a role of linc complexes in nuclear membrane spacing . fundamental cellular processes may rely on the proper structural organization of the nuclear membrane system . the coupling of the nuclear membranes may be particularly important for membrane remodeling and fusion events , such as the breakdown and reformation of the ne during open mitosis in metazoan cells , or the assembly of new npcs during interphase . interphase npc biogenesis occurs by insertion of new npcs into the intact double membrane of the ne . membrane fusion , in general , necessitates a very close proximity of two membranes which at the sites of npc insertion into the ne might be assisted by linc complexes that sew inm and onm together . in line with this
, human sun1 was found to locally cluster around npcs and to be required for the equal distribution of npcs in the ne . in vitro studies revealed that the formation of aqueous pores in the ne was prohibited in the absence of sun1 , suggesting a function in the membrane fusion step of npc biogenesis . consistently , the assembly of npcs during ne reformation at the end of mitosis , which could in principle mechanistically differ from interphase assembly and must not necessarily require membrane fusion , was unaffected by sun1 depletion . similar to npcs , the spbs of yeast are gigantic , multi - subunit protein complexes inserted into the double membrane of the ne . spbs are the main microtubule - organizing centers of yeast cells , and their integration into the ne is critical for the formation of the intra - nuclear spindle during mitosis . in s.
cerevisiae , spbs are inserted into the ne as a late step of their duplication in g1 and remain embedded throughout the cell cycle , whereas spbs of s. pombe become integrated into the ne only temporarily during mitosis . also the insertion of spbs thus necessitates the fusion between the inm and the onm and likely poses strict requirements on nuclear membrane structure . sun and kash domain proteins are integral components of spbs in both budding and fission yeast , and their importance for spb structure and function is well established . mps3 was found to influence the lipid composition of the nuclear membranes , potentially by recruiting lipid - modifying enzymes to the ne , which was proposed to modulate physical membrane properties required for spb insertion . as an underlying principle , linc complexes may simply generate and/or maintain ne structure , in particular the even spacing of the nuclear membranes . their molecular architecture the extended coiled coil region in combination with the robustly coupled sun - kash assembly renders the complexes ideally suited for this task . or might linc complexes even play a more direct role ? both linc and viral fusion complexes are trimeric protein assemblies , which at least at certain steps during the fusion process
influenza hemagglutinin and linc complexes further share their composition of extended , trimeric coiled coils and globular head domains . however , it is currently unclear whether the extended luminal coiled coil regions of sun domain proteins are indeed capable of undergoing such changes . clearly , much remains to be discovered with respect to structure , function and dynamics of these versatile lincs in the ne . | [
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since the inception of our specialty , radiologists have served as consultants to physicians of various disciplines , and we continue to diagnose , inform treatment decisions and guide management across the spectrum of medical disease . while the role of the radiologist in the multidisciplinary approach to care is little changed over time , our communication with ordering providers has evolved dramatically . widely available technologies , including the picture archiving communication system ( pacs ) , appropriateness criteria , and critical results communication systems , amongst others , have had the potential to decrease personalized , one - on - one interactions with referring providers .
the means by which we fulfill our consultant role have changed , whereby information / result delivery is facilitated but free exchange of pertinent details is limited in any single clinical scenario .
a variety of radiology consultation services have been described in the literature , aimed at facilitating interactions between radiologists and clinical providers to optimize patient care .
consultation model types include clinical decision support , patient - centric , subspecialty interpretation , and/or some combination of these . in the clinical decision support model ,
radiologists are consulted by referring clinicians for assistance in radiologic procedure selection and planning . in the patient - centric model ,
patients are encouraged to interact directly with radiologists , for discussion of results or questions related to their imaging evaluation .
the second opinion / subspecialty interpretation is generally sought out by referring clinicians or healthcare teams , but in some instances , is available directly to patients ( in the latter instance , typically for a fee ) ( table 1 ) ( 1 ) . in oncology care in particular ,
nuanced , yet critical clinical details may not be readily available in study indications from electronic systems . in tumor board conferences and formal or informal consultation
, radiologists play an integral role in multidisciplinary discussions centered on diagnosis , staging , approach to tissue sampling and definitive versus palliative management . at referral centers ,
outside imaging studies are reviewed in consultation to broaden the team 's understanding of the prior workup , minimize duplication of services , and plan subsequent treatments .
imaging studies performed at the home institution are also re - examined to answer specific clinical questions , which may be related to patient symptomatology , underlying tumor genomics , targeted therapy selection , predicted treatment response and/or prognosis .
radiologists as consultants have both an opportunity and an obligation to demonstrate true added value to optimized treatment plans . to explore the utility and impact of radiology consultation services in the academic setting ,
this article will further describe existing consultation models and the circumstances that precipitated their development .
in addition , the contributions of a consultant radiologist in breast cancer care are reviewed as the archetype of radiology consultation services provided to oncology practitioners .
the concept of a radiology consultation service was first reported in 1979 , when shuman and heilman ( 2 ) described their 2 year experience offering formal radiologic consults to clinicians anticipating complex radiologic work - ups .
referring clinicians would utilize the standard consultation form used for their services , which was then forwarded to the radiology resident assigned to consult that month .
the resident would carefully review the patient 's medical record , discuss issues with the appropriate services , and when indicated , perform a physical exam , and examine all prior pertinent imaging studies .
a comprehensive radiologic evaluation plan was devised , discussed with the appropriate attending radiologist , and then conveyed to the requesting clinician .
the impetus for starting this service arose from the rising cost of healthcare , with often indiscriminate use of expensive imaging tests , and the simultaneous explosion of technological advances in radiology , as well as marked growth of interventional radiology procedures .
the authors reasoned that radiologists are most knowledgeable about varied imaging modalities and their best utilization , as well as the capabilities of radiologic intervention , and should thus be consulted when making imaging decisions regarding complex cases .
they describe the mixed responses of both the referring clinicians ( some disliking the intrusion into their diagnostic arena ) , and the radiology residents and staff ( some of whom were uneasy with the expansion of clinical interactions ) , but promoted the concept as deserving of further evaluation .
several other academic radiology departments have instituted various forms of consultation , using the clinical decision support model
, in some cases , mandating approval , and in others , offering it as an optional service and educational tool .
baker ( 3 ) analyzed several different scenarios where consult radiologists would provide guidance to clinicians in selecting appropriate radiologic studies and/or in planning imaging workups , and showed utility when compared to a control group without access to radiologic consultation . in a pilot study of patients presenting with biliary tract disease , non - emergent gastrointestinal bleeding or abdominal mass , they reported that actively structuring the diagnostic evaluation by a consultant radiologist resulted in a 64% reduction in time to diagnosis and 32% reduction in number of studies performed ( 3 ) . by embedding a radiology consultant in daily medical rounds on an acute care medical ward ,
whereby all radiology requests were discussed daily , they reported significantly fewer radiologic examinations were performed , while house staff were educated on proper utilization of imaging tests , a potential means of cost containment ( 4 ) . in a third study , on an acute care surgical ward , a consultant radiologist reviewed all radiographic studies performed , discussing their limitations , pertinence and cost , and future examination were discussed . over the study period ,
the number of ct scans , ultrasound studies and barium studies decreased significantly , and the average length of patient stay decreased by 2.8 days ( 5 ) .
( 6 ) reported a study whereby all body ct scans required consultation with a radiologist prior to scheduling .
they noted 95% cooperation by referring clinicians , and described benefits of obtaining more detailed clinical information , thus providing improved scanning protocols , fewer post - ct diagnostic tests , and improved rapport between the referring clinicians and radiologists . with the ever present reminder of cost containment looming
, the role of the radiologist as a consultant offering clinical decision support has been further investigated using several study designs , including optional vs. mandatory , assisting house staff vs. all referring clinicians , incorporated into rounds vs. a stand - alone service ( 789 ) . in most institutions it was generally well received , but has had variable success in decreasing the number of studies ordered .
( 8) found that mandatory radiology precertification in an inpatient setting did not reduce radiology resource utilization .
they noted that although participating physicians were supportive , internal medicine ward physicians did not reduce their test ordering , and radiologists , trained in the interpretation of exams , rather than assessment of appropriateness , were reluctant to perform precertification .
the authors suggested that practice guidelines might help standardize the consultation process , and that financial incentives may motivate physicians to reduce ancillary tests . with the advent of pacs , direct interactions with radiologists in reading rooms has declined , in one study , by 82% for general radiography , and 44% for cross sectional imaging studies , despite an increase in overall volume ( 10 ) .
as consultation has been shown to improve cost containment and allow for a more rapid diagnosis , improved communication between referring clinicians and radiologists is imperative .
tillack and borgstede ( 11 ) looked at the impact of embedding radiology reading rooms in the clinic , noting a significantly greater number of visits by referring providers to embedded reading rooms ( though the study was slightly confounded by comparing embedded versus non - embedded reading rooms of different specialties , with inherent differences in consultation patterns ) .
the present emphasis on value ( over volume ) in health imaging is strongly touted by the american college of radiology ( acr ) , as noted in imaging 3.0 , which encourages radiologists to assist providers with clinical decision support , to discuss results and provide actionable , meaningful reports ( 12 ) .
it is felt that clinical decision support helps to eliminate unnecessary examinations and increases the radiologists ' relevance , as well as provides radiologists with better detailed patient information , which allows for more directed protocols and accurate coding for billing .
in 1966 , sherman ( 13 ) asked , is it not really the patient we are obligated to serve above all others ? , and suggested that patients ( not their clinicians ) are entitled to results of their exams . in 1990 , the beginning of malpractice litigation that alleged failure of communication of radiologic results was brought to light in the american college of radiology ( acr ) bulletin ( 14 ) .
berlin noted that soon after the expansion of radiologist 's duty to directly convey significant radiologic findings to the referring clinician , their role was further extended to include direct communication to patients , fueled by the judicial system , the federal government , the medical community and patients ( 15 ) .
he cites numerous lawsuits where radiologists were found to be negligent for not communicating significant results directly to patients .
the united states government 's involvement is evident in the mammography quality standards act , published in october 1992 , mandating that all patients receive a summary of their mammographic findings , in layman 's terms , within 30 days of their mammogram .
berlin further notes that both the american medical association and acr have language in their bylaws and guidelines , describing the responsibility of the radiologist ( and all consulting physicians ) to communicate results directly to patients , and answer any questions they might have .
however , he goes on to discuss the various difficulties that might arise , including selecting the format of transmissions , the affect on relationships with referring physicians , placing the radiologist in the unenviable position of being asked to advise on treatment options in which he / she is not involved and possibly less knowledgeable .
several older studies that surveyed patients undergoing radiologic imaging reported patient preferences to hear imaging results from the radiologists interpreting their exams .
a small study of 79 cancer patients undergoing ct imaging at a dedicated cancer center reported that 70% wanted the radiologist to disclose their results ( 16 ) . in a survey of patient preferences of 261 consecutive patients undergoing radiologic evaluation in a large university hospital ,
the overwhelming majority ( 92% for normal findings , 87% for abnormal findings ) also preferred to get results from the radiologist at the time of the procedure , rather than later , from their referring clinician ( 17 ) .
a follow - up survey of referring clinicians and radiologists concluded that radiologists and referring physicians both tended to support the proposition that , if asked by patients , radiologists should disclose the results of their imaging studies ( 18 ) .
however , given the uncertainties of many , direct patient radiologist communication has not become the standard of care in most general radiology practices .
recently , the consultation radiologist role has resurfaced in the literature , as a means of increasing the radiologists ' visibility to patients . in most scenarios ,
the ordering physician conveys results to the patient , who is often unaware that a radiologist has played any role in providing his or her care .
several institutions have revisited the exploration of patient preferences in receiving results of radiologic studies .
a survey of 129 patients undergoing ct or mri at an academic medical center outpatient facility revealed that speed of report delivery was the most important factor in patient satisfaction , without an overwhelming preference for which physician provided the results ( 19 ) . in another study of 86 patients undergoing ct or ultrasound evaluation , patients preferred hearing results from both their referring clinicians and the interpreting radiologist , and found the latter consultation beneficial ( 20 ) .
however , at least 2 other studies noted that most patients preferred to hear from their referring clinicians .
642 responses to a survey of patients undergoing ct or mri revealed that for both normal and abnormal results , the preferred mode of communication was a phone call from the referring physician ( 34.1% and 49.8% , respectively ) ( 21 ) .
patients also preferred detailed radiology reports , with some requesting access to key images . in a similar study of patients undergoing ct or mri at an academic medical center ( 77% ) or a county hospital ( 23% ) , among 617 surveys ,
63% revealed preference for receiving results from their ordering providers ; 64% desired access to their imaging report , and 85% wished to see their images ( 22 ) .
mangano et al . ( 23 ) have piloted a patient - centered radiology consultation area , where patients meet with a radiologist to discuss their imaging study results .
the impetus in piloting this service was to increase the visibility of the radiologist , and increase awareness of the critical role imagers play in healthcare .
this has been among the goals of several acr outreach campaigns , including the face of radiology campaign ( 24 ) .
another academic medical center created a unique consultation service , devised to address the ambiguous information legally mandated to be shared with patients following mammograms that reveal dense breast tissue .
following mammography , patients are presented with data regarding breast density and cancer , though without evidence - based guidelines for further evaluation .
sullivan et al . ( 25 ) report their experience in providing consultation services to these patients ( and referring clinicians ) , by employing a registered radiology assistant ( rra ) , who they define as a
mid - level provider who has received advanced education and clinical experience in radiology .
the rra receives the requests , collects pertinent information and prepares the consultation , thus improving efficiency and optimizing the radiologist 's time spent in direct consultation with the patient .
although the consultation service was only utilized by 138 of 7131 ( 2% ) of their dense breast patient population ( plus an additional 14 patients whom heard about the service from other sources ) , over 70% felt sure of their best choice for additional screening and over 78% felt they had gained enough information to make a decision regarding screening following their consultations .
the authors concluded that their service provides education , and ultimately improves quality of care .
the subspecialty interpretation model is based on a general consensus that subspecialists provide better care in their area of expertise than generalists .
although a study evaluating interpretation of pediatric plain radiographs at a rural hospital did not show a statistically significant difference in interpretation between pediatric radiologists and generalists , the authors note that the subspecialists were more accurate ( 26 ) . in another study ,
the detection of extracapsular extension of prostate cancer on mri was significantly affected by the reader 's subspecialty experience ( 27 ) .
several tertiary care centers have looked at the quantitative effect of radiology consultations on management of cancer patients .
one center reported the addition of significant new information in 49% of patients reviewed at their division of oncology radiology conference , resulting in major changes in management in 37% ( 28 ) .
two different centers specifically looked at reinterpretation of imaging studies in head and neck cancer patients .
the first reported a change in interpretation of 41% , with statistically significant changes in tnm staging in 34% of patients , which altered treatment plans in 98% and affected prognosis in 95% ( 29 ) .
changes were attributed to lack of subspecialization in head and neck imaging at the outside institution , and to often incomplete clinical information of the original interpreting radiologist . in a similar study ,
fellowship trained neuroradiologists provided formal second opinions on head and neck imaging studies , resulting in changes in stage in 56% and management changes in 38% , noting 93% accuracy based on pathologic staging as gold standard ( 30 ) .
a recent study at our cancer center evaluated the influence of a second opinion / subspecialty consultation on the surgical management of breast cancer patients ( 31 ) .
they reported changes in 11.7% of patients undergoing surgery for breast cancer . given the variability in quality of outside studies and their interpretations , and the differences that a comprehensive evaluation may make in a patient 's prognosis and treatment strategy , review of outside studies by a specialized oncologic imager is critically important .
changes in patient care due to direct subspecialty radiology consultation have recently been reported in disciplines other than oncology .
( 32 ) reviewed over 2000 second opinion subspecialty consultations in musculoskeletal radiology , and noted clinically important differences in interpretation in 26.2% overall , and discrepant interpretations in 36.3% of oncologic cases . based on the final pathologic diagnosis
, they reported that second opinion consultations were correct in 82.0% of cases where discrepancies were likely to change patient management .
dickerson et al . ( 33 ) also reported significant alterations in surgical decision making when acute care surgeons collaborated directly and in - person with radiologists .
they reported that surgeons ' diagnostic impressions and medical and/or surgical planning were altered in 43% of cases , and concluded that the in - person collaboration promoted a shared mental model that facilitates the exchange of complex information . of note ,
some departments have taken a different approach , choosing to issue a formal dictation of outside studies rather than a verbal consultation and bill for a second opinion read .
yousem ( 34 ) chose to upload outside neuroradiology studies to their electronic archive and provide an official dictation , noting the benefits to the referring clinicians including less hassle in bringing studies to radiologists , increased ease in making images available to operating rooms and conference rooms ( via pacs ) , provision of subspecialty interpretations , access to the outside preoperative studies when evaluating for residual disease postoperatively , decreased interruption to workflow ( as studies are now read from a pacs system work list ) , and monetary reimbursement . given their success in neuroradiology , they have now initiated this approach throughout their department .
at our tertiary care cancer center , imaging plays an integral role in optimizing patient care by guiding decisions regarding diagnosis , staging , tissue sampling , trial eligibility and treatment .
since pacs enables remote access to imaging studies , radiologists are commonly called upon to lend expertise in viewing both our own and outside imaging studies .
a large study at our own institution over a decade ago looked at the volume and impact of second opinion consultations , noting an overall increase in daily workload of 18% ( 35 ) .
the impact on workflow , finances , and compensation in terms of relative value units were all considered .
based on the strict reimbursement requirements ( which include a written report and documentation of medical necessity ) , as well as the variable quality of outside imaging studies and time needed to thoroughly review and redictate a study , it was deemed not cost effective to issue formal reports on outside consultation cases . however , referring clinicians strongly favored and valued this service , therefore institute administrators deemed it worthwhile to contract with the imaging department to provide consultation services by a radiologist located within the clinic .
our decision to participate was not only a matter of convenience , profit , and productivity , but one that has been shown to greatly benefit clinicians and patients alike .
monetary compensation for this dedicated radiologist allowed the hiring of an additional full time employee , allowing for fewer interruptions to the radiologists interpreting our home institution 's studies , and incidentally increasing productivity overall .
although our survey only captured consultations on outside studies or on our studies that required further comparison , the consult radiologist is responsible for reviewing both on - site studies , as well as those from associated institutions , and is available to perform tumor measurements for clinical trial patients . at our institution
, the success of the consultation service and increasing patient volume has resulted in expanded general and subspecialty oncologic imaging consultation services .
the consult radiologist provides several services , including ( though not limited to ) clinical decision support , interpretations , advice regarding biopsy or treatment planning , and determination of measurable disease to confirm trial eligibility .
given the ease of access and face - to - face interaction , we have noted increased dependence on our interpretation of all studies , including studies from both outside and inside the network , and also our daily services from within the institution .
most importantly , however , the interactions between imagers and oncologists , surgeons , radiation oncologists , nurses and physician assistants , have grown more collegial , and in fact have facilitated research opportunities / collaborative efforts .
thus , radiologists have become more integrated into the multidisciplinary approach which is so critical in caring for cancer patients ( 36 ) .
( 37 ) describe this integrated consultation service as only a part of their vision for the role of imaging in optimizing cancer care .
they support the use of imaging in pre - clinical studies to facilitate translational research , and also note the importance of effective , specialized training programs for future oncologic imagers .
a brief evaluation of various treatment decisions in a breast cancer patient 's medical journey elucidates the critical role that radiologists play as consultants to surgeons , radiation oncologists and medical oncologists . after a breast cancer diagnosis
although a patient may opt for a lumpectomy versus mastectomy by personal choice , the radiologist , upon evaluation of the breast imaging studies , will assist the surgeon in determining whether breast conservation is feasible .
additional sites or wider extent of disease seen with mammography , ultrasound , or mri , may preclude planned breast conservation surgery .
the breast imager is not infrequently consulted by the radiation oncologist when planning radiation therapy for breast cancer patients .
the presence of internal mammary lymphadenopathy may change the radiation field , and therefore the radiologist is sought out to provide an opinion regarding the presence of internal mammary lymph nodes and their exact location on mri and ct , so that radiation treatment can be accurately planned .
patients with a diagnosis of inflammatory breast cancer undergo additional staging with pet - ct imaging , which in a small study at our institution altered the radiation treatment planning in 24% ( fig .
1 ) ( 38 ) . knowledge of a patient 's breast tumor pathology will also affect the consult radiologist 's approach to disease evaluation and recommendations for future imaging . in cases of invasive lobular cancer , which are often mammographically occult , sonography or mri may be important in better defining tumor margins .
given the knowledge of different patterns of metastatic disease spread with lobular breast cancer ( versus invasive ductal cancer ) , a subspecialized oncologic radiologist is more attentive to subtle peritoneal thickening , bowel wall thickening or adnexal changes , that may herald early disease spread , thus suggesting biopsies or close follow - up ( fig .
the radiologist is often consulted by the oncologist to evaluate changes , especially those not readily evaluated with standard response criteria ( such as response evaluation criteria in solid tumors [ recist ] ) .
for example , the presence of increased sclerosis in a breast cancer patient with known osseous metastases at a site of previously radiologically occult disease may actually represent response , rather than disease progression as described in the university of texas md anderson cancer center criteria ( mda criteria ) , and must be viewed in light of other changes ( fig .
3 ) ( 39 ) . many , increasing , radiologic response criteria are not necessarily known to many referring clinicians .
the combination of imaging expertise , knowledge of disease presentation and patterns of spread , as well as responses to therapeutic options with their potential toxicities and complications make the consultant radiologist vitally important to patient care in the oncologic setting .
it is often the radiologist who will first detect signs of drug toxicity or will note disease that will preclude the use of certain medications . as newer therapies have been developed , response criteria have changed to address the varied appearances of tumor response , and different patterns of drug toxicity have been noted .
therefore , it is critical that radiologists interact personally with oncologists and other members of the oncology care team to be made aware of the newest therapies .
together , care teams can learn about their resultant imaging findings , as well as their potential complications and toxicities .
this relationship is mutually beneficial , affording imagers the ability to stay abreast of continuously changing therapeutic advances and the opportunity to acquire information about individual patients and their specific radiologic queries , as well as providing referring clinicians with radiologic expertise to help provide the best care to their patients .
in the last several years , there has been much focus on the value of imaging in healthcare , with campaigns sponsored by the acr , and multiple publications urging radiologists to take a more active role in reaching out to clinicians and patients as a consultant ( 404142 ) .
this report summarizes several different models of consultation services which address various end goals including optimization of interdisciplinary care foremost . at our tertiary center
, a hybrid model has been incorporated to serve the specific needs of our patients and providers , aimed at cultivating these relationships and leveraging the strengths of our cancer imaging practice . in coming years
, it will be important to quantify the added value of radiology consultation , as well as secure buy - in from the stakeholders on a broader scale . | the purpose of the article is to describe the various radiology consultation models in the era of precision medicine . since the inception of our specialty , radiologists have served as consultants to physicians of various disciplines .
a variety of radiology consultation services have been described in the literature , including clinical decision support , patient - centric , subspecialty interpretation , and/or some combination of these . in oncology care in particular ,
case complexity often merits open dialogue with clinical providers . to explore the utility and impact of radiology consultation services in the academic setting ,
this article will further describe existing consultation models and the circumstances that precipitated their development .
the hybrid model successful at our tertiary cancer center is discussed .
in addition , the contributions of a consultant radiologist in breast cancer care are reviewed as the archetype of radiology consultation services provided to oncology practitioners . | INTRODUCTION
The Clinical Decision Support Model
The Patient-Centric Model
The Second Opinion/Subspecialty Interpretation Model
Hybrid Radiologic Consultation
Case Study in Breast Cancer Consultation
CONCLUSION | since the inception of our specialty , radiologists have served as consultants to physicians of various disciplines , and we continue to diagnose , inform treatment decisions and guide management across the spectrum of medical disease . while the role of the radiologist in the multidisciplinary approach to care is little changed over time , our communication with ordering providers has evolved dramatically . widely available technologies , including the picture archiving communication system ( pacs ) , appropriateness criteria , and critical results communication systems , amongst others , have had the potential to decrease personalized , one - on - one interactions with referring providers . the means by which we fulfill our consultant role have changed , whereby information / result delivery is facilitated but free exchange of pertinent details is limited in any single clinical scenario . a variety of radiology consultation services have been described in the literature , aimed at facilitating interactions between radiologists and clinical providers to optimize patient care . consultation model types include clinical decision support , patient - centric , subspecialty interpretation , and/or some combination of these . in the clinical decision support model ,
radiologists are consulted by referring clinicians for assistance in radiologic procedure selection and planning . in the patient - centric model ,
patients are encouraged to interact directly with radiologists , for discussion of results or questions related to their imaging evaluation . the second opinion / subspecialty interpretation is generally sought out by referring clinicians or healthcare teams , but in some instances , is available directly to patients ( in the latter instance , typically for a fee ) ( table 1 ) ( 1 ) . in oncology care in particular ,
nuanced , yet critical clinical details may not be readily available in study indications from electronic systems . in tumor board conferences and formal or informal consultation
, radiologists play an integral role in multidisciplinary discussions centered on diagnosis , staging , approach to tissue sampling and definitive versus palliative management . at referral centers ,
outside imaging studies are reviewed in consultation to broaden the team 's understanding of the prior workup , minimize duplication of services , and plan subsequent treatments . radiologists as consultants have both an opportunity and an obligation to demonstrate true added value to optimized treatment plans . to explore the utility and impact of radiology consultation services in the academic setting ,
this article will further describe existing consultation models and the circumstances that precipitated their development . in addition , the contributions of a consultant radiologist in breast cancer care are reviewed as the archetype of radiology consultation services provided to oncology practitioners . the concept of a radiology consultation service was first reported in 1979 , when shuman and heilman ( 2 ) described their 2 year experience offering formal radiologic consults to clinicians anticipating complex radiologic work - ups . referring clinicians would utilize the standard consultation form used for their services , which was then forwarded to the radiology resident assigned to consult that month . the resident would carefully review the patient 's medical record , discuss issues with the appropriate services , and when indicated , perform a physical exam , and examine all prior pertinent imaging studies . the impetus for starting this service arose from the rising cost of healthcare , with often indiscriminate use of expensive imaging tests , and the simultaneous explosion of technological advances in radiology , as well as marked growth of interventional radiology procedures . the authors reasoned that radiologists are most knowledgeable about varied imaging modalities and their best utilization , as well as the capabilities of radiologic intervention , and should thus be consulted when making imaging decisions regarding complex cases . they describe the mixed responses of both the referring clinicians ( some disliking the intrusion into their diagnostic arena ) , and the radiology residents and staff ( some of whom were uneasy with the expansion of clinical interactions ) , but promoted the concept as deserving of further evaluation . several other academic radiology departments have instituted various forms of consultation , using the clinical decision support model
, in some cases , mandating approval , and in others , offering it as an optional service and educational tool . baker ( 3 ) analyzed several different scenarios where consult radiologists would provide guidance to clinicians in selecting appropriate radiologic studies and/or in planning imaging workups , and showed utility when compared to a control group without access to radiologic consultation . in a pilot study of patients presenting with biliary tract disease , non - emergent gastrointestinal bleeding or abdominal mass , they reported that actively structuring the diagnostic evaluation by a consultant radiologist resulted in a 64% reduction in time to diagnosis and 32% reduction in number of studies performed ( 3 ) . in a third study , on an acute care surgical ward , a consultant radiologist reviewed all radiographic studies performed , discussing their limitations , pertinence and cost , and future examination were discussed . over the study period ,
the number of ct scans , ultrasound studies and barium studies decreased significantly , and the average length of patient stay decreased by 2.8 days ( 5 ) . ( 6 ) reported a study whereby all body ct scans required consultation with a radiologist prior to scheduling . they noted 95% cooperation by referring clinicians , and described benefits of obtaining more detailed clinical information , thus providing improved scanning protocols , fewer post - ct diagnostic tests , and improved rapport between the referring clinicians and radiologists . with the ever present reminder of cost containment looming
, the role of the radiologist as a consultant offering clinical decision support has been further investigated using several study designs , including optional vs. mandatory , assisting house staff vs. all referring clinicians , incorporated into rounds vs. a stand - alone service ( 789 ) . they noted that although participating physicians were supportive , internal medicine ward physicians did not reduce their test ordering , and radiologists , trained in the interpretation of exams , rather than assessment of appropriateness , were reluctant to perform precertification . with the advent of pacs , direct interactions with radiologists in reading rooms has declined , in one study , by 82% for general radiography , and 44% for cross sectional imaging studies , despite an increase in overall volume ( 10 ) . tillack and borgstede ( 11 ) looked at the impact of embedding radiology reading rooms in the clinic , noting a significantly greater number of visits by referring providers to embedded reading rooms ( though the study was slightly confounded by comparing embedded versus non - embedded reading rooms of different specialties , with inherent differences in consultation patterns ) . the present emphasis on value ( over volume ) in health imaging is strongly touted by the american college of radiology ( acr ) , as noted in imaging 3.0 , which encourages radiologists to assist providers with clinical decision support , to discuss results and provide actionable , meaningful reports ( 12 ) . it is felt that clinical decision support helps to eliminate unnecessary examinations and increases the radiologists ' relevance , as well as provides radiologists with better detailed patient information , which allows for more directed protocols and accurate coding for billing . in 1990 , the beginning of malpractice litigation that alleged failure of communication of radiologic results was brought to light in the american college of radiology ( acr ) bulletin ( 14 ) . berlin noted that soon after the expansion of radiologist 's duty to directly convey significant radiologic findings to the referring clinician , their role was further extended to include direct communication to patients , fueled by the judicial system , the federal government , the medical community and patients ( 15 ) . he cites numerous lawsuits where radiologists were found to be negligent for not communicating significant results directly to patients . the united states government 's involvement is evident in the mammography quality standards act , published in october 1992 , mandating that all patients receive a summary of their mammographic findings , in layman 's terms , within 30 days of their mammogram . berlin further notes that both the american medical association and acr have language in their bylaws and guidelines , describing the responsibility of the radiologist ( and all consulting physicians ) to communicate results directly to patients , and answer any questions they might have . however , he goes on to discuss the various difficulties that might arise , including selecting the format of transmissions , the affect on relationships with referring physicians , placing the radiologist in the unenviable position of being asked to advise on treatment options in which he / she is not involved and possibly less knowledgeable . several older studies that surveyed patients undergoing radiologic imaging reported patient preferences to hear imaging results from the radiologists interpreting their exams . a small study of 79 cancer patients undergoing ct imaging at a dedicated cancer center reported that 70% wanted the radiologist to disclose their results ( 16 ) . in a survey of patient preferences of 261 consecutive patients undergoing radiologic evaluation in a large university hospital ,
the overwhelming majority ( 92% for normal findings , 87% for abnormal findings ) also preferred to get results from the radiologist at the time of the procedure , rather than later , from their referring clinician ( 17 ) . a follow - up survey of referring clinicians and radiologists concluded that radiologists and referring physicians both tended to support the proposition that , if asked by patients , radiologists should disclose the results of their imaging studies ( 18 ) . however , given the uncertainties of many , direct patient radiologist communication has not become the standard of care in most general radiology practices . recently , the consultation radiologist role has resurfaced in the literature , as a means of increasing the radiologists ' visibility to patients . in most scenarios ,
the ordering physician conveys results to the patient , who is often unaware that a radiologist has played any role in providing his or her care . a survey of 129 patients undergoing ct or mri at an academic medical center outpatient facility revealed that speed of report delivery was the most important factor in patient satisfaction , without an overwhelming preference for which physician provided the results ( 19 ) . in another study of 86 patients undergoing ct or ultrasound evaluation , patients preferred hearing results from both their referring clinicians and the interpreting radiologist , and found the latter consultation beneficial ( 20 ) . 642 responses to a survey of patients undergoing ct or mri revealed that for both normal and abnormal results , the preferred mode of communication was a phone call from the referring physician ( 34.1% and 49.8% , respectively ) ( 21 ) . patients also preferred detailed radiology reports , with some requesting access to key images . in a similar study of patients undergoing ct or mri at an academic medical center ( 77% ) or a county hospital ( 23% ) , among 617 surveys ,
63% revealed preference for receiving results from their ordering providers ; 64% desired access to their imaging report , and 85% wished to see their images ( 22 ) . ( 23 ) have piloted a patient - centered radiology consultation area , where patients meet with a radiologist to discuss their imaging study results . the impetus in piloting this service was to increase the visibility of the radiologist , and increase awareness of the critical role imagers play in healthcare . this has been among the goals of several acr outreach campaigns , including the face of radiology campaign ( 24 ) . another academic medical center created a unique consultation service , devised to address the ambiguous information legally mandated to be shared with patients following mammograms that reveal dense breast tissue . ( 25 ) report their experience in providing consultation services to these patients ( and referring clinicians ) , by employing a registered radiology assistant ( rra ) , who they define as a
mid - level provider who has received advanced education and clinical experience in radiology . the rra receives the requests , collects pertinent information and prepares the consultation , thus improving efficiency and optimizing the radiologist 's time spent in direct consultation with the patient . the subspecialty interpretation model is based on a general consensus that subspecialists provide better care in their area of expertise than generalists . although a study evaluating interpretation of pediatric plain radiographs at a rural hospital did not show a statistically significant difference in interpretation between pediatric radiologists and generalists , the authors note that the subspecialists were more accurate ( 26 ) . in another study ,
the detection of extracapsular extension of prostate cancer on mri was significantly affected by the reader 's subspecialty experience ( 27 ) . several tertiary care centers have looked at the quantitative effect of radiology consultations on management of cancer patients . one center reported the addition of significant new information in 49% of patients reviewed at their division of oncology radiology conference , resulting in major changes in management in 37% ( 28 ) . changes were attributed to lack of subspecialization in head and neck imaging at the outside institution , and to often incomplete clinical information of the original interpreting radiologist . in a similar study ,
fellowship trained neuroradiologists provided formal second opinions on head and neck imaging studies , resulting in changes in stage in 56% and management changes in 38% , noting 93% accuracy based on pathologic staging as gold standard ( 30 ) . a recent study at our cancer center evaluated the influence of a second opinion / subspecialty consultation on the surgical management of breast cancer patients ( 31 ) . they reported changes in 11.7% of patients undergoing surgery for breast cancer . given the variability in quality of outside studies and their interpretations , and the differences that a comprehensive evaluation may make in a patient 's prognosis and treatment strategy , review of outside studies by a specialized oncologic imager is critically important . changes in patient care due to direct subspecialty radiology consultation have recently been reported in disciplines other than oncology . based on the final pathologic diagnosis
, they reported that second opinion consultations were correct in 82.0% of cases where discrepancies were likely to change patient management . dickerson et al . given their success in neuroradiology , they have now initiated this approach throughout their department . at our tertiary care cancer center , imaging plays an integral role in optimizing patient care by guiding decisions regarding diagnosis , staging , tissue sampling , trial eligibility and treatment . since pacs enables remote access to imaging studies , radiologists are commonly called upon to lend expertise in viewing both our own and outside imaging studies . a large study at our own institution over a decade ago looked at the volume and impact of second opinion consultations , noting an overall increase in daily workload of 18% ( 35 ) . based on the strict reimbursement requirements ( which include a written report and documentation of medical necessity ) , as well as the variable quality of outside imaging studies and time needed to thoroughly review and redictate a study , it was deemed not cost effective to issue formal reports on outside consultation cases . however , referring clinicians strongly favored and valued this service , therefore institute administrators deemed it worthwhile to contract with the imaging department to provide consultation services by a radiologist located within the clinic . monetary compensation for this dedicated radiologist allowed the hiring of an additional full time employee , allowing for fewer interruptions to the radiologists interpreting our home institution 's studies , and incidentally increasing productivity overall . although our survey only captured consultations on outside studies or on our studies that required further comparison , the consult radiologist is responsible for reviewing both on - site studies , as well as those from associated institutions , and is available to perform tumor measurements for clinical trial patients . at our institution
, the success of the consultation service and increasing patient volume has resulted in expanded general and subspecialty oncologic imaging consultation services . the consult radiologist provides several services , including ( though not limited to ) clinical decision support , interpretations , advice regarding biopsy or treatment planning , and determination of measurable disease to confirm trial eligibility . given the ease of access and face - to - face interaction , we have noted increased dependence on our interpretation of all studies , including studies from both outside and inside the network , and also our daily services from within the institution . most importantly , however , the interactions between imagers and oncologists , surgeons , radiation oncologists , nurses and physician assistants , have grown more collegial , and in fact have facilitated research opportunities / collaborative efforts . thus , radiologists have become more integrated into the multidisciplinary approach which is so critical in caring for cancer patients ( 36 ) . ( 37 ) describe this integrated consultation service as only a part of their vision for the role of imaging in optimizing cancer care . they support the use of imaging in pre - clinical studies to facilitate translational research , and also note the importance of effective , specialized training programs for future oncologic imagers . a brief evaluation of various treatment decisions in a breast cancer patient 's medical journey elucidates the critical role that radiologists play as consultants to surgeons , radiation oncologists and medical oncologists . after a breast cancer diagnosis
although a patient may opt for a lumpectomy versus mastectomy by personal choice , the radiologist , upon evaluation of the breast imaging studies , will assist the surgeon in determining whether breast conservation is feasible . the breast imager is not infrequently consulted by the radiation oncologist when planning radiation therapy for breast cancer patients . the presence of internal mammary lymphadenopathy may change the radiation field , and therefore the radiologist is sought out to provide an opinion regarding the presence of internal mammary lymph nodes and their exact location on mri and ct , so that radiation treatment can be accurately planned . patients with a diagnosis of inflammatory breast cancer undergo additional staging with pet - ct imaging , which in a small study at our institution altered the radiation treatment planning in 24% ( fig . 1 ) ( 38 ) . knowledge of a patient 's breast tumor pathology will also affect the consult radiologist 's approach to disease evaluation and recommendations for future imaging . in cases of invasive lobular cancer , which are often mammographically occult , sonography or mri may be important in better defining tumor margins . given the knowledge of different patterns of metastatic disease spread with lobular breast cancer ( versus invasive ductal cancer ) , a subspecialized oncologic radiologist is more attentive to subtle peritoneal thickening , bowel wall thickening or adnexal changes , that may herald early disease spread , thus suggesting biopsies or close follow - up ( fig . the radiologist is often consulted by the oncologist to evaluate changes , especially those not readily evaluated with standard response criteria ( such as response evaluation criteria in solid tumors [ recist ] ) . for example , the presence of increased sclerosis in a breast cancer patient with known osseous metastases at a site of previously radiologically occult disease may actually represent response , rather than disease progression as described in the university of texas md anderson cancer center criteria ( mda criteria ) , and must be viewed in light of other changes ( fig . 3 ) ( 39 ) . the combination of imaging expertise , knowledge of disease presentation and patterns of spread , as well as responses to therapeutic options with their potential toxicities and complications make the consultant radiologist vitally important to patient care in the oncologic setting . as newer therapies have been developed , response criteria have changed to address the varied appearances of tumor response , and different patterns of drug toxicity have been noted . therefore , it is critical that radiologists interact personally with oncologists and other members of the oncology care team to be made aware of the newest therapies . this relationship is mutually beneficial , affording imagers the ability to stay abreast of continuously changing therapeutic advances and the opportunity to acquire information about individual patients and their specific radiologic queries , as well as providing referring clinicians with radiologic expertise to help provide the best care to their patients . in the last several years , there has been much focus on the value of imaging in healthcare , with campaigns sponsored by the acr , and multiple publications urging radiologists to take a more active role in reaching out to clinicians and patients as a consultant ( 404142 ) . this report summarizes several different models of consultation services which address various end goals including optimization of interdisciplinary care foremost . at our tertiary center
, a hybrid model has been incorporated to serve the specific needs of our patients and providers , aimed at cultivating these relationships and leveraging the strengths of our cancer imaging practice . in coming years
, it will be important to quantify the added value of radiology consultation , as well as secure buy - in from the stakeholders on a broader scale . | [
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] |
rheumatoid arthritis ( ra ) is a chronic , autoimmune , systemic , inflammatory disease mainly affecting the joints characterized by pain , swelling , and stiffness , resulting in progressive joint destruction , deformity , and loss of function .
it is less prevalent in mainland china and in hong kong , with a reported prevalence of 0.37% and 0.35% , respectively [ 2 , 3 ] .
although the etiology of ra remains largely unknown , both genetic and nongenetic causes have been implicated .
inflammation is initially localized in the synovial lining where there is synovial cell proliferation ( pannus formation ) and infiltration by inflammatory cells .
the pannus invades and destroys cartilage and bone , leading to irreversible joint destruction and deformity .
erosions of the feet joints on extremity magnetic resonance imaging ( mri ) are found in up to 91% of patients early in the course of the disease .
currently , the classification of ra is based on the 1987 american college of rheumatology ( acr ) clinical classification criteria using history , physical examination , and laboratory and radiographic findings ; this set of criteria has been criticized for lack of sensitivity in early disease .
a new set of joint acr - european league against rheumatism ( eular ) criteria for ra classification has recently been published .
the goals of treatment of ra are to alleviate symptoms , slow or stop disease progression , and reduce disability .
the current ra treatment armamentarium comprises both synthetic and biologic disease - modifying anti - rheumatic drugs ( dmards ) . in 2005 , the hong kong society of rheumatology ( hksr ) published consensus recommendations on the use of anti - tumor necrosis factor ( tnf)- agents in the treatment of ra , ankylosing spondylitis , and psoriatic arthritis . however , consensus recommendations on overall ra management had yet to be developed .
given the availability of newer biologic agents for ra treatment , the hksr initiated the development of these consensus recommendations , which are aimed at providing guidance to hong kong physicians on appropriate , literature - based management of this condition , specifically on the indications and monitoring of the novel biologic dmards .
the consensus recommendations were developed using the eular recommendations for the management of early arthritis as a guide , along with local expert opinion . clinical evidence supporting these recommendations were categorized as to level of evidence based on the classification system in table 1 .
table 1strength of clinical evidencecategory aat least one rct or meta - analyses of rcts , or reviews if these contain category a referencescategory bat least one controlled trial without randomization or at least one other type of experimental study , or extrapolated recommendations from rcts or meta - analysescategory cnon - experimental descriptive studies , such as comparative studies , correlational studies , and case - control studies , which are extrapolated from rcts , non - randomized controlled studies , or other experimental studiescategory dexpert committee reports or opinions or clinical experience of respected authorities .
recommendation 1 : general principlesearly ra treatment improves the rate of clinical response and ultimate outcome . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors .
treatment must be individualized , based on physicians ' and patients ' preference , and medical contraindications , among others .
recommendation 1 : general principlesearly ra treatment improves the rate of clinical response and ultimate outcome . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors .
treatment must be individualized , based on physicians ' and patients ' preference , and medical contraindications , among others .
early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors .
treatment must be individualized , based on physicians ' and patients ' preference , and medical contraindications , among others .
evidence from published clinical trials shows better outcomes in early arthritis when treatment is initiated early ; considerably more radiographic damage occurs when therapy is delayed ( category a ) [ 911 ] . in patients with more severe disease ,
it is important to achieve and maintain low disease activity as early as possible to prevent structural damage , thus necessitating a more intense treatment approach .
clinical trial data show that initial intensive treatment produces better outcomes than monotherapy in early arthritis ( category a ) [ 9 , 1214 ] .
however , it is important to bear in mind that , given the variety of available treatments and the heterogeneity of patients , the management strategy should ultimately be tailored to the individual patient , taking into account factors such as individual preferences , contraindications , and side effects .
recommendation 2 : patient assessment
anti - cyclic citrullinated peptide ( anti - ccp ) antibody testing , ultrasound , and mri may be utilized in the early diagnosis of ra .
these tests are not recommended as routine tests but should be used for specific indications only .
recommendation 2 : patient assessment
anti - cyclic citrullinated peptide ( anti - ccp ) antibody testing , ultrasound , and mri may be utilized in the early diagnosis of ra .
these tests are not recommended as routine tests but should be used for specific indications only .
anti - cyclic citrullinated peptide ( anti - ccp ) antibody testing , ultrasound , and mri may be utilized in the early diagnosis of ra .
these tests are not recommended as routine tests but should be used for specific indications only .
anti - ccp testing may help establish an early diagnosis in patients who do not fulfill the 1987 acr criteria , or for atypical cases .
the anti - ccp antibodies have been shown to be more specific than the rheumatoid factor ( rf ) for diagnosing ra and possibly better predict radiographic progression of joint erosion ( category c ) ; anti - ccp testing has been incorporated into the new eular classification criteria to enhance its specificity for early disease .
ultrasound and mri are suitable for early diagnosis of bony erosions in patients who do not fulfill the 1987 acr criteria , or as a guide for joint aspiration and injection in difficult cases .
both these imaging techniques are more sensitive than radiography for detecting cartilage and bone abnormalities and can even identify earlier stages of the disease ( category b ) [ 16 , 17 ] . however ,
despite their benefits in early diagnosis of ra , these imaging techniques and testing for anti - ccp antibodies are significantly more costly and , therefore , should not be used routinely for every patient who presents with joint symptoms .
disease activity score using 28 joint counts ( das28 ) should be utilized in the assessment of ra disease activity .
clinicians should try to compute the das28 at regular intervals , preferably during each clinic visit .
high disease activity ( hda ) : das28 > 5.1moderate disease activity ( mda ) : das28 = 3.25.1low disease activity ( lda ) : das28 = 2.63.2remission : das28 < 2.6 disease activity score using 28 joint counts ( das28 ) should be utilized in the assessment of ra disease activity .
clinicians should try to compute the das28 at regular intervals , preferably during each clinic visit .
high disease activity ( hda ) : das28 > 5.1moderate disease activity ( mda ) : das28 = 3.25.1low disease activity ( lda ) : das28 = 2.63.2remission : das28 < 2.6 high disease activity ( hda ) : das28 > 5.1 moderate disease activity ( mda ) : das28 = 3.25.1 low disease activity ( lda ) : das28 = 2.63.2 remission : das28 < 2.6 the das28 is a useful instrument in clinical practice for objectively evaluating disease progression and response to therapy ( category b ) .
it includes four variables : ( 1 ) number of tender joints from among 28 joints , ( 2 ) number of swollen joints from among 28 joints , ( 3 ) erythrocyte sedimentation rate ( esr ) , and ( 4 ) patient 's assessment of general health status .
c - reactive protein ( crp ) may be used as an alternative to esr in the calculation of the das28 score , using predetermined formulas ( category b ) .
the das28 score ranges from 0 to 9.4 and can be used to readily classify disease activity levels into high , moderate , low , and remission .
factors indicating an unfavorable prognosis of ra include chronic smoking , high titres of anti - ccp or rf , radiologic erosion at onset , positive family history , hda , severe functional limitation , and extra - articular manifestations ( e.g. , rheumatoid nodules ) .
factors indicating an unfavorable prognosis of ra include chronic smoking , high titres of anti - ccp or rf , radiologic erosion at onset , positive family history , hda , severe functional limitation , and extra - articular manifestations ( e.g. , rheumatoid nodules ) .
numerous reports exist of a relationship between smoking and increased severity of ra ( category c ) [ 20 , 21 ] .
the presence of anti - ccp antibodies is a strong predictor of radiological damage and progression ( category c ) .
in addition , hda at baseline , rf , radiologic damage at disease onset , and presence of extra - articular manifestations are also predictors of poor outcome ( category c ) .
the acr 2008 recommendations further confirm these prognostic factors as commonly used clinically important predictors of poor ra outcome .
possible assessment tools include the health assessment questionnaire ( haq ) and the medical outcomes study short - form health survey ( sf-36 ) .
possible assessment tools include the health assessment questionnaire ( haq ) and the medical outcomes study short - form health survey ( sf-36 ) .
since improving disability , function , and health - related quality of life are important goals for patients , regular assessments of these should become an integral part of ra management .
the commonly used assessment tools , the haq and sf-36 , assess the impact of disease in terms of patient - centered outcomes .
the haq , the most commonly used health status measure , primarily determines functional disability , while the sf-36 assesses limitations in physical activities , as well as emotional status of patients .
recommendation 3 : treatment
the goal of treatment is disease remission ( i.e. , das28 < 2.6 ) , regardless of whether the patient has early or established ra . it should be emphasized that treatment of early ra results in better response rates and a higher probability of drug - free remission .
recommendation 3 : treatment
the goal of treatment is disease remission ( i.e. , das28 < 2.6 ) , regardless of whether the patient has early or established ra . it should be emphasized that treatment of early ra results in better response rates and a higher probability of drug - free remission .
the goal of treatment is disease remission ( i.e. , das28 < 2.6 ) , regardless of whether the patient has early or established ra .
it should be emphasized that treatment of early ra results in better response rates and a higher probability of drug - free remission . since there is no cure for ra
, remission has become the accepted treatment goal to arrest joint damage and reduce the likelihood of long - term disability ( category b ) [ 9 , 11 ] .
clinical trials of both early [ 1214 ] and established ra have demonstrated better remission rates and radiographic progression with early intensive treatment than monotherapy or routine care ( category a ) .
treatment with synthetic dmards should be initiated as soon as possible after a diagnosis of ra is made .
methotrexate should be given for a duration of no less than 3 months at the maximally tolerated dose .
patients without poor prognostic factors ( i.e. , with no erosions , are rf - negative , with low crp levels , or with low disease activity ) or those who can not tolerate methotrexate may receive other dmards , such as leflunomide , sulfasalazine , hydroxychloroquine , or injectable gold .
corticosteroids , given orally , intramuscularly , or intra - articularly , may be used as bridging therapy as appropriate . a combination of dmards , or methotrexate combined with an anti - tnf- agent ,
however , the cost - effectiveness of the latter approach has yet to be determined .
treatment with synthetic dmards should be initiated as soon as possible after a diagnosis of ra is made .
methotrexate should be given for a duration of no less than 3 months at the maximally tolerated dose .
patients without poor prognostic factors ( i.e. , with no erosions , are rf - negative , with low crp levels , or with low disease activity ) or those who can not tolerate methotrexate may receive other dmards , such as leflunomide , sulfasalazine , hydroxychloroquine , or injectable gold .
corticosteroids , given orally , intramuscularly , or intra - articularly , may be used as bridging therapy as appropriate . a combination of dmards , or methotrexate combined with an anti - tnf- agent ,
however , the cost - effectiveness of the latter approach has yet to be determined .
several clinical trials have clearly established the short- and long - term efficacy and the relatively beneficial safety profile of methotrexate in ra ( category a ) [ 9 , 29 , 30 ] .
methotrexate is considered the anchor drug , and there is widespread acceptance for its use as initial therapy in most ra patients ( category a ) [ 9 , 31 , 32 ] .
alternative first - line treatments include sulfasalazine , which shows similar efficacy to methotrexate , and hydroxychloroquine , which has very good tolerability among the dmards ( category b ) [ 11 , 26 , 33 ] .
combination treatment with two or more dmards , or of methotrexate and an anti - tnf- agent , has been shown to confer greater efficacy than monotherapy and , thus , is appropriate for patients with serious disease or poor prognostic factors ( category a ) [ 9 , 12 , 13 , 3436 ] .
however , the cost - effectiveness of first - line intervention with biologic agents in treatment - nave patients remains uncertain ( category c ) .
suboptimal treatment response is defined as failure to achieve remission ( i.e. , das28 < 2.6 ) after 3 months of methotrexate at a dose of at least 15 mg / week .
such patients ( i.e. , das28 2.6 despite methotrexate ) should receive step - up therapy , i.e. , combination therapy of methotrexate plus another agent : methotrexate plus leflunomide , methotrexate plus sulfasalazine plus hydroxychloroquine , or methotrexate plus a biologic agent .
suboptimal treatment response is defined as failure to achieve remission ( i.e. , das28 < 2.6 ) after 3 months of methotrexate at a dose of at least 15 mg / week .
such patients ( i.e. , das28 2.6 despite methotrexate ) should receive step - up therapy , i.e. , combination therapy of methotrexate plus another agent : methotrexate plus leflunomide , methotrexate plus sulfasalazine plus hydroxychloroquine , or methotrexate plus a biologic agent . for patients who continue to present with active disease despite methotrexate , addition of another conventional dmard or biologic agent should be considered ( category a ) [ 9 , 11 , 26 ] . in one randomized , placebo - controlled trial ,
the addition of leflunomide to existing methotrexate therapy resulted in significant clinical benefit in patients with persistently active ra ( category a ) ; however , given the recent fda report of fulminant hepatitis in leflunomide users , meticulous monitoring of liver function is warranted in patients administered this drug . in another randomized trial ,
combination therapy with methotrexate , sulfasalazine , and hydroxychloroquine resulted in better remission rates than single treatment in early ra ( category a ) .
similarly , other randomized controlled trials have demonstrated better clinical efficacy when tnf- inhibitors ( etanercept , adalimumab , and infliximab ) were given concomitant with methotrexate , following inadequate response to methotrexate alone ( category a ) [ 4143 ] .
the acr 2008 recommendations suggest a variety of dual and triple drug combinations , incorporating methotrexate , depending on the patient 's level of disease activity , disease duration , and prognostic features ( category b ) .
patients who require methotrexate plus a biologic agent may be administered any one of the following combinations : methotrexate plus an anti - tnf- agent , methotrexate plus tocilizumab , methotrexate plus abatacept , or methotrexate plus rituximab .
the choice of biologic will depend on various factors , including patient 's and physician 's preferences , availability of funding , and medical history .
patients who require methotrexate plus a biologic agent may be administered any one of the following combinations : methotrexate plus an anti - tnf- agent , methotrexate plus tocilizumab , methotrexate plus abatacept , or methotrexate plus rituximab .
the choice of biologic will depend on various factors , including patient 's and physician 's preferences , availability of funding , and medical history .
the value of the combination of methotrexate and anti - tnf- agents in improving radiographic , clinical , and functional outcomes is well established ( category a ) [ 12 , 13 , 36 , 4143 ] . in recent years
, several novel biologic drugs with different mechanisms of action have emerged as potential additions to rheumatologic pharmacotherapy .
these include the interleukin ( il)-6 inhibitor tocilizumab , the selective t - cell co - stimulation modulator abatacept , and the chimeric , anti - cd20 monoclonal antibody rituximab .
several recent randomized , double - blind , placebo - controlled trials have demonstrated superior therapeutic efficacy when each of these agents was co - administered with methotrexate in ra patients [ 4451 ] .
trials involving tocilizumab have shown decreased disease activity and better remission rates ( category a ) [ 4446 ] .
one trial showed tocilizumab monotherapy to be superior to methotrexate monotherapy in dmard / methotrexate - nave patients ( category a ) ; another demonstrated that tocilizumab with methotrexate delayed radiographic progression of structural joint damage significantly more than methotrexate alone in patients who respond inadequately to methotrexate ( category a ) .
a clinical trial using abatacept demonstrated improvements in arthritis signs and symptoms , physical function , and quality of life at 1 year , and inhibition of radiological progression for up to 2 years ( category a ) .
another clinical trial of rituximab showed improvements in disease activity , fatigue , disability , and health - related quality of life ( category a ) .
anti - tnf failure patients ( failure of das28 to improve by 1.2 or < 5.1 over 16 weeks ) may be administered any one of the following : another anti - tnf- agent , tocilizumab , abatacept , or rituximab .
anti - tnf failure patients ( failure of das28 to improve by 1.2 or < 5.1 over 16 weeks ) may be administered any one of the following : another anti - tnf- agent , tocilizumab , abatacept , or rituximab .
there is less evidence to support the efficacy of rituximab in seronegative ra patients . despite their efficacy in ra
, anti - tnf- agents elicit an inadequate response in a substantial number of patients . in such patients , alternative biologic agents
evidence shows that patients who are refractory to anti - tnf- therapy gain significant clinical and functional benefits from tocilizumab , rituximab , or abatacept ( category a ) . however , rituximab may not be equally efficacious in all ra patients .
a recent pooled analysis of two - phase iii rituximab trials has shown that patients who are seropositive ( anti - rf and anti - ccp positive ) are significantly more likely to achieve a clinical response than those who are seronegative ( category a ) .
these findings suggest that the serological status of patients should be taken into consideration when choosing these biologic agents .
recommendation 4 : safety considerationsprior to using a biologic agent , the clinician should screen the patient for both active and latent tuberculosis infection .
patients with active tuberculosis should be adequately treated with the standard regimen before reconsideration of biologic treatment .
patients who screen positive for latent tuberculosis infection ( i.e. , with a positive purified protein derivative ( ppd ) test , defined as an induration of 10 mm in diameter ) should be given isoniazid treatment for 9 months .
if the indication for biologic use is not urgent , isoniazid should be given for 4 weeks , to assess tolerability , before administration of the biologic agent .
for patients who test ppd - negative , there is insufficient evidence to recommend annual retesting unless there is recent exposure to tuberculosis or the risk of transmission is high .
recommendation 4 : safety considerations prior to using a biologic agent , the clinician should screen the patient for both active and latent tuberculosis infection .
patients with active tuberculosis should be adequately treated with the standard regimen before reconsideration of biologic treatment .
patients who screen positive for latent tuberculosis infection ( i.e. , with a positive purified protein derivative ( ppd ) test , defined as an induration of 10 mm in diameter ) should be given isoniazid treatment for 9 months .
if the indication for biologic use is not urgent , isoniazid should be given for 4 weeks , to assess tolerability , before administration of the biologic agent .
for patients who test ppd - negative , there is insufficient evidence to recommend annual retesting unless there is recent exposure to tuberculosis or the risk of transmission is high .
numerous reports indicate an increased risk of developing active tuberculosis soon after initiating anti - tnf- agents ( category a ) . furthermore , in patients with a past history of tuberculosis , abatacept treatment may reactivate the infection as it is known to compromise t - cell immunity .
this has led to the recommendation for routine screening for active and latent tuberculosis infection whenever treatment with these biologic agents is considered ( category d ) [ 54 , 55 ] . while an induration of 10 mm in diameter has been determined to be appropriate to signify a positive ppd test for patients with ra , a lower cut - off of 5 mm has been utilized to define latent tuberculosis in patients who are significantly immunocompromised , such as those with human immunodeficiency virus ( hiv ) infection or who are transplant recipients ( category d ) .
patients with evidence of latent tuberculosis infection should receive prophylactic tuberculosis treatment with isoniazid for 9 months before commencing biologic agents ( category b ) [ 54 , 55 ] .
rifampicin as monotherapy for 4 months is recommended for patients resistant to , or intolerant of , isoniazid , or for whom isoniazid is contraindicated ( category d ) .
chronic carriage of hepatitis b and c virus ( hbv and hcv , respectively ) is a relative contraindication for the use of anti - tnf- agents .
active hepatitis has to be excluded and baseline hbv dna or hcv rna levels should be checked for chronic carriers of hbv and hcv .
appropriate antiviral therapy , as determined by the co - managing hepatologist , is indicated .
there is little information regarding the safety of tocilizumab and abatacept in chronic viral hepatitis carriers .
chronic carriage of hepatitis b and c virus ( hbv and hcv , respectively ) is a relative contraindication for the use of anti - tnf- agents .
active hepatitis has to be excluded and baseline hbv dna or hcv rna levels should be checked for chronic carriers of hbv and hcv .
appropriate antiviral therapy , as determined by the co - managing hepatologist , is indicated .
there is little information regarding the safety of tocilizumab and abatacept in chronic viral hepatitis carriers .
another important safety consideration when making therapeutic decisions with biologics is hepatitis b and c status .
the anti - tnf- agents are relatively contraindicated in carriers of hbv and hcv because of reports of viral reactivation , which can lead to serious complications ( category b ) [ 26 , 56 , 57 ] .
if there are no alternatives , patients should be warned of the risk of serious hepatitis reactivation prior to administration of an anti - tnf- agent . in clinical trials of hemic malignancies
, rituximab has been shown to reactivate occult hepatitis b ( i.e. , hbsag - negative but anti - hbc - igg - positive ) [ 58 , 59 ] ; it is , therefore , contraindicated in chronic hepatitis b carriers and in patients with past hepatitis b infection .
hence , all candidate patients for these drugs should be screened for hbv and hcv infections before commencing treatment .
in particular , the status of both hbsag and anti - hbc - igg should be determined before rituximab treatment .
there is insufficient information on the safety of tocilizumab and abatacept in chronic viral hepatitis carriers .
history of solid tumor is not a contraindication for use of anti - tnf- agents if there is no recurrence in the past 5 years .
anti - tnf- agents should be avoided in patients with a history of lymphoproliferative disorders .
history of solid tumor is not a contraindication for use of anti - tnf- agents if there is no recurrence in the past 5 years .
anti - tnf- agents should be avoided in patients with a history of lymphoproliferative disorders .
although etanercept has been associated with an increased incidence of solid malignancies in one clinical trial ( category a ) , anti - tnf- therapy is generally not associated with an increased occurrence of solid tumors . according to a swedish population - based study ,
the relative risks of solid cancers in anti - tnf- treated patients were similar to those of other ra patients ( category c ) .
hence , anti - tnf- agents are not contraindicated in patients with solid tumor history if there is no recurrence in the past 5 years .
however , they should be avoided in patients with prior lymphoproliferative disorders because of significantly increased risk of lymphomas ( category c ) [ 26 , 62 ] .
use of anti - tnf- agents is contraindicated in patients with severe heart failure , demyelinating disorders , or lupus - like features .
use of anti - tnf- agents is contraindicated in patients with severe heart failure , demyelinating disorders , or lupus - like features .
treatment of moderate - to - severe heart failure patients with anti - tnf- agents has been associated with either no clinical benefit or worsening of heart failure outcomes ( category a ) .
furthermore , these agents are known to induce autoimmune diseases such as demyelinating disorders and lupus and , as such , should be avoided in patients with these underlying conditions ( category b ) [ 26 , 65 ] .
killed vaccines ( e.g. , influenza vaccine , pneumococcal vaccine ) , if required , should be given prior to administration of biologic agent .
killed vaccines ( e.g. , influenza vaccine , pneumococcal vaccine ) , if required , should be given prior to administration of biologic agent . because the level of immune response to vaccinations may be impaired , live vaccines are contraindicated during biologic therapy ( category d ) .
alternatively , inactivated vaccines can be safely administered , typically at least 2 to 4 weeks , before initiating biologic therapy .
patients should be very closely monitored for infective complications , including opportunistic infections.patients should be monitored for symptoms of tuberculosis infection , both within and outside the respiratory system .
chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms.complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked.fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate.look out for new neurological symptoms in patients administered rituximab.look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration.adverse events , especially serious adverse events , should be reported to the hksr biologics registry .
patients should be very closely monitored for infective complications , including opportunistic infections.patients should be monitored for symptoms of tuberculosis infection , both within and outside the respiratory system .
chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms.complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked.fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate.look out for new neurological symptoms in patients administered rituximab.look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration.adverse events , especially serious adverse events , should be reported to the hksr biologics registry .
patients should be monitored for symptoms of tuberculosis infection , both within and outside the respiratory system .
chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms .
complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked .
fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate .
look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration .
adverse events , especially serious adverse events , should be reported to the hksr biologics registry .
registries and databases have reported an increased risk of serious bacterial infections with the use of biologic dmards ( category a ) [ 6668 ] .
in particular , the rates of pneumonia and serious skin and soft tissue infections are increased .
once biologic therapy is initiated , patients should , therefore , undergo regular safety assessments .
laboratory testing , including lymphocyte count for rituximab and neutrophil count for tocilizumab , are warranted as treatment with each of these agents is associated with rapid depletion of peripheral b - cells and neutropenia [ 4446 , 69 ] , respectively ( category a ) .
treatment with biologic agents , particularly tocilizumab , can alter the serum lipid profile , resulting in increased low density lipoprotein ( ldl ) , and total cholesterol levels ( category b ) . hence , it is important to monitor lipid levels and administer statin therapy , when required .
tocilizumab administration has been associated with bowel perforation and subsequent peritonitis and , therefore , should be closely monitored in patients with a history of diverticulitis or intestinal ulceration ( category b ) .
recommendation 5 : cardiovascular risk factors and bone mineral densitypatients with ra should be screened for risk factors for cardiovascular ( cv ) disease and for osteoporosis .
recommendation 5 : cardiovascular risk factors and bone mineral densitypatients with ra should be screened for risk factors for cardiovascular ( cv ) disease and for osteoporosis .
patients with ra should be screened for risk factors for cardiovascular ( cv ) disease and for osteoporosis .
ra patients are more prone to cv disease : the standardized incidence ratio of myocardial infarction and stroke is 1.5 to 1.7 times that of the incidence in the general population ( category a ) [ 7174 ] .
this is attributable to an increased prevalence of traditional cv risk factors and persistent elevation of inflammatory cytokines in this population .
early identification , adequate management , and ongoing monitoring of risk factors are necessary to reduce this excess cv risk . the latest eular recommendations for cv risk management in patients with ra and other forms of inflammatory arthritis states that the first principle of management is to assess and control all components of total cv risk , which includes providing appropriate , evidenced - based advice with regard to smoking , physical activity , diet , weight , and blood pressure , as well as aggressive suppression of the inflammatory process to further lower risk ( category d ) .
similarly , ra patients are more likely to develop osteoporosis and fragility fractures ( category a ) .
recommendation 1 : general principlesearly ra treatment improves the rate of clinical response and ultimate outcome . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors .
treatment must be individualized , based on physicians ' and patients ' preference , and medical contraindications , among others .
recommendation 1 : general principlesearly ra treatment improves the rate of clinical response and ultimate outcome . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors .
treatment must be individualized , based on physicians ' and patients ' preference , and medical contraindications , among others .
early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors .
treatment must be individualized , based on physicians ' and patients ' preference , and medical contraindications , among others .
evidence from published clinical trials shows better outcomes in early arthritis when treatment is initiated early ; considerably more radiographic damage occurs when therapy is delayed ( category a ) [ 911 ] . in patients with more severe disease ,
it is important to achieve and maintain low disease activity as early as possible to prevent structural damage , thus necessitating a more intense treatment approach .
clinical trial data show that initial intensive treatment produces better outcomes than monotherapy in early arthritis ( category a ) [ 9 , 1214 ] .
however , it is important to bear in mind that , given the variety of available treatments and the heterogeneity of patients , the management strategy should ultimately be tailored to the individual patient , taking into account factors such as individual preferences , contraindications , and side effects .
recommendation 2 : patient assessment
anti - cyclic citrullinated peptide ( anti - ccp ) antibody testing , ultrasound , and mri may be utilized in the early diagnosis of ra .
these tests are not recommended as routine tests but should be used for specific indications only .
recommendation 2 : patient assessment
anti - cyclic citrullinated peptide ( anti - ccp ) antibody testing , ultrasound , and mri may be utilized in the early diagnosis of ra .
these tests are not recommended as routine tests but should be used for specific indications only .
anti - cyclic citrullinated peptide ( anti - ccp ) antibody testing , ultrasound , and mri may be utilized in the early diagnosis of ra .
these tests are not recommended as routine tests but should be used for specific indications only .
anti - ccp testing may help establish an early diagnosis in patients who do not fulfill the 1987 acr criteria , or for atypical cases .
the anti - ccp antibodies have been shown to be more specific than the rheumatoid factor ( rf ) for diagnosing ra and possibly better predict radiographic progression of joint erosion ( category c ) ; anti - ccp testing has been incorporated into the new eular classification criteria to enhance its specificity for early disease .
ultrasound and mri are suitable for early diagnosis of bony erosions in patients who do not fulfill the 1987 acr criteria , or as a guide for joint aspiration and injection in difficult cases .
both these imaging techniques are more sensitive than radiography for detecting cartilage and bone abnormalities and can even identify earlier stages of the disease ( category b ) [ 16 , 17 ] . however ,
despite their benefits in early diagnosis of ra , these imaging techniques and testing for anti - ccp antibodies are significantly more costly and , therefore , should not be used routinely for every patient who presents with joint symptoms .
disease activity score using 28 joint counts ( das28 ) should be utilized in the assessment of ra disease activity .
clinicians should try to compute the das28 at regular intervals , preferably during each clinic visit .
high disease activity ( hda ) : das28 > 5.1moderate disease activity ( mda ) : das28 = 3.25.1low disease activity ( lda ) : das28 = 2.63.2remission : das28 < 2.6 disease activity score using 28 joint counts ( das28 ) should be utilized in the assessment of ra disease activity .
clinicians should try to compute the das28 at regular intervals , preferably during each clinic visit .
high disease activity ( hda ) : das28 > 5.1moderate disease activity ( mda ) : das28 = 3.25.1low disease activity ( lda ) : das28 = 2.63.2remission : das28 < 2.6 high disease activity ( hda ) : das28 > 5.1 moderate disease activity ( mda ) : das28 = 3.25.1 low disease activity ( lda ) : das28 = 2.63.2 remission : das28 < 2.6 the das28 is a useful instrument in clinical practice for objectively evaluating disease progression and response to therapy ( category b ) .
it includes four variables : ( 1 ) number of tender joints from among 28 joints , ( 2 ) number of swollen joints from among 28 joints , ( 3 ) erythrocyte sedimentation rate ( esr ) , and ( 4 ) patient 's assessment of general health status .
c - reactive protein ( crp ) may be used as an alternative to esr in the calculation of the das28 score , using predetermined formulas ( category b ) .
the das28 score ranges from 0 to 9.4 and can be used to readily classify disease activity levels into high , moderate , low , and remission .
factors indicating an unfavorable prognosis of ra include chronic smoking , high titres of anti - ccp or rf , radiologic erosion at onset , positive family history , hda , severe functional limitation , and extra - articular manifestations ( e.g. , rheumatoid nodules ) .
factors indicating an unfavorable prognosis of ra include chronic smoking , high titres of anti - ccp or rf , radiologic erosion at onset , positive family history , hda , severe functional limitation , and extra - articular manifestations ( e.g. , rheumatoid nodules ) .
numerous reports exist of a relationship between smoking and increased severity of ra ( category c ) [ 20 , 21 ] .
the presence of anti - ccp antibodies is a strong predictor of radiological damage and progression ( category c ) .
in addition , hda at baseline , rf , radiologic damage at disease onset , and presence of extra - articular manifestations are also predictors of poor outcome ( category c ) .
the acr 2008 recommendations further confirm these prognostic factors as commonly used clinically important predictors of poor ra outcome .
possible assessment tools include the health assessment questionnaire ( haq ) and the medical outcomes study short - form health survey ( sf-36 ) .
possible assessment tools include the health assessment questionnaire ( haq ) and the medical outcomes study short - form health survey ( sf-36 ) .
since improving disability , function , and health - related quality of life are important goals for patients , regular assessments of these should become an integral part of ra management .
the commonly used assessment tools , the haq and sf-36 , assess the impact of disease in terms of patient - centered outcomes .
the haq , the most commonly used health status measure , primarily determines functional disability , while the sf-36 assesses limitations in physical activities , as well as emotional status of patients .
recommendation 3 : treatment
the goal of treatment is disease remission ( i.e. , das28 < 2.6 ) , regardless of whether the patient has early or established ra . it should be emphasized that treatment of early ra results in better response rates and a higher probability of drug - free remission .
recommendation 3 : treatment
the goal of treatment is disease remission ( i.e. , das28 < 2.6 ) , regardless of whether the patient has early or established ra . it should be emphasized that treatment of early ra results in better response rates and a higher probability of drug - free remission .
the goal of treatment is disease remission ( i.e. , das28 < 2.6 ) , regardless of whether the patient has early or established ra .
it should be emphasized that treatment of early ra results in better response rates and a higher probability of drug - free remission . since there is no cure for ra
, remission has become the accepted treatment goal to arrest joint damage and reduce the likelihood of long - term disability ( category b ) [ 9 , 11 ] .
clinical trials of both early [ 1214 ] and established ra have demonstrated better remission rates and radiographic progression with early intensive treatment than monotherapy or routine care ( category a ) .
treatment with synthetic dmards should be initiated as soon as possible after a diagnosis of ra is made .
methotrexate should be given for a duration of no less than 3 months at the maximally tolerated dose .
patients without poor prognostic factors ( i.e. , with no erosions , are rf - negative , with low crp levels , or with low disease activity ) or those who can not tolerate methotrexate may receive other dmards , such as leflunomide , sulfasalazine , hydroxychloroquine , or injectable gold .
corticosteroids , given orally , intramuscularly , or intra - articularly , may be used as bridging therapy as appropriate . a combination of dmards , or methotrexate combined with an anti - tnf- agent ,
however , the cost - effectiveness of the latter approach has yet to be determined .
treatment with synthetic dmards should be initiated as soon as possible after a diagnosis of ra is made .
methotrexate should be given for a duration of no less than 3 months at the maximally tolerated dose .
patients without poor prognostic factors ( i.e. , with no erosions , are rf - negative , with low crp levels , or with low disease activity ) or those who can not tolerate methotrexate may receive other dmards , such as leflunomide , sulfasalazine , hydroxychloroquine , or injectable gold .
corticosteroids , given orally , intramuscularly , or intra - articularly , may be used as bridging therapy as appropriate . a combination of dmards , or methotrexate combined with an anti - tnf- agent ,
however , the cost - effectiveness of the latter approach has yet to be determined .
several clinical trials have clearly established the short- and long - term efficacy and the relatively beneficial safety profile of methotrexate in ra ( category a ) [ 9 , 29 , 30 ] .
methotrexate is considered the anchor drug , and there is widespread acceptance for its use as initial therapy in most ra patients ( category a ) [ 9 , 31 , 32 ] .
alternative first - line treatments include sulfasalazine , which shows similar efficacy to methotrexate , and hydroxychloroquine , which has very good tolerability among the dmards ( category b ) [ 11 , 26 , 33 ] .
combination treatment with two or more dmards , or of methotrexate and an anti - tnf- agent , has been shown to confer greater efficacy than monotherapy and , thus , is appropriate for patients with serious disease or poor prognostic factors ( category a ) [ 9 , 12 , 13 , 3436 ] .
however , the cost - effectiveness of first - line intervention with biologic agents in treatment - nave patients remains uncertain ( category c ) .
suboptimal treatment response is defined as failure to achieve remission ( i.e. , das28 < 2.6 ) after 3 months of methotrexate at a dose of at least 15 mg / week .
such patients ( i.e. , das28 2.6 despite methotrexate ) should receive step - up therapy , i.e. , combination therapy of methotrexate plus another agent : methotrexate plus leflunomide , methotrexate plus sulfasalazine plus hydroxychloroquine , or methotrexate plus a biologic agent .
suboptimal treatment response is defined as failure to achieve remission ( i.e. , das28 < 2.6 ) after 3 months of methotrexate at a dose of at least 15 mg / week .
such patients ( i.e. , das28 2.6 despite methotrexate ) should receive step - up therapy , i.e. , combination therapy of methotrexate plus another agent : methotrexate plus leflunomide , methotrexate plus sulfasalazine plus hydroxychloroquine , or methotrexate plus a biologic agent . for patients who continue to present with active disease despite methotrexate , addition of another conventional dmard or biologic agent should be considered ( category a ) [ 9 , 11 , 26 ] . in one randomized , placebo - controlled trial ,
the addition of leflunomide to existing methotrexate therapy resulted in significant clinical benefit in patients with persistently active ra ( category a ) ; however , given the recent fda report of fulminant hepatitis in leflunomide users , meticulous monitoring of liver function is warranted in patients administered this drug . in another randomized trial ,
combination therapy with methotrexate , sulfasalazine , and hydroxychloroquine resulted in better remission rates than single treatment in early ra ( category a ) .
similarly , other randomized controlled trials have demonstrated better clinical efficacy when tnf- inhibitors ( etanercept , adalimumab , and infliximab ) were given concomitant with methotrexate , following inadequate response to methotrexate alone ( category a ) [ 4143 ] .
the acr 2008 recommendations suggest a variety of dual and triple drug combinations , incorporating methotrexate , depending on the patient 's level of disease activity , disease duration , and prognostic features ( category b ) .
patients who require methotrexate plus a biologic agent may be administered any one of the following combinations : methotrexate plus an anti - tnf- agent , methotrexate plus tocilizumab , methotrexate plus abatacept , or methotrexate plus rituximab .
the choice of biologic will depend on various factors , including patient 's and physician 's preferences , availability of funding , and medical history .
patients who require methotrexate plus a biologic agent may be administered any one of the following combinations : methotrexate plus an anti - tnf- agent , methotrexate plus tocilizumab , methotrexate plus abatacept , or methotrexate plus rituximab .
the choice of biologic will depend on various factors , including patient 's and physician 's preferences , availability of funding , and medical history .
the value of the combination of methotrexate and anti - tnf- agents in improving radiographic , clinical , and functional outcomes is well established ( category a ) [ 12 , 13 , 36 , 4143 ] . in recent years
, several novel biologic drugs with different mechanisms of action have emerged as potential additions to rheumatologic pharmacotherapy .
these include the interleukin ( il)-6 inhibitor tocilizumab , the selective t - cell co - stimulation modulator abatacept , and the chimeric , anti - cd20 monoclonal antibody rituximab .
several recent randomized , double - blind , placebo - controlled trials have demonstrated superior therapeutic efficacy when each of these agents was co - administered with methotrexate in ra patients [ 4451 ] .
trials involving tocilizumab have shown decreased disease activity and better remission rates ( category a ) [ 4446 ] .
one trial showed tocilizumab monotherapy to be superior to methotrexate monotherapy in dmard / methotrexate - nave patients ( category a ) ; another demonstrated that tocilizumab with methotrexate delayed radiographic progression of structural joint damage significantly more than methotrexate alone in patients who respond inadequately to methotrexate ( category a ) .
a clinical trial using abatacept demonstrated improvements in arthritis signs and symptoms , physical function , and quality of life at 1 year , and inhibition of radiological progression for up to 2 years ( category a ) .
another clinical trial of rituximab showed improvements in disease activity , fatigue , disability , and health - related quality of life ( category a ) .
anti - tnf failure patients ( failure of das28 to improve by 1.2 or < 5.1 over 16 weeks ) may be administered any one of the following : another anti - tnf- agent , tocilizumab , abatacept , or rituximab .
anti - tnf failure patients ( failure of das28 to improve by 1.2 or < 5.1 over 16 weeks ) may be administered any one of the following : another anti - tnf- agent , tocilizumab , abatacept , or rituximab .
there is less evidence to support the efficacy of rituximab in seronegative ra patients . despite their efficacy in ra
, anti - tnf- agents elicit an inadequate response in a substantial number of patients . in such patients , alternative biologic agents
evidence shows that patients who are refractory to anti - tnf- therapy gain significant clinical and functional benefits from tocilizumab , rituximab , or abatacept ( category a ) . however , rituximab may not be equally efficacious in all ra patients .
a recent pooled analysis of two - phase iii rituximab trials has shown that patients who are seropositive ( anti - rf and anti - ccp positive ) are significantly more likely to achieve a clinical response than those who are seronegative ( category a ) .
these findings suggest that the serological status of patients should be taken into consideration when choosing these biologic agents .
recommendation 4 : safety considerationsprior to using a biologic agent , the clinician should screen the patient for both active and latent tuberculosis infection .
patients with active tuberculosis should be adequately treated with the standard regimen before reconsideration of biologic treatment .
patients who screen positive for latent tuberculosis infection ( i.e. , with a positive purified protein derivative ( ppd ) test , defined as an induration of 10 mm in diameter ) should be given isoniazid treatment for 9 months .
if the indication for biologic use is not urgent , isoniazid should be given for 4 weeks , to assess tolerability , before administration of the biologic agent .
for patients who test ppd - negative , there is insufficient evidence to recommend annual retesting unless there is recent exposure to tuberculosis or the risk of transmission is high .
recommendation 4 : safety considerations prior to using a biologic agent , the clinician should screen the patient for both active and latent tuberculosis infection .
patients with active tuberculosis should be adequately treated with the standard regimen before reconsideration of biologic treatment .
patients who screen positive for latent tuberculosis infection ( i.e. , with a positive purified protein derivative ( ppd ) test , defined as an induration of 10 mm in diameter ) should be given isoniazid treatment for 9 months .
if the indication for biologic use is not urgent , isoniazid should be given for 4 weeks , to assess tolerability , before administration of the biologic agent .
for patients who test ppd - negative , there is insufficient evidence to recommend annual retesting unless there is recent exposure to tuberculosis or the risk of transmission is high .
numerous reports indicate an increased risk of developing active tuberculosis soon after initiating anti - tnf- agents ( category a ) . furthermore , in patients with a past history of tuberculosis , abatacept treatment may reactivate the infection as it is known to compromise t - cell immunity .
this has led to the recommendation for routine screening for active and latent tuberculosis infection whenever treatment with these biologic agents is considered ( category d ) [ 54 , 55 ] . while an induration of 10 mm in diameter has been determined to be appropriate to signify a positive ppd test for patients with ra , a lower cut - off of 5 mm has been utilized to define latent tuberculosis in patients who are significantly immunocompromised , such as those with human immunodeficiency virus ( hiv ) infection or who are transplant recipients ( category d ) .
patients with evidence of latent tuberculosis infection should receive prophylactic tuberculosis treatment with isoniazid for 9 months before commencing biologic agents ( category b ) [ 54 , 55 ] .
rifampicin as monotherapy for 4 months is recommended for patients resistant to , or intolerant of , isoniazid , or for whom isoniazid is contraindicated ( category d ) .
chronic carriage of hepatitis b and c virus ( hbv and hcv , respectively ) is a relative contraindication for the use of anti - tnf- agents .
active hepatitis has to be excluded and baseline hbv dna or hcv rna levels should be checked for chronic carriers of hbv and hcv .
appropriate antiviral therapy , as determined by the co - managing hepatologist , is indicated .
there is little information regarding the safety of tocilizumab and abatacept in chronic viral hepatitis carriers .
chronic carriage of hepatitis b and c virus ( hbv and hcv , respectively ) is a relative contraindication for the use of anti - tnf- agents .
active hepatitis has to be excluded and baseline hbv dna or hcv rna levels should be checked for chronic carriers of hbv and hcv .
appropriate antiviral therapy , as determined by the co - managing hepatologist , is indicated .
there is little information regarding the safety of tocilizumab and abatacept in chronic viral hepatitis carriers .
another important safety consideration when making therapeutic decisions with biologics is hepatitis b and c status .
the anti - tnf- agents are relatively contraindicated in carriers of hbv and hcv because of reports of viral reactivation , which can lead to serious complications ( category b ) [ 26 , 56 , 57 ] .
if there are no alternatives , patients should be warned of the risk of serious hepatitis reactivation prior to administration of an anti - tnf- agent . in clinical trials of hemic malignancies
, rituximab has been shown to reactivate occult hepatitis b ( i.e. , hbsag - negative but anti - hbc - igg - positive ) [ 58 , 59 ] ; it is , therefore , contraindicated in chronic hepatitis b carriers and in patients with past hepatitis b infection .
hence , all candidate patients for these drugs should be screened for hbv and hcv infections before commencing treatment .
in particular , the status of both hbsag and anti - hbc - igg should be determined before rituximab treatment .
there is insufficient information on the safety of tocilizumab and abatacept in chronic viral hepatitis carriers .
history of solid tumor is not a contraindication for use of anti - tnf- agents if there is no recurrence in the past 5 years .
anti - tnf- agents should be avoided in patients with a history of lymphoproliferative disorders .
history of solid tumor is not a contraindication for use of anti - tnf- agents if there is no recurrence in the past 5 years .
anti - tnf- agents should be avoided in patients with a history of lymphoproliferative disorders .
although etanercept has been associated with an increased incidence of solid malignancies in one clinical trial ( category a ) , anti - tnf- therapy is generally not associated with an increased occurrence of solid tumors . according to a swedish population - based study ,
the relative risks of solid cancers in anti - tnf- treated patients were similar to those of other ra patients ( category c ) .
hence , anti - tnf- agents are not contraindicated in patients with solid tumor history if there is no recurrence in the past 5 years .
however , they should be avoided in patients with prior lymphoproliferative disorders because of significantly increased risk of lymphomas ( category c ) [ 26 , 62 ] .
use of anti - tnf- agents is contraindicated in patients with severe heart failure , demyelinating disorders , or lupus - like features .
use of anti - tnf- agents is contraindicated in patients with severe heart failure , demyelinating disorders , or lupus - like features .
treatment of moderate - to - severe heart failure patients with anti - tnf- agents has been associated with either no clinical benefit or worsening of heart failure outcomes ( category a ) .
furthermore , these agents are known to induce autoimmune diseases such as demyelinating disorders and lupus and , as such , should be avoided in patients with these underlying conditions ( category b ) [ 26 , 65 ] .
killed vaccines ( e.g. , influenza vaccine , pneumococcal vaccine ) , if required , should be given prior to administration of biologic agent .
killed vaccines ( e.g. , influenza vaccine , pneumococcal vaccine ) , if required , should be given prior to administration of biologic agent . because the level of immune response to vaccinations may be impaired , live vaccines are contraindicated during biologic therapy ( category d ) .
alternatively , inactivated vaccines can be safely administered , typically at least 2 to 4 weeks , before initiating biologic therapy .
patients should be very closely monitored for infective complications , including opportunistic infections.patients should be monitored for symptoms of tuberculosis infection , both within and outside the respiratory system .
chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms.complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked.fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate.look out for new neurological symptoms in patients administered rituximab.look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration.adverse events , especially serious adverse events , should be reported to the hksr biologics registry .
patients should be very closely monitored for infective complications , including opportunistic infections.patients should be monitored for symptoms of tuberculosis infection , both within and outside the respiratory system .
chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms.complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked.fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate.look out for new neurological symptoms in patients administered rituximab.look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration.adverse events , especially serious adverse events , should be reported to the hksr biologics registry .
patients should be monitored for symptoms of tuberculosis infection , both within and outside the respiratory system .
chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms .
complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked .
fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate .
look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration .
adverse events , especially serious adverse events , should be reported to the hksr biologics registry .
registries and databases have reported an increased risk of serious bacterial infections with the use of biologic dmards ( category a ) [ 6668 ] .
in particular , the rates of pneumonia and serious skin and soft tissue infections are increased .
once biologic therapy is initiated , patients should , therefore , undergo regular safety assessments .
laboratory testing , including lymphocyte count for rituximab and neutrophil count for tocilizumab , are warranted as treatment with each of these agents is associated with rapid depletion of peripheral b - cells and neutropenia [ 4446 , 69 ] , respectively ( category a ) .
treatment with biologic agents , particularly tocilizumab , can alter the serum lipid profile , resulting in increased low density lipoprotein ( ldl ) , and total cholesterol levels ( category b ) . hence , it is important to monitor lipid levels and administer statin therapy , when required .
tocilizumab administration has been associated with bowel perforation and subsequent peritonitis and , therefore , should be closely monitored in patients with a history of diverticulitis or intestinal ulceration ( category b ) .
recommendation 5 : cardiovascular risk factors and bone mineral densitypatients with ra should be screened for risk factors for cardiovascular ( cv ) disease and for osteoporosis .
recommendation 5 : cardiovascular risk factors and bone mineral densitypatients with ra should be screened for risk factors for cardiovascular ( cv ) disease and for osteoporosis .
patients with ra should be screened for risk factors for cardiovascular ( cv ) disease and for osteoporosis .
ra patients are more prone to cv disease : the standardized incidence ratio of myocardial infarction and stroke is 1.5 to 1.7 times that of the incidence in the general population ( category a ) [ 7174 ] .
this is attributable to an increased prevalence of traditional cv risk factors and persistent elevation of inflammatory cytokines in this population .
early identification , adequate management , and ongoing monitoring of risk factors are necessary to reduce this excess cv risk . the latest eular recommendations for cv risk management in patients with ra and other forms of inflammatory arthritis states that the first principle of management is to assess and control all components of total cv risk , which includes providing appropriate , evidenced - based advice with regard to smoking , physical activity , diet , weight , and blood pressure , as well as aggressive suppression of the inflammatory process to further lower risk ( category d ) .
similarly , ra patients are more likely to develop osteoporosis and fragility fractures ( category a ) .
these recommendations by the hksr summarize the latest evidence pertaining to ra management ( table 2 ) , with a focus on the use of novel , biologic dmards . while methotrexate is still held up as the primary therapeutic option for most ra patients , new biologic agents currently available are recognized as effective and safe treatment modalities , particularly in patients with a suboptimal response to traditional dmards . given that most of these biologics have only very recently come into use , and that they been associated with the development of infective and other complications
, these recommendations emphasize the importance of vigilant monitoring for side effects , notably serious bacterial and viral infections , in patients administered these agents .
table 2panel recommendations on ra management summaryrecommendation 1 : general principles early ra treatment improves the outcome .
early , aggressive therapy is indicated for patients with serious disease and/or poor prognostic factorsrecommendation 2 : patient assessment ( a ) anti - ccp antibody testing , ultrasound and mri may be utilized to aid early diagnosis of ra but they are not recommended for routine use ( b ) das 28 should be utilized in the assessment of ra disease activity .
clinicians should try to compute the das28 at regular intervals ( c ) factors indicating an unfavorable prognosis of ra include chronic smoking , high titres of anti - ccp or rf , radiologic erosion at onset , positive family history , hda , severe functional limitation , and extra - articular manifestations ( e.g. , rheumatoid nodules ) ( d ) clinicians should regularly assess the extent of their patients ' disability and functional capacityrecommendation 3 : treatment ( a ) the goal of treatment is disease remission ( i.e. , das28 < 2.6 ) ( b ) treatment with synthetic dmards should be initiated as soon as possible after a diagnosis of ra is made .
a combination of dmards , or mtx combined with an anti - tnf- agent , may be considered in patients with very serious disease and poor prognostic factors ( c ) suboptimal treatment response is defined as failure to achieve remission after 3 months of mtx at its maximally tolerated dose .
such patients should receive step - up therapy , i.e. , combination therapy of mtx plus another agent ( e.g. , lef , ssz / hcq , biologic agent ) ( d ) patients who require mtx plus a biologic agent may be administered any one of the following combinations : mtx plus an anti - tnf- agent , tocilizumab , abatacept , or rituximab ( e ) anti - tnf therapy failure patients may be administered another anti - tnf- agent , tocilizumab , abatacept or rituximabrecommendation 4 : safety considerations ( a ) prior to using a biologic agent , patients should be screened for tuberculosis infection .
patients who screen positive for latent tuberculosis infection receive isoniazid treatment for 9 months
active hepatitis has to be excluded and baseline hbv dna or hcv rna levels should be checked for chronic carriers .
rituximab is contraindicated in chronic hepatitis b or c carriers ( c ) patients should be regularly monitored for side effects .
investigations such as chest radiograph , complete blood counts , lymphocyte count , liver and renal function tests , and lipid level should be assessed at regular intervalsrecommendation 5 : cardiovascular risk factors and bone mineral density patients with ra should be screened for risk factors for cv disease and for osteoporosis . once detected , these conditions should be managed as appropriate panel recommendations on ra management summary these recommendations will be reviewed periodically , in light of new published evidence and adverse event reports . as these recommendations evolve and gradually become more explicit , they will provide even more specific guidance to rheumatologists and other physicians who treat patients with ra , ultimately leading to improved clinical outcomes . | given the recent availability of novel biologic agents for the treatment of rheumatoid arthritis ( ra ) , the hong kong society of rheumatology has developed consensus recommendations on the management of ra , which aim at providing guidance to local physicians on appropriate , literature - based management of this condition , specifically on the indications and monitoring of the biologic disease - modifying anti - rheumatic drugs ( dmards ) .
the recommendations were developed using the european league against rheumatism ( eular ) recommendations for the management of early arthritis as a guide , along with local expert opinion .
as significant joint damage occurs early in the course of ra , initiating therapy early is key to minimizing further damage and disability .
patients with serious disease or poor prognosis should receive early , aggressive therapy . because of its good efficacy and safety profile , methotrexate is considered the standard first - line dmard for most treatment - nave ra patients .
patients with a suboptimal response to methotrexate monotherapy should receive step - up ( combination ) therapy with either the synthetic or biologic dmards . in recent years
, combinations of methotrexate with tocilizumab , abatacept , or rituximab have emerged as effective therapies in patients who are unresponsive to traditional dmards or the anti - tumor necrosis factor ( tnf)- agents . as biologic agents can increase the risk of infections such as tuberculosis and reactivation of viral hepatitis , screening for the presence of latent tuberculosis and chronic viral hepatitis carrier state
is recommended before initiating therapy . | Introduction
Methods
Results
Panel recommendations on RA management
Conclusion | erosions of the feet joints on extremity magnetic resonance imaging ( mri ) are found in up to 91% of patients early in the course of the disease . currently , the classification of ra is based on the 1987 american college of rheumatology ( acr ) clinical classification criteria using history , physical examination , and laboratory and radiographic findings ; this set of criteria has been criticized for lack of sensitivity in early disease . a new set of joint acr - european league against rheumatism ( eular ) criteria for ra classification has recently been published . the current ra treatment armamentarium comprises both synthetic and biologic disease - modifying anti - rheumatic drugs ( dmards ) . in 2005 , the hong kong society of rheumatology ( hksr ) published consensus recommendations on the use of anti - tumor necrosis factor ( tnf)- agents in the treatment of ra , ankylosing spondylitis , and psoriatic arthritis . given the availability of newer biologic agents for ra treatment , the hksr initiated the development of these consensus recommendations , which are aimed at providing guidance to hong kong physicians on appropriate , literature - based management of this condition , specifically on the indications and monitoring of the novel biologic dmards . the consensus recommendations were developed using the eular recommendations for the management of early arthritis as a guide , along with local expert opinion . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors . however , it is important to bear in mind that , given the variety of available treatments and the heterogeneity of patients , the management strategy should ultimately be tailored to the individual patient , taking into account factors such as individual preferences , contraindications , and side effects . ultrasound and mri are suitable for early diagnosis of bony erosions in patients who do not fulfill the 1987 acr criteria , or as a guide for joint aspiration and injection in difficult cases . the presence of anti - ccp antibodies is a strong predictor of radiological damage and progression ( category c ) . a combination of dmards , or methotrexate combined with an anti - tnf- agent ,
however , the cost - effectiveness of the latter approach has yet to be determined . combination treatment with two or more dmards , or of methotrexate and an anti - tnf- agent , has been shown to confer greater efficacy than monotherapy and , thus , is appropriate for patients with serious disease or poor prognostic factors ( category a ) [ 9 , 12 , 13 , 3436 ] . however , the cost - effectiveness of first - line intervention with biologic agents in treatment - nave patients remains uncertain ( category c ) . , das28 2.6 despite methotrexate ) should receive step - up therapy , i.e. , das28 2.6 despite methotrexate ) should receive step - up therapy , i.e. in one randomized , placebo - controlled trial ,
the addition of leflunomide to existing methotrexate therapy resulted in significant clinical benefit in patients with persistently active ra ( category a ) ; however , given the recent fda report of fulminant hepatitis in leflunomide users , meticulous monitoring of liver function is warranted in patients administered this drug . patients who require methotrexate plus a biologic agent may be administered any one of the following combinations : methotrexate plus an anti - tnf- agent , methotrexate plus tocilizumab , methotrexate plus abatacept , or methotrexate plus rituximab . patients who require methotrexate plus a biologic agent may be administered any one of the following combinations : methotrexate plus an anti - tnf- agent , methotrexate plus tocilizumab , methotrexate plus abatacept , or methotrexate plus rituximab . in recent years
, several novel biologic drugs with different mechanisms of action have emerged as potential additions to rheumatologic pharmacotherapy . one trial showed tocilizumab monotherapy to be superior to methotrexate monotherapy in dmard / methotrexate - nave patients ( category a ) ; another demonstrated that tocilizumab with methotrexate delayed radiographic progression of structural joint damage significantly more than methotrexate alone in patients who respond inadequately to methotrexate ( category a ) . anti - tnf failure patients ( failure of das28 to improve by 1.2 or < 5.1 over 16 weeks ) may be administered any one of the following : another anti - tnf- agent , tocilizumab , abatacept , or rituximab . anti - tnf failure patients ( failure of das28 to improve by 1.2 or < 5.1 over 16 weeks ) may be administered any one of the following : another anti - tnf- agent , tocilizumab , abatacept , or rituximab . in such patients , alternative biologic agents
evidence shows that patients who are refractory to anti - tnf- therapy gain significant clinical and functional benefits from tocilizumab , rituximab , or abatacept ( category a ) . for patients who test ppd - negative , there is insufficient evidence to recommend annual retesting unless there is recent exposure to tuberculosis or the risk of transmission is high . furthermore , in patients with a past history of tuberculosis , abatacept treatment may reactivate the infection as it is known to compromise t - cell immunity . this has led to the recommendation for routine screening for active and latent tuberculosis infection whenever treatment with these biologic agents is considered ( category d ) [ 54 , 55 ] . while an induration of 10 mm in diameter has been determined to be appropriate to signify a positive ppd test for patients with ra , a lower cut - off of 5 mm has been utilized to define latent tuberculosis in patients who are significantly immunocompromised , such as those with human immunodeficiency virus ( hiv ) infection or who are transplant recipients ( category d ) . patients with evidence of latent tuberculosis infection should receive prophylactic tuberculosis treatment with isoniazid for 9 months before commencing biologic agents ( category b ) [ 54 , 55 ] . the anti - tnf- agents are relatively contraindicated in carriers of hbv and hcv because of reports of viral reactivation , which can lead to serious complications ( category b ) [ 26 , 56 , 57 ] . if there are no alternatives , patients should be warned of the risk of serious hepatitis reactivation prior to administration of an anti - tnf- agent . anti - tnf- agents should be avoided in patients with a history of lymphoproliferative disorders . anti - tnf- agents should be avoided in patients with a history of lymphoproliferative disorders . according to a swedish population - based study ,
the relative risks of solid cancers in anti - tnf- treated patients were similar to those of other ra patients ( category c ) . however , they should be avoided in patients with prior lymphoproliferative disorders because of significantly increased risk of lymphomas ( category c ) [ 26 , 62 ] . treatment of moderate - to - severe heart failure patients with anti - tnf- agents has been associated with either no clinical benefit or worsening of heart failure outcomes ( category a ) . chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms.complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked.fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate.look out for new neurological symptoms in patients administered rituximab.look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration.adverse events , especially serious adverse events , should be reported to the hksr biologics registry . chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms.complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked.fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate.look out for new neurological symptoms in patients administered rituximab.look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration.adverse events , especially serious adverse events , should be reported to the hksr biologics registry . the latest eular recommendations for cv risk management in patients with ra and other forms of inflammatory arthritis states that the first principle of management is to assess and control all components of total cv risk , which includes providing appropriate , evidenced - based advice with regard to smoking , physical activity , diet , weight , and blood pressure , as well as aggressive suppression of the inflammatory process to further lower risk ( category d ) . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors . early , aggressive therapy is required for patients with serious disease and/or poor prognostic factors . anti - ccp testing may help establish an early diagnosis in patients who do not fulfill the 1987 acr criteria , or for atypical cases . ultrasound and mri are suitable for early diagnosis of bony erosions in patients who do not fulfill the 1987 acr criteria , or as a guide for joint aspiration and injection in difficult cases . the presence of anti - ccp antibodies is a strong predictor of radiological damage and progression ( category c ) . a combination of dmards , or methotrexate combined with an anti - tnf- agent ,
however , the cost - effectiveness of the latter approach has yet to be determined . combination treatment with two or more dmards , or of methotrexate and an anti - tnf- agent , has been shown to confer greater efficacy than monotherapy and , thus , is appropriate for patients with serious disease or poor prognostic factors ( category a ) [ 9 , 12 , 13 , 3436 ] . however , the cost - effectiveness of first - line intervention with biologic agents in treatment - nave patients remains uncertain ( category c ) . , das28 2.6 despite methotrexate ) should receive step - up therapy , i.e. in one randomized , placebo - controlled trial ,
the addition of leflunomide to existing methotrexate therapy resulted in significant clinical benefit in patients with persistently active ra ( category a ) ; however , given the recent fda report of fulminant hepatitis in leflunomide users , meticulous monitoring of liver function is warranted in patients administered this drug . patients who require methotrexate plus a biologic agent may be administered any one of the following combinations : methotrexate plus an anti - tnf- agent , methotrexate plus tocilizumab , methotrexate plus abatacept , or methotrexate plus rituximab . patients who require methotrexate plus a biologic agent may be administered any one of the following combinations : methotrexate plus an anti - tnf- agent , methotrexate plus tocilizumab , methotrexate plus abatacept , or methotrexate plus rituximab . in recent years
, several novel biologic drugs with different mechanisms of action have emerged as potential additions to rheumatologic pharmacotherapy . these include the interleukin ( il)-6 inhibitor tocilizumab , the selective t - cell co - stimulation modulator abatacept , and the chimeric , anti - cd20 monoclonal antibody rituximab . one trial showed tocilizumab monotherapy to be superior to methotrexate monotherapy in dmard / methotrexate - nave patients ( category a ) ; another demonstrated that tocilizumab with methotrexate delayed radiographic progression of structural joint damage significantly more than methotrexate alone in patients who respond inadequately to methotrexate ( category a ) . anti - tnf failure patients ( failure of das28 to improve by 1.2 or < 5.1 over 16 weeks ) may be administered any one of the following : another anti - tnf- agent , tocilizumab , abatacept , or rituximab . anti - tnf failure patients ( failure of das28 to improve by 1.2 or < 5.1 over 16 weeks ) may be administered any one of the following : another anti - tnf- agent , tocilizumab , abatacept , or rituximab . in such patients , alternative biologic agents
evidence shows that patients who are refractory to anti - tnf- therapy gain significant clinical and functional benefits from tocilizumab , rituximab , or abatacept ( category a ) . for patients who test ppd - negative , there is insufficient evidence to recommend annual retesting unless there is recent exposure to tuberculosis or the risk of transmission is high . for patients who test ppd - negative , there is insufficient evidence to recommend annual retesting unless there is recent exposure to tuberculosis or the risk of transmission is high . furthermore , in patients with a past history of tuberculosis , abatacept treatment may reactivate the infection as it is known to compromise t - cell immunity . while an induration of 10 mm in diameter has been determined to be appropriate to signify a positive ppd test for patients with ra , a lower cut - off of 5 mm has been utilized to define latent tuberculosis in patients who are significantly immunocompromised , such as those with human immunodeficiency virus ( hiv ) infection or who are transplant recipients ( category d ) . patients with evidence of latent tuberculosis infection should receive prophylactic tuberculosis treatment with isoniazid for 9 months before commencing biologic agents ( category b ) [ 54 , 55 ] . the anti - tnf- agents are relatively contraindicated in carriers of hbv and hcv because of reports of viral reactivation , which can lead to serious complications ( category b ) [ 26 , 56 , 57 ] . if there are no alternatives , patients should be warned of the risk of serious hepatitis reactivation prior to administration of an anti - tnf- agent . use of anti - tnf- agents is contraindicated in patients with severe heart failure , demyelinating disorders , or lupus - like features . use of anti - tnf- agents is contraindicated in patients with severe heart failure , demyelinating disorders , or lupus - like features . treatment of moderate - to - severe heart failure patients with anti - tnf- agents has been associated with either no clinical benefit or worsening of heart failure outcomes ( category a ) . chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms.complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked.fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate.look out for new neurological symptoms in patients administered rituximab.look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration.adverse events , especially serious adverse events , should be reported to the hksr biologics registry . chest x - ray should be performed every 3 months during the first year of therapy and annually thereafter , or when the patient develops symptoms.complete blood count , renal / liver function tests , lymphocyte count ( for rituximab ) , and neutrophil count ( for tocilizumab ) should be regularly checked.fasting lipid levels should be regularly surveyed ( particularly for tocilizumab ) and statins should be given whenever appropriate.look out for new neurological symptoms in patients administered rituximab.look out for gastrointestinal symptoms in patients administered tocilizumab , especially those with a history of diverticulitis or intestinal ulceration.adverse events , especially serious adverse events , should be reported to the hksr biologics registry . the latest eular recommendations for cv risk management in patients with ra and other forms of inflammatory arthritis states that the first principle of management is to assess and control all components of total cv risk , which includes providing appropriate , evidenced - based advice with regard to smoking , physical activity , diet , weight , and blood pressure , as well as aggressive suppression of the inflammatory process to further lower risk ( category d ) . these recommendations by the hksr summarize the latest evidence pertaining to ra management ( table 2 ) , with a focus on the use of novel , biologic dmards . while methotrexate is still held up as the primary therapeutic option for most ra patients , new biologic agents currently available are recognized as effective and safe treatment modalities , particularly in patients with a suboptimal response to traditional dmards . early , aggressive therapy is indicated for patients with serious disease and/or poor prognostic factorsrecommendation 2 : patient assessment ( a ) anti - ccp antibody testing , ultrasound and mri may be utilized to aid early diagnosis of ra but they are not recommended for routine use ( b ) das 28 should be utilized in the assessment of ra disease activity . a combination of dmards , or mtx combined with an anti - tnf- agent , may be considered in patients with very serious disease and poor prognostic factors ( c ) suboptimal treatment response is defined as failure to achieve remission after 3 months of mtx at its maximally tolerated dose . such patients should receive step - up therapy , i.e. , lef , ssz / hcq , biologic agent ) ( d ) patients who require mtx plus a biologic agent may be administered any one of the following combinations : mtx plus an anti - tnf- agent , tocilizumab , abatacept , or rituximab ( e ) anti - tnf therapy failure patients may be administered another anti - tnf- agent , tocilizumab , abatacept or rituximabrecommendation 4 : safety considerations ( a ) prior to using a biologic agent , patients should be screened for tuberculosis infection . | [
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] |
lyme disease ( ld ) and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections ( pandas ) are mutually exclusive disease states which share similarities but also important differences .
correct initial assessment is important since treatment for each may differ and delay may result in worsening symptoms , as evidenced in ld.1,2 in this current overview , ld and pandas are juxtaposed through a review of their respective infectious etiologies , clinical presentations , mechanisms of disease development , courses of illness , and treatment options .
ld is attributed to infection from borrelia burgdorferi and potential coinfections transmitted to humans via the ixodes scapularis tick bite.3 a recent report from the centers for disease control and prevention ( cdc)3 shows an annual increase of reported cases despite increased public awareness and preventative measures . from 1992 to 2006 , most reported cases occurred during the summer months ; average annual rates peaked for 5- to 9-year - olds and for 55- to 59-year - olds , with rates increasing disproportionately among males .
proportion of cases developing disseminated disease states has not decreased.3 additionally , natural history and epidemiology of coinfections are not fully known , and some clinicians may have limited experience in recognizing and managing them.4 pandas is attributed to the relatively abrupt onset and recurrence of pediatric obsessive - compulsive disorders ( ocd ) and neurologic tic disorders ( as defined by the outdated diagnostic and statistical manual of mental disorders : dsm - iv [ dsm - iv]5 criteria ) following bacterial infection streptococcus pyogenes of group a beta - hemolytic streptococcus ( gahs ) . sore throat and flu - like symptoms often precedes the neuropsychiatric sequelae .
five diagnostic criteria , outlined in table 1 , are needed to diagnose pandas.6,7 incidence per 100 child - years of acute sore throat , gahs swab - positive pharyngitis , and serologically confirmed gahs pharyngitis were 33 , 13 , and 8 , respectively , in 5- to 12-years - olds according to one study.8,9 overall prevalence or percentage of infected children who then develop pandas is not yet known .
it was first described in a landmark study by swedo and grant7 in 1998 but since its inception , pandas has become a controversial subject in the medical literature and across the internet.1012
although b. burgdorferi and gahs are vastly different microorganisms , their ability to evade the immune system and invade a wide variety of tissues , including the coveted central nervous system ( cns ) , is a paradigm of survival .
the mechanism with which it then results in diverse somatic symptoms and neuropsychiatric sequelae underscores the need for experienced clinicians , laboratory testing , and early treatment .
ld is caused by an infection from the bacterial spirochete b. burgdorferi and potential coinfections from agents of anaplasmosis , babesiosis , and ehrlichiosis carried by the primary tick vector ixodes scapularis , which bites its human host to transmit microorganisms.4,13,14 the white - footed mouse is a commonly cited b. burgdorferi host , but at least ten other wild and domestic mammalian species harbor b. burgdorferi , including dogs , horses , cows , rabbits , and raccoons . 15 various species of borrelia with numerous antigenic heterogeneity have been implicated in ld.13,14
b. burgdorferi was initially isolated and described in the 1970s during an epidemic of pediatric arthritic cases in the northeastern united states ( us);16,17 however , similar descriptions have been observed in europe since the 1800s.18,19 today b. burgdorferi is reported worldwide , possibly distributed via migratory birds.20,21 b. burgdorferi is a pleomorphic bacterial spirochete enclosed in a cell cylinder covered with multiple periplasmic flagella , surrounded by an outer membrane sheath;22 it exists in elongated , atypical , or cystic forms.23 after the tick bite injects b. burgdorferi and potential coinfections into the host , ideally the innate immune cells engulf the spirochete , digesting it enzymatically , which generally succeeds in killing the invading organisms .
however , an unknown number of b. burgdorferi may survive for days and even weeks after initial infection and continue the invasion , evading humoral immunity possibly by manipulating antigenic surface proteins or a weakened host immune response.2426
b. burgdorferi has not yet been found to cause tissue damage by releasing toxins or proteases itself , but may in fact over - activate the host immune system , which may then lead to inflammation and tissue damage .
significance of b. burgdorferi adhesive properties with regard to host cells has also been reported.27 cellular immunity and secretion of its factors have been well characterized in the murine model , but the exact mechanism in humans is not well known.26 gahs in pandas is a spherical , gram - positive , non - motile organism and the most common bacterial cause of acute pharyngitis ( strep throat ) in children.9,28 numerous serotypes of gahs have varying degrees of disease activity with classification based on antigenic surface proteins m and t. they are the infectious agents of scarlet fever , acute rheumatic fever ( arf ) , glomerulonephritis , toxic shock syndrome , and necrotizing fasciitis , amongst others .
its armament of antigenic surface proteins and pyrogenic exotoxins , as well as its ability to lyse its way systemically and evade the immune system effectively , have been well characterized .
m protein , for example , is the major virulence factor preventing phagocytosis , multiplying rapidly in human tissue and initiating the disease process .
more than 80 types of s. pyogenes m proteins alone have been isolated.29 the serotype(s ) in pandas is not yet known .
gahs is generally spread by direct personal contact , most likely through droplets of saliva or nasal secretions .
crowding increases transmission , and outbreaks are common through chronic asymptomatic carriers and in institutional settings , such as the military , daycare centers , and within households .
human contamination of food has also been reported.3032 resurgence of invasive streptococcal diseases and the continued presence of arf in the us predicates continued surveillance.33,34
b. burgdorferi may spread systemically localizing in somatic regions such as skin and joints,23,35,36 but can also maintain the ability to remain in a dormant , remissive state sequestered in collagen tissues , evading the immune system .
37 successful immune response clears the infection , but continued activation due to persistent infection may lead to chronic inflammation , lesion development , and subsequent multisystemic disease formation.38,39 arthritis , for example , is generally attributed to chronic neutrophilic activation , whereas carditis is associated with macrophytic and t lymphocytic activities in murine studies.40,41 somatic signs and symptoms in ld children resemble those seen in adults.42 findings most commonly reported to the cdc were erythema chronicum migrans ( ecm ) , arthritic , neurologic , and cardiac abnormalities .
early stages may present with ecm , which is often described as a bulls - eye rash , but the cdc reports 31% had none.3 variability in gross appearance of ecm has also been noted , including homogeneous erythema , multiple annular lesions , and vesicular or centrally - ulcerated dermal pathology .
43,44 aucott et al46 reported misdiagnoses occurred with greater frequency in patients with objective extracutaneous manifestations without ecm than in patients with ecm ( 83% vs 23% ; p = 0.004 ) .
of misdiagnosed cases , 41% received antibiotics not recommended for ld treatment and 30% were given steroids46 which have been shown to decrease patient response to antibiotics.47 additionally , number of ecm or the disappearance of rash may not be indicative of disseminated state of disease.48,49 other ld somatic signs and symptoms may include fatigue , arthralgia(s ) , cardiopathy , hepatitis , or splenomegaly .
50,51 patients may also present with flu - like symptoms such as sore throat , nonproductive cough , fever and chills , or lymphadenopathy .
somatic disease may also be migratory and episodic over several weeks.50 of note , children over 10 years of age with arthritis and cardiopulmonary symptoms were more likely to be diagnosed with carditis.52 ld children with arthralgias have been misdiagnosed with septic arthritis or juvenile rheumatoid arthritis , resulting in delayed treatment.53 of patients who do not receive proper early treatment , more than half may go on to develop recurrent arthralgias,16 and children may not receive proper treatment for over a year.54 in contrast to ld , somatic signs and symptoms of pandas may begin as streptococcal pharyngitis or strep throat which may later manifest as ocd or neurologic tics.6 pharyngitis may also present with a fever greater than 38c and with cervical lymphadenopathy .
examination of oral mucosa may show erythema or exudates present on tonsillopharyngeal regions along with palatal petechiae.32 reports of children with stomach pains , emesis , and other upper respiratory illnesses such as new - onset asthma , sinus infections , and severe recurrent ear infections have also been noted.32,55 although gahs is reportedly a cause of acute pharyngitis in up to 30% of children,9 viral or other bacterial etiologies may need further consideration .
symptomatic presentation of ld and pandas may cross paths in the nervous system . from the original port of entry , whether it be punctured epidermis or upper respiratory tract , invading microorganisms may make their way systemically , potentially resulting in neuropsychiatric pathology impacting the child s quality of life , school performance , and relationships with family and friends .
untreated ld may develop neurologic sequelae in up to 15% of cases.56 in ld , the mechanism b. burgdorferi utilizes to evade immune defenses and enter the nervous system has been the focus of intense research .
differences in disease severity57 have been attributed to genomic variations of bacterial strains58 or host responses as observed in murine models.41 animal studies have shown spirochete load is not associated with severity of illness59,60 suggesting b. burgdorferi may not be directly involved in neuronal damage .
spirochetes have also been observed localized in collagenous areas and along perivascular spaces in the human brain.1 entry of b. burgdorferi into the cns may be attributed to its adherence along the endothelial lining of blood vessels resulting in an immune response releasing cytokines , initiating fibrinolysis , and recruiting leukocytes , causing damage to the blood brain barrier .
other recent studies have suggested , at least in part , ligand - gated or paracellular routes of transmigration without endothelial basement membrane pathology.6165 groundbreaking in vivo murine studies by norman et al66 and moriarty et al67 used genetically engineered fluorescent b. burgdorferi strains expressing green fluorescent proteins to visualize their movements .
they filmed in real time b. burgdorferi glowingly tether , drag , and adhere to the vessel wall of endothelium along much of their length .
in addition , stationary adhesions were usually followed by extravasations at intercellular junctions.66,67 in humans , cortical regions sans blood brain barrier include the posterior pituitary gland ( site of hormones oxytocin and vasopressin release ) , pineal gland ( site of hormone melatonin release and control of circadian rhythm ) , median eminence of the hypothalamus ( site of pituitary hormones release ) , and the area postrema ( site eliciting nausea and vomiting in response to serum toxins ) .
68 but , because murine brain lacks collagen , relevance to human pathology may be limited.1 within the cerebral cortex , pleomorphic b. burgdorferi may also exist in alternate forms , possibly explaining a prolonged latent stage and persistent infection in neuroborreliosis . using atomic force and dark - field microscopy in postmortem studies of patients with chronic lyme neuroborreliosis , miklossy et
al23 collaborated with the us army to photograph atypical and cystic states which were then successfully cultured in growth media .
weis et al69 attributed cellular damage to robust induction of cytokines by b. burgdorferi antigens , up to 500-fold greater than escherichia coli .
evidence suggests macrophages of innate immunity ingest b. burgdorferi , activate cellular immunity , and through cytosolic signaling undergo programmed cell death.70 ramesh et al71,72 studied ex vivo and in vivo nonhuman primates stereotactically infected with b. burgdorferi directly into the brain .
their findings suggest b. burgdorferi induces inflammatory mediators leading to glial and neuronal apoptosis consistent with the bystander effect .
neuronal and schwann cell apoptosis in dorsal root ganglia may be a mechanism whereby b. burgdorferi affects the peripheral nervous system as well .
subsequent research by myers et al73 confirmed proinflammatory cytokines released from resident microglia were implicated as mediators to neuronal apoptosis via the p53 pathway .
however , antineuronal antibodies suggestive of molecular mimicry have also been debated.7476 interestingly , newell et al77 concluded rogue nonantigen - primed b - cell proliferation failing to apoptose after tlr - dependent b - cell polyclonal activation may be a mechanism to chronic inflammation .
genetic mhc variants in patients may determine t - cell receptor - dependent b - cell death.77 development of anti - neuronal antibodies from renegade b cells associated with b. burgdorferi patient - human leukocyte antigen ( hla ) haplotyping is not yet known .
the majority of young patients do not develop problems if treated promptly and appropriately ; however , b. burgdorferi has been observed to exhibit cns dissemination within 2 weeks of active disease.78 a study of ld children by belman et al79 reported the most frequent symptom was headache and the most common sign was facial palsy .
less common were sleep disturbances and papilledema associated with increased intracranial pressure ; peripheral nervous system involvement was infrequent .
other findings were mild encephalopathy , lymphocytic meningitis , and cranial neuropathy , as well as anecdotal reports of pseudotumor cerebri - like disease.80 ld psychiatric manifestations such as behavioral changes and memory deficits may have its greatest impact on school performance and quality of life .
intellectual functioning may be normal but auditory or visual sequential processing pathology have been reported.8183 in a well - designed controlled study investigating cognitive impairment in children having already received antibiotic therapy ( previous medication type , dose , and treatment duration not reported ) , a significant number continued to experience problems .
neurocognitive testing revealed frequent and severe headaches ( 100% ) , brain fog ( 88% ) , short - term memory loss ( 94% ) , word- finding problems ( 82% ) , distractibility ( 82% ) , schoolwork deterioration ( 94% ) , irritability / depression ( 94% ) , insomnia ( 82% ) , and sensitivity to sound ( 58% ) and/or light ( 74%).54 another study reported ld children with oppositional behavior , anxiety disorders , and attention - deficit / hyperactivity disorder ( adhd).82 of special interest , a report by riedel et al84 described an ld child presenting with tourette s syndrome , a neurologic tic disorder also seen in pandas , which resolved with antibiotic treatment .
although psychiatric manifestations of pediatric ld appear to have little or no mortality risk , tager et al54 reported 40% had suicidal thoughts and parents indicated 11% had made a suicide gesture .
however , larger , more in - depth studies are needed to better assess suicide risk in this patient population .
gahs infection in pandas is a well - characterized bacterium implicated in other neuropsychiatric disease states.8587 in the case of pandas , passive antibody transfer in murine models,88 maternal history of autoimmune disease,89 and reports of positive antineuronal antibodies in patient sera90 suggest molecular mimicry , at least in part , as a cause of disorder development .
movement of immune products into the cns has been illustrated in animal models . in mouse studies of nascent autoimmune cns lesions , bartholomus et al91 filmed in real time effector t cells trekking their way upstream against tides of vascular flow and their subsequent diapedesis across the blood
stimulated effector t cells then produced proinflammatory mediators , resulting in tissue invasion and inflammatory infiltration .
lipopolysaccharide epitopes or ligand epinephrine may alter permeability , allowing cellular immunity to penetrate into the brain.92 once inside the cerebral cortex , antibodies may also cross - react with neuronal cells .
kirvan et al90 reported antibodies from pandas serum reacted in vitro with caudate and putamen neuronal lysoganglioside gm1 , inducing calcium / calmodulin - dependent protein ( cam ) kinase ii activity .
removal of immunoglobulins from patient serum extinguished cam kinase ii cell signaling , and cerebrospinal fluid ( csf ) reactivity was then successfully blocked by gahs cell wall epitope n - acetyl - beta- d - glucosamine .
resulting neuropsychiatric pathology seen in pandas meeting the 1998 criteria7 is episodic and acute ocd and/or neurologic tics ( defined by the outdated dsm - iv criteria ) in temporal relation with gahs infection .
generally well - adjusted children may develop changes within days and resolution up to 8 weeks later.93 however , the summarized definition of ocd as defined by the dsm - iv - tr,94 the latest version of the manual replacing the dsm - iv are recurrent and persistent thoughts , impulses , or images which are intrusive and inappropriate , causing distress and anxiety beyond excessive worries about real - life problems .
children may attempt to ignore , suppress , or neutralize them with other thoughts or actions .
resulting compulsions are repetitive behaviors or mental acts performed ritualistically in response to obsessions , the purpose of which is to prevent or reduce distress or actions .
children would not necessarily have insight into their pathology which can be time consuming and may interfere with normal daily activities.94 an example of this is a child who brushes his teeth exactly ten strokes several times daily , believing it keeps the wind from blowing his parents away .
the most current dsm - iv - tr defines obsessive - compulsive disorder as not having disturbances attributed to a general medical condition ( 300.3 ) .
therefore most appropriate diagnosis may be anxiety disorder due to [ streptococcal infection ] , with obsessive - compulsive symptoms ( 293.84 ) which also requires clinical coding on axis iii of the multiaxial diagnostic assessment . in the case of neurologic tic disorders ,
they are defined as vocal or motor repetitions which are rapid , sudden , repetitive , nonrhythmic but stereotyped ( eg , eye blinking , coughing , sniffing , throat clearing ) .
tourette s disorder ( 307.23 ) , chronic motor or vocal tic disorder ( 307.22 ) , and transient tic disorder ( 307.21 ) exclude general medical conditions .
therefore , most appropriate diagnosis may be tic disorder not otherwise specified ( 307.20)94 with congruent axis iii coding .
the american psychiatric association established a task force to update and release the new dsm-5 in 2013.95 definition of general medical condition may need further clarification .
a study by bernstein et al96 found urinary urgency , hyperactivity , impulsivity , deterioration in handwriting , separation anxiety , and decline in school performance as significant traits in the initial neuropsychiatric episode .
other traits in the sentinel event included inattention , mood swings , and oppositional defiant behavior .
most common obsessions were aggression and contamination , and most common compulsions were washing , cleaning , and checking rituals .
most common symptoms associated with exacerbations were labile emotions , decline in school performance , personality change , bedtime fears / rituals , and restlessness .
in addition , motor hyperactivity and adventitious movements were not infrequently reported and should be distinguished from sydenham s chorea , a criterion for arf which necessitates antibiotic treatment because of its association with endocarditis .
use of prophylactic antimicrobials in pandas remains controversial.7,97 an interesting case of an 8-year - old boy with pandas who developed ocd and an eating disorder was recently described . when asked about his ritualistic behavior
statements of his internal running monologues were you must do the hand thing before you eat or the food will poison you , and your mommy is a criminal and contaminating your favorite things .
the child only walked on his father s right - hand side so as not to give off fat cells to people walking by .
the boy also developed signs of paranoia , believing hospital staff were evil and trying to poison him .
he recognized these fears were not real but coming from his imagination.98 another report described a child with pandas who experienced catatonic episodes but whose cognition , comprehension , and receptive language were otherwise intact .
subsequent magnetic resonance imaging showed swelling in both the caudate and the putamen with disruption of the blood
brain barrier resulting in vasogenic edema.99 reports of children having homicidal thoughts directed against their parents and others55 may require further investigation .
objective laboratory testing may be of benefit in distinguishing between ld and pandas and in differential diagnosing .
although ld is a clinical assessment , enzyme - linked immunosorbent assay ( elisa ) and western blots can confirm diagnoses but may not be necessary for patients with ecm.100 for those without dermal pathology , diagnoses by experienced ld clinicians , in conjunction with high - quality laboratory testing , are important to prevent treatment delays .
csf studies have also been examined101103 but with anecdotal reports of ld misdiagnosed as malignancies,80 a high index of suspicion from experienced ld clinicians remains the gold standard . in the case of pandas ,
confirmation of gahs infection is generally performed with the rapid antigen detection test ( radt ) in the physician s office . however , throat culture is considered the gold standard and may require up to 2 days for confirmation .
both have similar sensitivities of over 85% and even higher specificities.9 interestingly , the sensitivity of radt may not be a fixed value but may vary with disease severity . even among pediatric patients with a high centor score ,
sensitivity of radt remains too low to support the use of radt without culture confirmation of negative results.106 ancillary studies such as serologies of antistreptolysin o and deoxyribonuclease - b antibodies,7,96,99,107110 csf from lumbar puncture,98,109,110 and radiographic findings6,7,99,111114 have also been helpful in differential diagnosing but may not be required in fulfilling criteria for pandas.6,7
primary treatment for children with ld utilizes antimicrobials in various doses , durations , and routes of administration.115118 ld may be refractory to initial care because of coinfections , incorrect diagnoses , improper medications , or patient genetic variations.56,82,119,120 treatment delays may compromise patient care . according to one study , average number of physicians consulted before correct diagnosis was 3.80 , and
mean time from parent - reported symptom onset to diagnosis was 47.3 weeks.54 primary treatment for pandas has been less clear . since its establishment as
a subcategory of gahs - induced neuropsychiatric disorder remains controversial , protocols are provisional at best .
irregardless of pandas diagnosis , children with evidence of gahs infection based on clinical scoring systems or objective testing may necessitate antibiotic use to prevent disseminated disease states such as arf.9,121 in the case of pandas , antibiotic treatment alone may be efficacious.122 prophylactic use remains controversial.95,123 aside from medications , reports of intravenous immunoglobulin ( ivig ) or plasma exchange have also been noted.97,124,125 in one study , double - blind , randomized controlled trials of ivig , plasma exchange ( not blinded ) , or placebo showed statistically significant improvements in obsessive - compulsive behaviors , neurologic tics ( plasma exchange only ) , global impairment , anxiety , global severity , and emotional lability . however , longitudinal studies with longer follow - up are needed to determine remission rates or rebound effects.124 surgery for adenoid and tonsil removal alone or in combination with medications may also be effective.55,98,113,126,127 secondary support for both ld and pandas may include use of psychotropics , therapy , education , and accommodations .
medications for ocd , neurological tics , depression , anxiety , and adhd have shown to benefit children and may not be required long - term.110,128,129 cognitive behavioral therapy,130,131 supportive therapy , and academic accommodations are also useful . in the us ,
individuals with disabilities education act , individualized education program , and section 504 of the rehabilitation act of 1973 have been mobilized for ld schoolchildren to achieve their academic goals .
other educational assistance includes shorter school days , untimed tests , alternative testing methods , separate / quieter testing locations , modified home instruction programs , and elimination of unnecessary requirements.132
we have presented the first overview of both ld and pandas detailing microbial etiology , disease development , clinical overlap , and treatment options . although ld as a clinical diagnosis is relatively established , pandas as a distinct subgroup remains controversial .
gahs infection occurring temporally with ocd or neurologic tics may be coincidental , given the high incidence of gahs in this age group , and symptom etiology or exacerbation may be due to stress of the illness versus the infection itself.6 however , pandas research suggestive of molecular mimicry , clinical improvement with ivig or plasmapheresis , and lack of cardiac involvement supports the existence of pandas as a distinct subgroup of gahs - induced neuropsychiatric disorders.112,113,133,134 inconsistencies in defining pandas may preclude development of treatment protocols and comparison between studies .
quantifying temporal timelines between infection and symptomatic presentation may also serve to standardize research protocols .
incorporating the most current dsm may update the pandas definition but could also redefine pandas itself . other possible investigative directions may include adult pandas,108,114,135 familial occurrences,107,136 or various infectious etiologies such as mycoplasma , which has also been associated with ocd , tourette s syndrome , parkinsonism , and dystonia.137139 differential and working diagnoses need continued consideration11,111 since not all symptom exacerbations are preceded by gahs infections ; viral infections or other illnesses could also trigger worsening of symptoms according to one study.7 in the interesting case of a 4-year - old boy with ld who developed a motor tic ( eye blinking ) coinciding with increased igg titres for b. burgdorferi on elisa , the child subsequently improved with antibiotic therapy .
infection with b. burgdorferi should be considered in cases of tourette s syndrome in endemic areas , according to one author.84 however , a detailed description of the boy s initial clinical presentation was lacking and gahs testing was not mentioned .
this may be attributed to publication coinciding with the first pandas study,7 both having occurred in february 1998 .
the latter may also present with an annular bulls - eye lesion termed erythema marginatum rheumaticum ( emr)140 grossly similar to that seen in ld.52,100,141 definitive diagnosis may be confirmed by punch biopsy and histologic studies.43,140,142 carditis , also observed in pediatric ld,52,143145 and emr are two major criteria in arf diagnosis.146,147 migratory arthritis usually involving large joints is often described in ld100,148,149 and is also , coincidentally , a third major criterion in arf.146,147 immunoneuropsychiatry has brought forth a plethora of questions regarding the immunomolecule effect on the human mind .
current research suggests anti - neuronal rather than antimicrobial vaccine development for hla haplotypes may be of future interest .
however , the ubiquitous mhc - i molecule was recently found to possibly regulate synaptic density during development in murine studies and to affect balance between excitatory and inhibitory plasticity in nascent neurons , a property critical for information processing in young brains.150 the potential to improve mentation with administration of anti - inflammatory medications would also be of future interest .
minocycline , a bacteriostatic tetracycline derivative with lipophilic properties and relatively high cns penetration , has been extensively investigated as an inhibitor of apoptosis.115,151159 its use in treating negative signs and symptoms of schizophrenia has also shown benefit.160162 interestingly , a correlation between sporadic clusters of schizophrenia and seasonal distribution of ixodes ticks attributed causality with intrauterine exposure to b. burgdorferi.163,164 the american college of rheumatology supports use of minocycline as a disease - modifying antirheumatic drug,165 irrespective of infectious etiology .
long - term minocycline use for its cns - penetrating , anti - inflammatory effects in children over the age of 8 with antibiotic - refractory ( or slowly resolving ) lyme arthritis166 would be of significant interest in future studies , allowing opposing viewpoints166,167 to claim victory and , most importantly , for children to receive proper treatment . recognition and validation of ld has come a long way since the essay ridiculing lime
patients was first published in annals of internal medicine.82,168 astute parents partnered with experienced clinicians make a formidable team in addressing the pediatric patient and treatment course .
as families attempt to receive care for their children , it is hoped they would not face ridicule from the medical community as well . | lyme disease ( ld ) is a complex , multisystemic illness .
as the most common vector- borne disease in the united states , ld is caused by bacterial spirochete borrelia burgdorferi sensu stricto , with potential coinfections from agents of anaplasmosis , babesiosis , and ehrlichiosis .
persistent symptoms and clinical signs reflect multiorgan involvement with episodes of active disease and periods of remission , not sparing the coveted central nervous system .
the capability of microorganisms to cause and exacerbate various neuropsychiatric pathology is also seen in pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections ( pandas ) , a recently described disorder attributed to bacterium streptococcus pyogenes of group a beta - hemolytic streptococcus in which neurologic tics and obsessive - compulsive disorders are sequelae of the infection . in the current overview , ld and
pandas are juxtaposed through a review of their respective infectious etiologies , clinical presentations , mechanisms of disease development , courses of illness , and treatment options .
future directions related to immunoneuropsychiatry are also discussed . | Introduction
The bacteria
Somatic signs and symptoms
Neuropsychiatric signs and symptoms
Laboratory testing and radiographic studies
Treatment
Future directions | lyme disease ( ld ) and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections ( pandas ) are mutually exclusive disease states which share similarities but also important differences . correct initial assessment is important since treatment for each may differ and delay may result in worsening symptoms , as evidenced in ld.1,2 in this current overview , ld and pandas are juxtaposed through a review of their respective infectious etiologies , clinical presentations , mechanisms of disease development , courses of illness , and treatment options . ld is attributed to infection from borrelia burgdorferi and potential coinfections transmitted to humans via the ixodes scapularis tick bite.3 a recent report from the centers for disease control and prevention ( cdc)3 shows an annual increase of reported cases despite increased public awareness and preventative measures . proportion of cases developing disseminated disease states has not decreased.3 additionally , natural history and epidemiology of coinfections are not fully known , and some clinicians may have limited experience in recognizing and managing them.4 pandas is attributed to the relatively abrupt onset and recurrence of pediatric obsessive - compulsive disorders ( ocd ) and neurologic tic disorders ( as defined by the outdated diagnostic and statistical manual of mental disorders : dsm - iv [ dsm - iv]5 criteria ) following bacterial infection streptococcus pyogenes of group a beta - hemolytic streptococcus ( gahs ) . it was first described in a landmark study by swedo and grant7 in 1998 but since its inception , pandas has become a controversial subject in the medical literature and across the internet.1012
although b. burgdorferi and gahs are vastly different microorganisms , their ability to evade the immune system and invade a wide variety of tissues , including the coveted central nervous system ( cns ) , is a paradigm of survival . the mechanism with which it then results in diverse somatic symptoms and neuropsychiatric sequelae underscores the need for experienced clinicians , laboratory testing , and early treatment . ld is caused by an infection from the bacterial spirochete b. burgdorferi and potential coinfections from agents of anaplasmosis , babesiosis , and ehrlichiosis carried by the primary tick vector ixodes scapularis , which bites its human host to transmit microorganisms.4,13,14 the white - footed mouse is a commonly cited b. burgdorferi host , but at least ten other wild and domestic mammalian species harbor b. burgdorferi , including dogs , horses , cows , rabbits , and raccoons . 15 various species of borrelia with numerous antigenic heterogeneity have been implicated in ld.13,14
b. burgdorferi was initially isolated and described in the 1970s during an epidemic of pediatric arthritic cases in the northeastern united states ( us);16,17 however , similar descriptions have been observed in europe since the 1800s.18,19 today b. burgdorferi is reported worldwide , possibly distributed via migratory birds.20,21 b. burgdorferi is a pleomorphic bacterial spirochete enclosed in a cell cylinder covered with multiple periplasmic flagella , surrounded by an outer membrane sheath;22 it exists in elongated , atypical , or cystic forms.23 after the tick bite injects b. burgdorferi and potential coinfections into the host , ideally the innate immune cells engulf the spirochete , digesting it enzymatically , which generally succeeds in killing the invading organisms . significance of b. burgdorferi adhesive properties with regard to host cells has also been reported.27 cellular immunity and secretion of its factors have been well characterized in the murine model , but the exact mechanism in humans is not well known.26 gahs in pandas is a spherical , gram - positive , non - motile organism and the most common bacterial cause of acute pharyngitis ( strep throat ) in children.9,28 numerous serotypes of gahs have varying degrees of disease activity with classification based on antigenic surface proteins m and t. they are the infectious agents of scarlet fever , acute rheumatic fever ( arf ) , glomerulonephritis , toxic shock syndrome , and necrotizing fasciitis , amongst others . crowding increases transmission , and outbreaks are common through chronic asymptomatic carriers and in institutional settings , such as the military , daycare centers , and within households . 37 successful immune response clears the infection , but continued activation due to persistent infection may lead to chronic inflammation , lesion development , and subsequent multisystemic disease formation.38,39 arthritis , for example , is generally attributed to chronic neutrophilic activation , whereas carditis is associated with macrophytic and t lymphocytic activities in murine studies.40,41 somatic signs and symptoms in ld children resemble those seen in adults.42 findings most commonly reported to the cdc were erythema chronicum migrans ( ecm ) , arthritic , neurologic , and cardiac abnormalities . of misdiagnosed cases , 41% received antibiotics not recommended for ld treatment and 30% were given steroids46 which have been shown to decrease patient response to antibiotics.47 additionally , number of ecm or the disappearance of rash may not be indicative of disseminated state of disease.48,49 other ld somatic signs and symptoms may include fatigue , arthralgia(s ) , cardiopathy , hepatitis , or splenomegaly . somatic disease may also be migratory and episodic over several weeks.50 of note , children over 10 years of age with arthritis and cardiopulmonary symptoms were more likely to be diagnosed with carditis.52 ld children with arthralgias have been misdiagnosed with septic arthritis or juvenile rheumatoid arthritis , resulting in delayed treatment.53 of patients who do not receive proper early treatment , more than half may go on to develop recurrent arthralgias,16 and children may not receive proper treatment for over a year.54 in contrast to ld , somatic signs and symptoms of pandas may begin as streptococcal pharyngitis or strep throat which may later manifest as ocd or neurologic tics.6 pharyngitis may also present with a fever greater than 38c and with cervical lymphadenopathy . examination of oral mucosa may show erythema or exudates present on tonsillopharyngeal regions along with palatal petechiae.32 reports of children with stomach pains , emesis , and other upper respiratory illnesses such as new - onset asthma , sinus infections , and severe recurrent ear infections have also been noted.32,55 although gahs is reportedly a cause of acute pharyngitis in up to 30% of children,9 viral or other bacterial etiologies may need further consideration . symptomatic presentation of ld and pandas may cross paths in the nervous system . from the original port of entry , whether it be punctured epidermis or upper respiratory tract , invading microorganisms may make their way systemically , potentially resulting in neuropsychiatric pathology impacting the child s quality of life , school performance , and relationships with family and friends . differences in disease severity57 have been attributed to genomic variations of bacterial strains58 or host responses as observed in murine models.41 animal studies have shown spirochete load is not associated with severity of illness59,60 suggesting b. burgdorferi may not be directly involved in neuronal damage . spirochetes have also been observed localized in collagenous areas and along perivascular spaces in the human brain.1 entry of b. burgdorferi into the cns may be attributed to its adherence along the endothelial lining of blood vessels resulting in an immune response releasing cytokines , initiating fibrinolysis , and recruiting leukocytes , causing damage to the blood brain barrier . they filmed in real time b. burgdorferi glowingly tether , drag , and adhere to the vessel wall of endothelium along much of their length . in addition , stationary adhesions were usually followed by extravasations at intercellular junctions.66,67 in humans , cortical regions sans blood brain barrier include the posterior pituitary gland ( site of hormones oxytocin and vasopressin release ) , pineal gland ( site of hormone melatonin release and control of circadian rhythm ) , median eminence of the hypothalamus ( site of pituitary hormones release ) , and the area postrema ( site eliciting nausea and vomiting in response to serum toxins ) . neuronal and schwann cell apoptosis in dorsal root ganglia may be a mechanism whereby b. burgdorferi affects the peripheral nervous system as well . the majority of young patients do not develop problems if treated promptly and appropriately ; however , b. burgdorferi has been observed to exhibit cns dissemination within 2 weeks of active disease.78 a study of ld children by belman et al79 reported the most frequent symptom was headache and the most common sign was facial palsy . less common were sleep disturbances and papilledema associated with increased intracranial pressure ; peripheral nervous system involvement was infrequent . other findings were mild encephalopathy , lymphocytic meningitis , and cranial neuropathy , as well as anecdotal reports of pseudotumor cerebri - like disease.80 ld psychiatric manifestations such as behavioral changes and memory deficits may have its greatest impact on school performance and quality of life . intellectual functioning may be normal but auditory or visual sequential processing pathology have been reported.8183 in a well - designed controlled study investigating cognitive impairment in children having already received antibiotic therapy ( previous medication type , dose , and treatment duration not reported ) , a significant number continued to experience problems . neurocognitive testing revealed frequent and severe headaches ( 100% ) , brain fog ( 88% ) , short - term memory loss ( 94% ) , word- finding problems ( 82% ) , distractibility ( 82% ) , schoolwork deterioration ( 94% ) , irritability / depression ( 94% ) , insomnia ( 82% ) , and sensitivity to sound ( 58% ) and/or light ( 74%).54 another study reported ld children with oppositional behavior , anxiety disorders , and attention - deficit / hyperactivity disorder ( adhd).82 of special interest , a report by riedel et al84 described an ld child presenting with tourette s syndrome , a neurologic tic disorder also seen in pandas , which resolved with antibiotic treatment . gahs infection in pandas is a well - characterized bacterium implicated in other neuropsychiatric disease states.8587 in the case of pandas , passive antibody transfer in murine models,88 maternal history of autoimmune disease,89 and reports of positive antineuronal antibodies in patient sera90 suggest molecular mimicry , at least in part , as a cause of disorder development . removal of immunoglobulins from patient serum extinguished cam kinase ii cell signaling , and cerebrospinal fluid ( csf ) reactivity was then successfully blocked by gahs cell wall epitope n - acetyl - beta- d - glucosamine . resulting neuropsychiatric pathology seen in pandas meeting the 1998 criteria7 is episodic and acute ocd and/or neurologic tics ( defined by the outdated dsm - iv criteria ) in temporal relation with gahs infection . generally well - adjusted children may develop changes within days and resolution up to 8 weeks later.93 however , the summarized definition of ocd as defined by the dsm - iv - tr,94 the latest version of the manual replacing the dsm - iv are recurrent and persistent thoughts , impulses , or images which are intrusive and inappropriate , causing distress and anxiety beyond excessive worries about real - life problems . the most current dsm - iv - tr defines obsessive - compulsive disorder as not having disturbances attributed to a general medical condition ( 300.3 ) . therefore most appropriate diagnosis may be anxiety disorder due to [ streptococcal infection ] , with obsessive - compulsive symptoms ( 293.84 ) which also requires clinical coding on axis iii of the multiaxial diagnostic assessment . tourette s disorder ( 307.23 ) , chronic motor or vocal tic disorder ( 307.22 ) , and transient tic disorder ( 307.21 ) exclude general medical conditions . a study by bernstein et al96 found urinary urgency , hyperactivity , impulsivity , deterioration in handwriting , separation anxiety , and decline in school performance as significant traits in the initial neuropsychiatric episode . other traits in the sentinel event included inattention , mood swings , and oppositional defiant behavior . most common obsessions were aggression and contamination , and most common compulsions were washing , cleaning , and checking rituals . most common symptoms associated with exacerbations were labile emotions , decline in school performance , personality change , bedtime fears / rituals , and restlessness . in addition , motor hyperactivity and adventitious movements were not infrequently reported and should be distinguished from sydenham s chorea , a criterion for arf which necessitates antibiotic treatment because of its association with endocarditis . when asked about his ritualistic behavior
statements of his internal running monologues were you must do the hand thing before you eat or the food will poison you , and your mommy is a criminal and contaminating your favorite things . objective laboratory testing may be of benefit in distinguishing between ld and pandas and in differential diagnosing . although ld is a clinical assessment , enzyme - linked immunosorbent assay ( elisa ) and western blots can confirm diagnoses but may not be necessary for patients with ecm.100 for those without dermal pathology , diagnoses by experienced ld clinicians , in conjunction with high - quality laboratory testing , are important to prevent treatment delays . in the case of pandas ,
confirmation of gahs infection is generally performed with the rapid antigen detection test ( radt ) in the physician s office . even among pediatric patients with a high centor score ,
sensitivity of radt remains too low to support the use of radt without culture confirmation of negative results.106 ancillary studies such as serologies of antistreptolysin o and deoxyribonuclease - b antibodies,7,96,99,107110 csf from lumbar puncture,98,109,110 and radiographic findings6,7,99,111114 have also been helpful in differential diagnosing but may not be required in fulfilling criteria for pandas.6,7
primary treatment for children with ld utilizes antimicrobials in various doses , durations , and routes of administration.115118 ld may be refractory to initial care because of coinfections , incorrect diagnoses , improper medications , or patient genetic variations.56,82,119,120 treatment delays may compromise patient care . irregardless of pandas diagnosis , children with evidence of gahs infection based on clinical scoring systems or objective testing may necessitate antibiotic use to prevent disseminated disease states such as arf.9,121 in the case of pandas , antibiotic treatment alone may be efficacious.122 prophylactic use remains controversial.95,123 aside from medications , reports of intravenous immunoglobulin ( ivig ) or plasma exchange have also been noted.97,124,125 in one study , double - blind , randomized controlled trials of ivig , plasma exchange ( not blinded ) , or placebo showed statistically significant improvements in obsessive - compulsive behaviors , neurologic tics ( plasma exchange only ) , global impairment , anxiety , global severity , and emotional lability . however , longitudinal studies with longer follow - up are needed to determine remission rates or rebound effects.124 surgery for adenoid and tonsil removal alone or in combination with medications may also be effective.55,98,113,126,127 secondary support for both ld and pandas may include use of psychotropics , therapy , education , and accommodations . medications for ocd , neurological tics , depression , anxiety , and adhd have shown to benefit children and may not be required long - term.110,128,129 cognitive behavioral therapy,130,131 supportive therapy , and academic accommodations are also useful . in the us ,
individuals with disabilities education act , individualized education program , and section 504 of the rehabilitation act of 1973 have been mobilized for ld schoolchildren to achieve their academic goals . other educational assistance includes shorter school days , untimed tests , alternative testing methods , separate / quieter testing locations , modified home instruction programs , and elimination of unnecessary requirements.132
we have presented the first overview of both ld and pandas detailing microbial etiology , disease development , clinical overlap , and treatment options . gahs infection occurring temporally with ocd or neurologic tics may be coincidental , given the high incidence of gahs in this age group , and symptom etiology or exacerbation may be due to stress of the illness versus the infection itself.6 however , pandas research suggestive of molecular mimicry , clinical improvement with ivig or plasmapheresis , and lack of cardiac involvement supports the existence of pandas as a distinct subgroup of gahs - induced neuropsychiatric disorders.112,113,133,134 inconsistencies in defining pandas may preclude development of treatment protocols and comparison between studies . incorporating the most current dsm may update the pandas definition but could also redefine pandas itself . other possible investigative directions may include adult pandas,108,114,135 familial occurrences,107,136 or various infectious etiologies such as mycoplasma , which has also been associated with ocd , tourette s syndrome , parkinsonism , and dystonia.137139 differential and working diagnoses need continued consideration11,111 since not all symptom exacerbations are preceded by gahs infections ; viral infections or other illnesses could also trigger worsening of symptoms according to one study.7 in the interesting case of a 4-year - old boy with ld who developed a motor tic ( eye blinking ) coinciding with increased igg titres for b. burgdorferi on elisa , the child subsequently improved with antibiotic therapy . infection with b. burgdorferi should be considered in cases of tourette s syndrome in endemic areas , according to one author.84 however , a detailed description of the boy s initial clinical presentation was lacking and gahs testing was not mentioned . this may be attributed to publication coinciding with the first pandas study,7 both having occurred in february 1998 . the latter may also present with an annular bulls - eye lesion termed erythema marginatum rheumaticum ( emr)140 grossly similar to that seen in ld.52,100,141 definitive diagnosis may be confirmed by punch biopsy and histologic studies.43,140,142 carditis , also observed in pediatric ld,52,143145 and emr are two major criteria in arf diagnosis.146,147 migratory arthritis usually involving large joints is often described in ld100,148,149 and is also , coincidentally , a third major criterion in arf.146,147 immunoneuropsychiatry has brought forth a plethora of questions regarding the immunomolecule effect on the human mind . however , the ubiquitous mhc - i molecule was recently found to possibly regulate synaptic density during development in murine studies and to affect balance between excitatory and inhibitory plasticity in nascent neurons , a property critical for information processing in young brains.150 the potential to improve mentation with administration of anti - inflammatory medications would also be of future interest . recognition and validation of ld has come a long way since the essay ridiculing lime
patients was first published in annals of internal medicine.82,168 astute parents partnered with experienced clinicians make a formidable team in addressing the pediatric patient and treatment course . | [
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the multipotent adult stem or progenitor cells reside at restricted locations to allow continuation of the cycle of life .
these tissue - specific stem cells have been identified in the bone marrow , brain , skin , retina , pancreas , intestinal crypt , and liver as well as in skeletal muscle .
depending on the origin , these adult stem cells exhibit the potency to differentiate into multiple cell types .
adult stem cells from muscle , for instance , predominantly become myogenic or hematopoietic while those from bone marrow are known to harbor hematopoietic , endothelial and mesenchymal ( msc ) stem cells .
mscs derived from the bone marrow stroma are multipotent , hypoimmunogenic [ 11 , 12 ] , and proliferate freely in vitro to undergo self - renewal and differentiation into multiple non - hematopoietic cell lineages such as chondrocytes , osteoblasts , adipocytes , and myoblasts , and cardiomyocytes , and neuronal cells [ 1416 ] .
this is attractive because , for example , unlike extraction of the neural stem cells or neural precursors that would require surgery , mscs are relatively easy to isolate and differentiate into neuronal cells , and therefore may be useful for tissue engineering strategies to repair and regenerate connective and excitable tissues .
other tissues such as adipose tissue , periosteum and synovial tissue have been shown to contain cells with properties similar to those of stem cells .
however , the limited life span and amount of cells as well as their heterogeneity pose difficulties in utilizing this particular cell population in the basic research and therapeutic applications [ 1720 ] .
generally , stem cells have the unique property of self - renewal without differentiation until and unless appropriate biological and physical signals are provided . in the context of tissue engineering ,
unlike engineering tissue constructs with differentiated cells , the multipotent mesenchymal stem cells ( mscs ) are hypoimminogenic , have higher proliferative capacity and provide excellent regenerative capability that will likely lead to desired integrity and functionality of the engineered tissue .
moreover , mscs make it possible to engineer multifunctional tissue constructs and also can be differentiated into phenotyically and functionally diverse lineages ( e.g. , osteoblasts and neuronal cells ) .
it is plausible that stem cell - based tissue implants can be designed for tissue formation and vascularization at the same time , which mimics typical physiological responses to tissue damage .
however , a clearer understanding and adequate control of stem cell differentiation into tissue - specific lineages would first have to be firmly established .
for example , development of the strategies to control stem cell behaviors and directing them to a specific lineage would require an understanding and manipulation of the cell - environment interactions .
determination of an optimal physicochemical microenvironment could lead to harvest the unique properties of the stem cells for self - renewal and tissue - specific differentiation that can be guided or perhaps directed by biochemical and physical cues .
rational approaches for stem cell - based engineering of functional tissues would then be realized and could be implemented , thus potentially overcoming the current challenges in regenerative medicine .
the therapeutic applications of stem cells would undoubtedly be enhanced by an efficient differentiation protocol that could prescribe the phenotype . upon implantation of an engineered tissue construct
, it may also be plausible to intervene externally and regulate the fate of stem cells . in this review paper
, we will first illustrate that the soluble factors and external physical forces ( i.e. , physicochemical cues ) have been combined , engineered and applied to induce synergistic msc differentiation
. we will next elucidate the potential mechanism(s ) that mediate the msc differentiation in response to the physicochemical cues . a detailed understanding of the lineage - dependent mechanisms
would be expected to prescribe the msc differentiation into specific tissue types using multiple cues .
first , the biological and pharmacological reagents ( e.g. , hormones ) have been used to induce stem cell differentiation into bone cells [ 2224 ] , chondrogenic cells , adipogenic cells [ 26 , 27 ] , and even functional neuronal cells [ 16 , 2830 ] .
further , mscs have also been shown to differentiate into the myogenic lineage [ 31 , 32 ] .
these recent advances in the msc manipulation demonstrate that the multipotent stem cells are capable of differentiating into excitable and non - excitable tissue phenotypes .
second , the msc differentiation may be regulated by mechanical cues , including changes in the cellular morphology [ 13 , 33 ] and matrix elasticity .
as the matrix stiffness increased , the focal adhesions could increase in size and also expression of the focal adhesion molecules .
this is consistent with the finding that the cell adhesion of several distinctive types may be involved , including focal complexes , focal adhesion , and fibrillar adhesion .
the cells may utilize the crosstalk between the extracellular matrix and the cytoskeleton , likely mediated by integrins , to determine the adhesion type .
other environmental factors ( e.g. , oxygen tension ) have also been incorporated to solicit synergistic stem cell differentiation .
for example , while a cyclic mechanical stretch can enhance bone formation [ 3742 ] , various types of shear stress can synergistically enhance msc osteodifferentiation [ 4347 ] .
low intensity ultrasound , another form of mechanical stress , has also shown to enhance the chondrogenic differentiation [ 48 , 49 ] . a recent study by haudenschild et al . reported the ability of mscs to distinguish between dynamic tensile and compressive loading by regulating distinct gene expression patterns . while dynamic tensile stresses induce both the fibroblastic and osteogenic markers , a compression upregulated the chondrogenic gene expressions .
moreover , our laboratory has recently found that an electrical stimulation facilitates osteodifferentiation , suggesting that physical stimuli can induce synergetic effects .
since the electrical and mechanical nature of the cell is now well established , we suggested that cellular and molecular responses to external physical stimuli would be cast more appropriately as electromechanical responses .
because there appear to be several physicochemical differentiation cues that promote stem cell differentiation , strategies to optimize these cues to facilitate or interfere with the intended fate of stem cells may be highly significant for regenerative tissue engineering and also in general stem cell biology .
optimization of at least three known cues would have to be determined based on multifactorial experimental design , requiring sophisticated statistical analyses .
however , the focus of this review is to illustrate synergistic stem cell differentiation in response to a combination of soluble factors and externally applied physical stimuli , which may additively or synergistically facilitate the intended msc differentiation . for this purpose
while additive differentiation implies effects ( e.g. , quantifiable tissue - specific markers ) produced by multiple cues should be the same as the sum of each cue used alone , synergistic differentiation would imply greater than the sum of effects produced by each cue when a combination of the physicochemical cues is applied . for example , a combination of soluble factors and an electrical stimulation induces synergistic osteodifferentiation , while the use of an electrical stimulation alone fails to differentiate mscs .
synergistic interaction may also lead to accelerate the pace at which the intended stem cell differentiation proceeds .
strategies to optimize various differentiation cues to facilitate and interfere with the intended fate of stem cells may be highly significant for regenerative tissue engineering and also in general stem cell biology .
in addition to demonstrating synergistic stem cell differentiation in response to a combination of soluble factors and externally applied physical stimuli , elucidation of the potential coupling mechanisms would have to be studied .
both mechanical and electrical stimulation has been applied separately and combined with soluble factors to facilitate msc differentiation .
for example , recent studies show that , when vascular endothelial growth factor ( vegf ) and a shear stress are used in a combinatorial manner , synergistic differentiation of endothelial progenitor cells into endothelial cells may be accomplished , and synergistic chondrogenic differentiation of mscs achieved by combining tgf-3 and a hydraulic pressure .
a combination of fluid shear stress and bone - like extracellular matrix , made of titanium ( ti ) fiber mesh discs , both in the presence or absence of osteogenic dexamethasone synergistically enhance the osteodifferentitation of mscs .
synergistic effects are also observed when a cyclic strain is combined with dexamethasone to enhance the osteogenic commitment of human mscs .
alternatively , soluble factors combined with an electrical stimulation induce synergistic osteodifferentiation , while the use of the electrical stimulation alone fails to differentiate mscs toward osteoblasts .
varying the modality of electrical stimulation , pulsatile biphasic electric field is now shown to enhance msc proliferation and upregulate the differentiation related genes , and an sinusoidal electrical stimulus ( 100 mv peak - to - peak at 4,000 hz ) appears to induce differentiation of rat pc12 and blastocyst - derived murine embryonic stem cells toward the neuronal lineage .
of potential mechanisms that might mediate the effects of multiple differentiation cues , we will focus on the three recent but likely mechanisms that have been carefully studied and documented .
first , calcium dynamics in mscs undergo noticeable changes that are lineage - dependent , suggesting that calcium plays a critical role in cell differentiation [ 5860 ] and could be used as a modulator for msc differentiation .
second , mitogen - activated protein ( map ) kinase - dependent signaling has emerged as a potent regulator of stem cell commitment and differentiation [ 61 , 62 ] . since mechanotransduction is likely associated with map kinase signaling , the potential coupling mechanisms to the map kinase signaling by the differentiation cues could offer beneficial strategies for tissue engineering . third , cellular biomechanical remodeling shows noticeably remarkable alterations that are also lineage - dependent .
moreover , a close correlation or interdependence between the [ ca]i and cytoskeletal reorganization is well known , established , and accepted , although the effects of altered and modulated [ ca]i oscillation on the cellular mechanics are yet to be fully elucidated .
it is interesting to note that , in response to osteogenic soluble factors or physical stimuli , the human msc cellular mechanics become indistinguishable from that of osteoblasts in about 10 days , and the maximal changes in the ca dynamics also takes about 14 days .
this may suggest that the biomechanical remodeling could precede the cellular and molecular identities of stem cell - derived osteoblasts , and that the microfilament - based biomechanical remodeling and [ ca]i oscillation are intimately connected .
mscs can differentiate down the lineages that are mechanically at the opposite ends of the spectrum ; mechanically strong osteoblasts versus soft neuronal cells .
there is no doubt that mscs will have to reorganize their cytoskeleton and membrane to comply with the intended differentiation . while methodologies are readily available to characterize changes in the calcium dynamics and the biochemical properties , more subtle questions yet to be fully answered include whether changes are simply a consequence of msc differentiation or perhaps a critical determinant to prescribe msc differentiation .
calcium ( ca ) is a ubiquitous second messenger and represents one of the most important biological signals .
the intracellular ca regulates important cellular and molecular processes such as proliferation , differentiation , cell biomechanics , cytoskeletal reorganization , gene expression and metabolism [ 21 , 65 , 66 ] . among many cell types ,
transient elevations in free cytosolic ca concentration ( i.e. , [ ca]i ) referred to as spikes are a nearly universal mode of signaling in both excitable and non - excitable cells [ 67 , 68 ] .
while the role of oscillatory ca signals has been extensively studied but not yet fully understood , it is evident that spatial and temporal patterns of ca dynamics ( e.g. , spiking amplitude and frequency , and spatial distribution ) are important characteristics of cellular regulatory pathways . [ ca]i oscillations are likely mediated by at least several cell type - dependent ca influx / efflux pathways .
first , ca entry across the plasma membrane can be mediated via voltage - operated ca channels , stretch - activated cation channels , and agonist - dependent channels [ 69 , 70 ] .
second , ca release from intracellular stores ( e.g. endoplasmic and sarcoplasmic reticulum , mitochondria ) can be controlled by inositol 1,4,5-triphosphate ( ip3)-gated channels and ryanodine receptors .
third , the excess ca is pumped back from cytosol to internal stores or extruded to extracellular medium by ca - atpase pumps .
while biochemical cascades , molecular and cellular interactions , and gene expression profiles can be controlled by altered intracellular ca dynamics , intracellular calcium activities themselves can be regulated by a variety of external physical stimuli , including electromechanical stimulation and substrate rigidity .
more specifically , spontaneous [ ca]i oscillations are readily observed in mscs [ 24 , 74 ] and can affect stem cell differentiation [ 58 , 59 ] . in response to a fluid flow , riddle et al .
have shown that shear stresses trigger flow rate - dependent increases in [ ca]i via the ip3 pathway in mscs .
moreover , the substrate rigidity now appears to alter [ ca]i oscillations via rhoa / rock activity , suggesting that the substrate rigidity - dependent msc differentiation may be mediated by altered [ ca]i oscillations .
our laboratory has recently demonstrated that [ ca]i oscillations in mscs are dramatically altered in response to the soluble factors and an electrical stimulation .
indeed , the physicochemical cues alter the calcium dynamics in mscs to resemble that found in terminally differentiated osteoblasts .
interestingly , a combination of osteogenic soluble factors with an electrical stimulation induced a synergistic differentiation of mscs into osteoblasts at rates that are significantly faster than those induced by the soluble factors alone .
however , application of an electrical stimulation by itself failed to induce msc differentiation , suggesting that an externally applied physical cue may amplify but not necessarily activate the mechanisms involved in osteodifferentiation .
multiple ca influx or efflux pathways are likely involved in the regulation of ca dynamics .
at least 4 testable ways are plausible in which the ca dynamics in mscs are modulated ( figure 1 ) .
first , because the ca channels are known to sustain [ ca]i oscillation , mscs should express these channels .
indeed , an abundance of the l - type channels fluorescently visualized in undifferentiated mscs ( data not shown ) indicates that these channels are involved in the regulation of the [ ca]i oscillation .
treatment of cells with verapamil ( a l - type inhibitor ) or depletion of extracellular ca decreased the [ ca]i oscillation to the level comparable to that found in terminally differentiated osteoblasts .
these findings led to formulation of a postulate that extracellular cais required to sustain the [ ca]i oscillation via the l - type ca channels . as mscs undergo osteodifferentiation , the [ ca]i oscillation is decreased .
therefore , the role of l - type ca channels would be expected to diminish , suggesting that these channels may be down regulated .
second , we have shown that , when the plc activity is inhibited , the [ ca]i oscillation is essentially abolished .
this leads to another hypothesis that , as mscs undergo osteodifferentiation , the plc activity is reduced .
concomitant reduction of l - type channels and plc activity may be coordinated to suppress the [ ca]i oscillation in mscs undergoing osteodifferentiation .
third , integrins have been shown to regulate the ca dynamics which , in turn , control integrin - mediated adhesion , and cell migration through phosphorylation of focal adhesion kinase ( fak ) .
this suggests a close association or correlation between differential focal adhesions formed in response to physicochemical cues and cell type - dependent [ ca]i oscillation .
fourth , mscs were found to secrete atp , and that autocrine and paracrine interactions involving purinergic receptors modulate [ ca]i oscillations .
interestingly , we recently detected and characterized atp release in response to an electrical stimulation .
taken together , the physical cues may alter ca dynamics mediated in part by atp secretion .
while several laboratories have shown evidence for induction of neuron - like cell , astrocytes , and glial cells [ 16 , 7982 ] , other reports suggest that in vitro differentiation protocols for bone marrow stromal cells disrupt actins and apparent neuronal morphology is therefore observed , or cellular makers in response to the neuroinductive factors could be attributed to cellular toxicity and changes in the cytoskeleton .
although the opposing findings should carefully be considered , the biochemical or substrate stiffness induction of neuron - like cells from human mscs does exhibit rapid [ ca]i oscillation .
in fact , the ca dynamics profiles found in these cells differ in many ways .
for example , the initial response to the neuroinductive factors suppressed the [ ca]i oscillation , similar to that found during osteodifferentiation .
however , mscs undergoing neurogenesis quickly regain the capability to sustain the [ ca]i oscillation in less than 7 days .
in addition , the [ ca]i oscillation profiles are also distinctively different ( figure 2 ) . those cells undergoing neurodifferentiation exhibit a pattern that consists of numerous ca spikes of both small and large amplitude .
this type of pattern is unique to the neurogenic cells and not found in any of the five cell types we have tested thus far , including undifferentiated human or rat mscs , p19 mouse embryonic stem cells , osteoblasts , fibroblasts , and even primary myocytes . unlike non - excitable cells however , the n - type cachannels are expected to play a major role in neuronal cells .
we detect essentially no n - type channels expressed in the undifferentiated stem cells , suggesting that the n - type channels are rapidly up - regulated .
in addition , the n - type channels are modulated by the extracellular signal - regulated kinase ( erk)-dependent phosphorylation .
since erk activation is also involved in stem cell differentiation , up - regulation of the n - type ca channels is expected as mscs undergo neurodifferentiation .
these findings provide evidence that the altered ca dynamics may be unique to the tissue type and perhaps be used as an early indicator to predict msc differentiation into the neurogenic phenotype .
the mitogen - activated protein ( map ) kinases are the serine / threonine kinases that respond to extracellular signals .
in addition to several important physiological responses , the role of map kinases in proliferation and differentiation has been well documented and established .
unregulated map kinase signaling , for example , has been implicated in many types of tumors [ 9092 ] .
it also appears that , at least for osteogenic differentiation , sustained map kinase activation promotes cell differentiation , suggesting an interesting hypothesis that a physical stimulation may be used to sustain and prolong the map kinase activation .
at least three major modules of the map kinase signaling cascades have been identified , including the extracellular signal - regulated kinase ( erk ) , the c - jun amino - terminal kinase / stress activated protein kinase ( jnk / sapk ) , and the p38 kinase .
of these three modules , the erk signaling pathway represents the most extensively studied mechanism .
although all three map kinase modules are involved in various biological processes , the erk pathway is associated particularly with cell proliferation and differentiation [ 9597 ] .
the jnk / sapk kinases have been shown to respond to external stress including heat shock , and cytokines .
interestingly , when cells are treated with mitogenic agents , the erks but not jnk / sapks are the dominant signaling pathways .
in contrast , cells exposed to a stress typically utilize the jnk / sapk pathway without significantly altering the erk activities [ 98 , 99 ] .
this observation raises a rather important hypothesis that , when cells are exposed to external physical stimuli in the presence of the mitogenic agents , the differential activation of map kinases could be combined , and synergistic biological responses such as cell proliferation and differentiation may be induced . it is noted here however that , in response to mechanical stimulation , the erk and p38 kinase cascades can be activated [ 100 , 101 ] , and tethering and rolling of neutrophils in shear flow activates integrins and induces firm adhesion through phosphorylation of the p38 kinase . unlike soluble factors that can readily bind to their receptors with a degree of specificity , the physical cues do not preferentially discriminate the targets . however , evidence has been gathered to suggest that there are potential candidates that may transduce the external physical stimuli .
for example , several mechanotranducers have been identified that include integrin - dependent focal adhesion [ 103107 ] and ion channels [ 108 , 109 ] , and integrin clustering has been shown to be responsible for transducing electrical stimulation [ 110 , 111 ] .
in addition to the critical role in formation of focal adhesions , integrins are also linked to cell proliferation , differentiation , migration , and apoptosis [ 112 , 113 ] .
moreover , integrins are essential for normal development of hematopoietic lineages and bone marrow by regulating cell proliferation and differentiation , and cardiomyocyte cell cycle depends on cell attachment via 1-integrins .
for example , neuronal differentiation involves down - regulation of integrins . at successive stage of the osteoblast lineages ,
cells show differential patterns of integrin expression . in spite of these diverse cellular responses regulated by integrins ,
it appears clear that the integrin - matrix interaction can activate map kinases , thus suggesting that map kinases act as one of the key molecules that connect integrins at the cell surface with adhesion - dependent gene expressions .
interestingly , some findings suggest that integrin can regulate activation of growth factor receptors by co - clustering and thereby stimulate map kinase signaling . another likely candidate is via g - proteins . indeed ,
activation of phospholipase c ( plc ) is a key event that modulates altered calcium dynamics and subsequent production of yet another secondary messenger , diacylglycerol ( dag ) .
dag can lead to activation of protein kinase c ( pkc ) that , in turn , activates map kinase cascades for proliferation and differentiation .
this g - protein mechanism has been demonstrated in human osteoblasts and human mscs using specific plc inhibitors ( e.g. , u73122 ) and also by blocking pkc ( e.g. , bisindolylmaleimidei ) .
soluble factors ( e.g. , dexamethasone ) typically regulate cell differentiation through multiple biochemical mechanisms ( figure 3 , thick arrows ) including map kinase signal transduction . the commitment of map kinases results in activation of various transcription factors ( e.g. , ap-1 , cbfa1/runx2 ) and , in the case of osteodifferentiation , up - regulation of osteoblast - specific genes . the first coupling mechanism ( figure 3(a ) )
this physical stimulus causes integrin redistribution , clustering , activation , and assembly of some focal adhesion proteins such as focal adhesion kinase ( fak ) , paxillin , vinculin , src [ 121 , 122 ] .
enzymatic activity of assembled proteins ( e.g. , fak or src ) causes further map kinase activation .
such assembly of adhesion proteins may mimic the integrin - matrix interactions , which are known to regulate the map kinase activities . in effect , the map kinase cascade is synergistically amplified and cell differentiation is facilitated .
alternatively , the second coupling mechanism involves activation of g - protein coupled receptors in the cell membrane ( figure 3(b ) ) that leads to the pkc pathway .
pkc activation is then effectively coupled to the map kinase cascades and thus promoting osteodifferentiation .
a cell has a remarkable capability to detect external physical forces of various modalities and adapt to the biomechanical environment by adjusting its mechanical properties to match those of the surrounding tissue .
clearly , cells do respond to the physical forces and mechanically remodel themselves in order to transduce the physical signals .
the concept of mechanotransduction relies on the bi - directional cross - talk between the cell and microenvironment [ 108 , 124 , 125 ] that modulates biochemistry in the cytoplasm and ultimately affects the nucleus .
cytoskeleton is thought to be one of the most significant cellular mechanical components and provides structural stability and elasticity to the cell undergoing multiple deformations without losing its integrity [ 127 , 128 ] .
in addition , the cytoskeleton has now been shown to mediate complex intracellular signaling pathways .
therefore , the cytoskeleton is referred to as a mechanotransducer that regulates the cytoskeleton - associated signaling cascades , such as rho family gtpases ( e.g. , rhoa , cdc42 , rac ) . in this way , the cytoskeleton mediates cell response to changing biomechanical environment ( e.g. , substrate stiffness , cell shape and deformation , external pressure , shear stress ) by structural rearrangement of the cytoskeleton itself , or alterations in gene expression profiles , cell adhesion , and secretion of extracellular matrix ( i.e. , bi - directional reciprocity ) [ 127 , 130 ] .
complex interactions between these multiple signaling molecules , likely triggered by external signals , cause activation of downstream target proteins , which could lead to a global structural rearrangement of the cytoskeleton or altered gene transcription profiles affecting cell adhesion , secretion of extracellular matrix components , and cell metabolic activity .
substantial structural and functional differences of cytoskeletons in cells of the same mesodermal origin ( myocytes , osteoblasts , endothelial cells , kidney cells , etc . )
the plasma membrane is also known to play an important role in determining the cellular biomechanics . due to the important role of the membrane in many cellular functions , it is likely involved in the intricate interplay of events accompanying cell differentiation . beside its function as a barrier from the outer environment , it participates in inward - outward trafficking , motility , cell adhesion to extracellular matrix , and cell - cell interaction [ 131 , 132 ] .
these and many other intracellular events are regulated by the membrane surface tension , which is maintained by several mechanisms including membrane reservoir and lipid material turnover [ 132 , 133 ] . generally , the membrane tension is determined by the lipid bilayer composition ( e.g. , cholesterol content ) , and the membrane interaction with the cytoskeleton via specific biomolecular links such as linker proteins .
we have recently shown that the plasma membrane attachment to the cytoskeleton in fully differentiated osteoblasts is much stronger than in undifferentiated human mscs .
functionally , a strong membrane - cytoskeleton adhesion should be beneficial to keep the structural integrity of osteoblasts subjected to continuous stress cycles . in human mscs , a lower membrane tension is observed that may better facilitate endo- and exocytosis and contributes to a higher sensitivity of these unqiue cells to various soluble biochemical environmental stimuli .
moreover , we have recently reported an interesting observation [ 21 , 63 ] that the cell type - dependent linkage between the membrane - cytoskeleton could be regulated by the linkers proteins [ 137141 ] , including ezrin , radixin , and moesin ( erm proteins ) .
for example , the erm proteins ' expression and distribution are clearly different in human mscs and osteoblasts .
interestingly , because the erm proteins are substrate for rhoa , the involvement of rhoa in stem cell differentiation in response to the extracellular environment may now be better understood and is likely to depend on the erm protein distribution and activation .
mechanotranduction may be mediated by the target molecules at the cell surface or by a
hire - wired tensegrity network of cytoskeleton [ 108 , 126 , 143 ] . in response to a physical force
, mechanically sensitive molecules such as integrins , ion channels , and g - protein coupled receptors could be activated and initiate downstream signaling cascades and alter gene expression [ 108 , 109 , 144 ] .
this view of mechanotranduction has been supported by evidence that includes integrin blocking by antibodies [ 111 , 145 ] and inhibiting stretch - activated cation channels ( saccs ) [ 146 , 147 ] .
alternatively , the tensegrity model postulates a rapid and coordinated response to a physical force by rearranging the cytoskeletal and nuclear elements [ 126 , 143 ] .
this model has gained much attention due to the fact that it provides an architectural description of mechanotransduction and that it can support a long range force transmission to influence the nucleus directly . based on our recent findings ,
we propose yet another alternative model that integrates the membrane - cytokseleton coupling into the overall cellular biomechanics .
incorporating afm - based microindentation technique to measure the cellular elasticity and assessing the membrane tension by optically pulling membrane tethers from the cell body [ 63 , 136 ] , the terminally differentiated cells ( e.g. , osteoblasts ) show a lower elastic modulus and formation of shorter membrane tethers .
these are in direct contrast to mscs in which the elastic modulus is much higher and the membrane tethers are significantly longer .
since the elasticity is primarily determined by actins , the actin stress fibers are found likely in large bundles in stem cells .
moreover , the membrane - cytoskeletal association can be probed by optically pulling the membrane tethers . significantly longer
tether lengths observed in stem cells are indicative of a weak association between the two components that determine the overall cellular biomechanics .
as illustrated in figure 4 , the plasma membrane is strongly associated with the underling meshwork of thin actin fibers through multiple linker proteins and focal adhesions in osteoblasts .
in contrast stem cells , especially in mscs , express thick actin stress fibers that are connected to the membrane only at few discreet sites ( e.g. , focal adhesions ) . as a result ,
the overall membrane - cytoskeleton adhesion in mscs is weaker and the optically extracted membrane tethers are therefore longer .
moreover , actin remodeling and physical coupling between the cell membrane and cytoskeleton , mediated by linker proteins , are now shown to be responsible for such dramatically different biomechanical properties .
indeed , recent work from our laboratory indicates that , when human mscs are transfected to transiently suppress the erm linker proteins ( ezrin , radixin , and moesin ) , osteogenic differentiation is either delayed or perhaps inhibited , and the cell membrane - cytoskeleton coupling is weakened .
for example , the transfected human mscs maintained similar biomechanical properties that are characteristics of undifferentiated stem cells , even though the osteogenic factors are present .
an additional observation indicates a decrease in the intracellular atp level and possibly atp release in response to an external physical stimulation , providing more evidence that the atp - dependent erm proteins may be a key player .
the erm proteins function not only as the connector linking the membrane with microfilaments but also are now known to regulate signaling . of particular interest
the role of rho - a in mechanically guided msc differentiation has been shown [ 33 , 34 ] .
the erm protein expression is cell type - dependent and also appears to have been redistributed when stem cells undergo differentiation .
moreover , the cellular biomechanical measurements indicate that the membrane - cytoskeleton coupling , as assessed by tether lengths , strengthens significantly during osteodifferentiation .
this is consistent with the erm proteins ' expression that promotes a stronger association between the cell membrane and cytoskeleton .
while this model is not intended to represent an exhaustive list of possibly numerous pathways , the differentiation cues may influence the cellular biomechanics through utilizing both ca - dependent and -independent pathways ( see figure 5 ) .
soluble factors either bind to the cell surface receptors or diffuse through the cell membrane , and alter gene expressions , modulate [ ca]i oscillation and reorganize cytoskeleton .
an external physical stimulus induces an increase in the [ ca]i mediated either by ca influx across the cell membrane or ca release from intracellular storage .
an elevated [ ca]i level is likely to cause depolymerization of f - actins and therefore an overall decrease in the cell elasticity , thus reinforcing the effects of the soluble factors .
recent studies suggest that , similar to a physical stimulus , the substrate matrix stiffness also can reorganize the actin cytoskeleton via the rhoa / rock pathway and alter the [ ca]i oscillation .
independent of an increase in the [ ca]i , the physical stimulus causes a depletion in the intracellular atp ( e.g. , atp release ) , which in turn leads to inhibition of erm proteins ' binding affinity and dissociation of the cytoskeleton from the membrane .
the resulting membrane separation from the cytoskeleton and a decrease in the membrane tension are attributed both to induced down - regulation of active erm linker proteins and actin depolymerization .
the linker proteins seem to play a significant role in modulation of the cell mechanics that is a critical factor in the regulation and even manipulation of stem cells .
it is interesting to know that an electrical stimulation alone fails to initiate msc osteodifferentiation but , when combined with the soluble factors , causes a synergistic stem cell differentiation . in the context of the proposed model
, an explanation can be provided that , as thick stress fibers appear less stable than thin microfilaments , the stress fibers are disassembled initially under an electrical exposure .
this brings the cell elastic and structural properties closer to those of fully differentiated osteoblasts , and induces the cell membrane dissociation from cytoskeleton , and consequently decreases the membrane tension to enhance endocytosis and transmembrane trafficking of the soluble factors . when cells are allowed to recover following the electrical exposure in presence of soluble factors
, a further rearrangement of actin and erm proteins will proceed along with expression of osteogenic markers .
in contrast , neuronal cells exhibit a very weak actin cytoskeleton and a relatively loose plasma membrane as indicated by the tether extraction experiments .
therefore , a higher physical stimulation might be required to facilitate neurogenic differentiation , which will maximally disrupt the actin cytoskeleton and inhibit erm linkers , suggesting a plausible approach for precisely controlling the physical parameters for selective manipulation of mechanical properties of a particular and pre - selected cell phenotype . taken together , optimal use of the physicochemical cues may lead the researcher to develop strategies for tissue engineering by manipulating cell differentiation , mobility , and cell incorporation into engineered scaffolds , and eventual maturation of tissue substitutes .
an in depth understanding of mechanisms that allow regulation of the cell biomechanical and biochemical properties will undoubtedly lead to a more effective development of therapeutics for regenerative medicine .
while biologists have long appreciated the important role for soluble factors in the regulation of stem cell differentiation , the mechanical and electrical stimuli are now firmly established to significantly influence the stem cell fate . therefore , the idea of combining multiple differentiation cues is gaining traction and could lead to an active research area of stem cells and perhaps unravel their intricate biological and physical properties .
future work should include execution and full analysis of multifactorial design experiments in which the presumed orthogonal cues ( e.g. , soluble biochemical and physical force are non - interactive ) might interact to produce higher order effects .
one example might be to vary the concentration of soluble factors that many laboratories currently use .
induction protocols using a lesser amount of soluble factors could be just as potent when combined with suitable physical cues . however , completion of the all possible combinatory experiments may be laborious , and it is unlikely that we can determine a set of combinational cues that is widely applicable irrespective of tissue phenotypes . rather , for an intended tissue phenotype
, the physicochemical cues would have to be optimized for this particular phenotype that is distinctively different from others morphologically and functionally ( e.g. , excitable versus non - excitable tissues ) . in spite of such difficulties , advances in the molecular , biophysical , and imaging tools
should allow a more detailed understanding of the coupling mechanisms that regulate stem cell differentiation in the near future .
establishment of new paradigms in which stem cells can be manipulated physicochemically will undoubtedly expedite the current effort for stem cell - based regenerative medicine by targeting specific combinations of physicochemical cues that optimally exploit the unique biological and biophysical properties of stem cells for the intended differentiation . | realization of the exciting potential for stem - cell - based biomedical and therapeutic applications , including tissue engineering , requires an understanding of the cell - cell and cell - environment interactions . to this end , recent efforts have been focused on the manipulation of adult stem cell differentiation using inductive soluble factors , designing suitable mechanical environments , and applying noninvasive physical forces . although each of these different approaches has been successfully applied to regulate stem cell differentiation , it would be of great interest and importance to integrate and optimally combine a few or all of the physicochemical differentiation cues to induce synergistic stem cell differentiation . furthermore , elucidation of molecular mechanisms that mediate the effects of multiple differentiation cues will enable the researcher to better manipulate stem cell behavior and response . | 1. Introduction
2. Synergistic MSC Differentiation
3. Elucidation of Mechanisms
4. Conclusions |
the multipotent adult stem or progenitor cells reside at restricted locations to allow continuation of the cycle of life . these tissue - specific stem cells have been identified in the bone marrow , brain , skin , retina , pancreas , intestinal crypt , and liver as well as in skeletal muscle . depending on the origin , these adult stem cells exhibit the potency to differentiate into multiple cell types . this is attractive because , for example , unlike extraction of the neural stem cells or neural precursors that would require surgery , mscs are relatively easy to isolate and differentiate into neuronal cells , and therefore may be useful for tissue engineering strategies to repair and regenerate connective and excitable tissues . however , the limited life span and amount of cells as well as their heterogeneity pose difficulties in utilizing this particular cell population in the basic research and therapeutic applications [ 1720 ] . in the context of tissue engineering ,
unlike engineering tissue constructs with differentiated cells , the multipotent mesenchymal stem cells ( mscs ) are hypoimminogenic , have higher proliferative capacity and provide excellent regenerative capability that will likely lead to desired integrity and functionality of the engineered tissue . it is plausible that stem cell - based tissue implants can be designed for tissue formation and vascularization at the same time , which mimics typical physiological responses to tissue damage . however , a clearer understanding and adequate control of stem cell differentiation into tissue - specific lineages would first have to be firmly established . for example , development of the strategies to control stem cell behaviors and directing them to a specific lineage would require an understanding and manipulation of the cell - environment interactions . determination of an optimal physicochemical microenvironment could lead to harvest the unique properties of the stem cells for self - renewal and tissue - specific differentiation that can be guided or perhaps directed by biochemical and physical cues . rational approaches for stem cell - based engineering of functional tissues would then be realized and could be implemented , thus potentially overcoming the current challenges in regenerative medicine . the therapeutic applications of stem cells would undoubtedly be enhanced by an efficient differentiation protocol that could prescribe the phenotype . in this review paper
, we will first illustrate that the soluble factors and external physical forces ( i.e. , physicochemical cues ) have been combined , engineered and applied to induce synergistic msc differentiation
. we will next elucidate the potential mechanism(s ) that mediate the msc differentiation in response to the physicochemical cues . a detailed understanding of the lineage - dependent mechanisms
would be expected to prescribe the msc differentiation into specific tissue types using multiple cues . , hormones ) have been used to induce stem cell differentiation into bone cells [ 2224 ] , chondrogenic cells , adipogenic cells [ 26 , 27 ] , and even functional neuronal cells [ 16 , 2830 ] . this is consistent with the finding that the cell adhesion of several distinctive types may be involved , including focal complexes , focal adhesion , and fibrillar adhesion . , oxygen tension ) have also been incorporated to solicit synergistic stem cell differentiation . since the electrical and mechanical nature of the cell is now well established , we suggested that cellular and molecular responses to external physical stimuli would be cast more appropriately as electromechanical responses . because there appear to be several physicochemical differentiation cues that promote stem cell differentiation , strategies to optimize these cues to facilitate or interfere with the intended fate of stem cells may be highly significant for regenerative tissue engineering and also in general stem cell biology . however , the focus of this review is to illustrate synergistic stem cell differentiation in response to a combination of soluble factors and externally applied physical stimuli , which may additively or synergistically facilitate the intended msc differentiation . , quantifiable tissue - specific markers ) produced by multiple cues should be the same as the sum of each cue used alone , synergistic differentiation would imply greater than the sum of effects produced by each cue when a combination of the physicochemical cues is applied . for example , a combination of soluble factors and an electrical stimulation induces synergistic osteodifferentiation , while the use of an electrical stimulation alone fails to differentiate mscs . synergistic interaction may also lead to accelerate the pace at which the intended stem cell differentiation proceeds . strategies to optimize various differentiation cues to facilitate and interfere with the intended fate of stem cells may be highly significant for regenerative tissue engineering and also in general stem cell biology . in addition to demonstrating synergistic stem cell differentiation in response to a combination of soluble factors and externally applied physical stimuli , elucidation of the potential coupling mechanisms would have to be studied . both mechanical and electrical stimulation has been applied separately and combined with soluble factors to facilitate msc differentiation . for example , recent studies show that , when vascular endothelial growth factor ( vegf ) and a shear stress are used in a combinatorial manner , synergistic differentiation of endothelial progenitor cells into endothelial cells may be accomplished , and synergistic chondrogenic differentiation of mscs achieved by combining tgf-3 and a hydraulic pressure . alternatively , soluble factors combined with an electrical stimulation induce synergistic osteodifferentiation , while the use of the electrical stimulation alone fails to differentiate mscs toward osteoblasts . varying the modality of electrical stimulation , pulsatile biphasic electric field is now shown to enhance msc proliferation and upregulate the differentiation related genes , and an sinusoidal electrical stimulus ( 100 mv peak - to - peak at 4,000 hz ) appears to induce differentiation of rat pc12 and blastocyst - derived murine embryonic stem cells toward the neuronal lineage . of potential mechanisms that might mediate the effects of multiple differentiation cues , we will focus on the three recent but likely mechanisms that have been carefully studied and documented . second , mitogen - activated protein ( map ) kinase - dependent signaling has emerged as a potent regulator of stem cell commitment and differentiation [ 61 , 62 ] . since mechanotransduction is likely associated with map kinase signaling , the potential coupling mechanisms to the map kinase signaling by the differentiation cues could offer beneficial strategies for tissue engineering . moreover , a close correlation or interdependence between the [ ca]i and cytoskeletal reorganization is well known , established , and accepted , although the effects of altered and modulated [ ca]i oscillation on the cellular mechanics are yet to be fully elucidated . it is interesting to note that , in response to osteogenic soluble factors or physical stimuli , the human msc cellular mechanics become indistinguishable from that of osteoblasts in about 10 days , and the maximal changes in the ca dynamics also takes about 14 days . this may suggest that the biomechanical remodeling could precede the cellular and molecular identities of stem cell - derived osteoblasts , and that the microfilament - based biomechanical remodeling and [ ca]i oscillation are intimately connected . calcium ( ca ) is a ubiquitous second messenger and represents one of the most important biological signals . the intracellular ca regulates important cellular and molecular processes such as proliferation , differentiation , cell biomechanics , cytoskeletal reorganization , gene expression and metabolism [ 21 , 65 , 66 ] . while the role of oscillatory ca signals has been extensively studied but not yet fully understood , it is evident that spatial and temporal patterns of ca dynamics ( e.g. , spiking amplitude and frequency , and spatial distribution ) are important characteristics of cellular regulatory pathways . while biochemical cascades , molecular and cellular interactions , and gene expression profiles can be controlled by altered intracellular ca dynamics , intracellular calcium activities themselves can be regulated by a variety of external physical stimuli , including electromechanical stimulation and substrate rigidity . more specifically , spontaneous [ ca]i oscillations are readily observed in mscs [ 24 , 74 ] and can affect stem cell differentiation [ 58 , 59 ] . our laboratory has recently demonstrated that [ ca]i oscillations in mscs are dramatically altered in response to the soluble factors and an electrical stimulation . indeed , the physicochemical cues alter the calcium dynamics in mscs to resemble that found in terminally differentiated osteoblasts . however , application of an electrical stimulation by itself failed to induce msc differentiation , suggesting that an externally applied physical cue may amplify but not necessarily activate the mechanisms involved in osteodifferentiation . indeed , an abundance of the l - type channels fluorescently visualized in undifferentiated mscs ( data not shown ) indicates that these channels are involved in the regulation of the [ ca]i oscillation . third , integrins have been shown to regulate the ca dynamics which , in turn , control integrin - mediated adhesion , and cell migration through phosphorylation of focal adhesion kinase ( fak ) . this suggests a close association or correlation between differential focal adhesions formed in response to physicochemical cues and cell type - dependent [ ca]i oscillation . while several laboratories have shown evidence for induction of neuron - like cell , astrocytes , and glial cells [ 16 , 7982 ] , other reports suggest that in vitro differentiation protocols for bone marrow stromal cells disrupt actins and apparent neuronal morphology is therefore observed , or cellular makers in response to the neuroinductive factors could be attributed to cellular toxicity and changes in the cytoskeleton . this type of pattern is unique to the neurogenic cells and not found in any of the five cell types we have tested thus far , including undifferentiated human or rat mscs , p19 mouse embryonic stem cells , osteoblasts , fibroblasts , and even primary myocytes . since erk activation is also involved in stem cell differentiation , up - regulation of the n - type ca channels is expected as mscs undergo neurodifferentiation . it also appears that , at least for osteogenic differentiation , sustained map kinase activation promotes cell differentiation , suggesting an interesting hypothesis that a physical stimulation may be used to sustain and prolong the map kinase activation . at least three major modules of the map kinase signaling cascades have been identified , including the extracellular signal - regulated kinase ( erk ) , the c - jun amino - terminal kinase / stress activated protein kinase ( jnk / sapk ) , and the p38 kinase . of these three modules , the erk signaling pathway represents the most extensively studied mechanism . the jnk / sapk kinases have been shown to respond to external stress including heat shock , and cytokines . this observation raises a rather important hypothesis that , when cells are exposed to external physical stimuli in the presence of the mitogenic agents , the differential activation of map kinases could be combined , and synergistic biological responses such as cell proliferation and differentiation may be induced . it is noted here however that , in response to mechanical stimulation , the erk and p38 kinase cascades can be activated [ 100 , 101 ] , and tethering and rolling of neutrophils in shear flow activates integrins and induces firm adhesion through phosphorylation of the p38 kinase . unlike soluble factors that can readily bind to their receptors with a degree of specificity , the physical cues do not preferentially discriminate the targets . for example , several mechanotranducers have been identified that include integrin - dependent focal adhesion [ 103107 ] and ion channels [ 108 , 109 ] , and integrin clustering has been shown to be responsible for transducing electrical stimulation [ 110 , 111 ] . in addition to the critical role in formation of focal adhesions , integrins are also linked to cell proliferation , differentiation , migration , and apoptosis [ 112 , 113 ] . moreover , integrins are essential for normal development of hematopoietic lineages and bone marrow by regulating cell proliferation and differentiation , and cardiomyocyte cell cycle depends on cell attachment via 1-integrins . in spite of these diverse cellular responses regulated by integrins ,
it appears clear that the integrin - matrix interaction can activate map kinases , thus suggesting that map kinases act as one of the key molecules that connect integrins at the cell surface with adhesion - dependent gene expressions . this g - protein mechanism has been demonstrated in human osteoblasts and human mscs using specific plc inhibitors ( e.g. such assembly of adhesion proteins may mimic the integrin - matrix interactions , which are known to regulate the map kinase activities . in effect , the map kinase cascade is synergistically amplified and cell differentiation is facilitated . alternatively , the second coupling mechanism involves activation of g - protein coupled receptors in the cell membrane ( figure 3(b ) ) that leads to the pkc pathway . a cell has a remarkable capability to detect external physical forces of various modalities and adapt to the biomechanical environment by adjusting its mechanical properties to match those of the surrounding tissue . clearly , cells do respond to the physical forces and mechanically remodel themselves in order to transduce the physical signals . the concept of mechanotransduction relies on the bi - directional cross - talk between the cell and microenvironment [ 108 , 124 , 125 ] that modulates biochemistry in the cytoplasm and ultimately affects the nucleus . cytoskeleton is thought to be one of the most significant cellular mechanical components and provides structural stability and elasticity to the cell undergoing multiple deformations without losing its integrity [ 127 , 128 ] . , substrate stiffness , cell shape and deformation , external pressure , shear stress ) by structural rearrangement of the cytoskeleton itself , or alterations in gene expression profiles , cell adhesion , and secretion of extracellular matrix ( i.e. complex interactions between these multiple signaling molecules , likely triggered by external signals , cause activation of downstream target proteins , which could lead to a global structural rearrangement of the cytoskeleton or altered gene transcription profiles affecting cell adhesion , secretion of extracellular matrix components , and cell metabolic activity . substantial structural and functional differences of cytoskeletons in cells of the same mesodermal origin ( myocytes , osteoblasts , endothelial cells , kidney cells , etc . ) due to the important role of the membrane in many cellular functions , it is likely involved in the intricate interplay of events accompanying cell differentiation . beside its function as a barrier from the outer environment , it participates in inward - outward trafficking , motility , cell adhesion to extracellular matrix , and cell - cell interaction [ 131 , 132 ] . in human mscs , a lower membrane tension is observed that may better facilitate endo- and exocytosis and contributes to a higher sensitivity of these unqiue cells to various soluble biochemical environmental stimuli . moreover , we have recently reported an interesting observation [ 21 , 63 ] that the cell type - dependent linkage between the membrane - cytoskeleton could be regulated by the linkers proteins [ 137141 ] , including ezrin , radixin , and moesin ( erm proteins ) . interestingly , because the erm proteins are substrate for rhoa , the involvement of rhoa in stem cell differentiation in response to the extracellular environment may now be better understood and is likely to depend on the erm protein distribution and activation . mechanotranduction may be mediated by the target molecules at the cell surface or by a
hire - wired tensegrity network of cytoskeleton [ 108 , 126 , 143 ] . in response to a physical force
, mechanically sensitive molecules such as integrins , ion channels , and g - protein coupled receptors could be activated and initiate downstream signaling cascades and alter gene expression [ 108 , 109 , 144 ] . this view of mechanotranduction has been supported by evidence that includes integrin blocking by antibodies [ 111 , 145 ] and inhibiting stretch - activated cation channels ( saccs ) [ 146 , 147 ] . incorporating afm - based microindentation technique to measure the cellular elasticity and assessing the membrane tension by optically pulling membrane tethers from the cell body [ 63 , 136 ] , the terminally differentiated cells ( e.g. moreover , actin remodeling and physical coupling between the cell membrane and cytoskeleton , mediated by linker proteins , are now shown to be responsible for such dramatically different biomechanical properties . indeed , recent work from our laboratory indicates that , when human mscs are transfected to transiently suppress the erm linker proteins ( ezrin , radixin , and moesin ) , osteogenic differentiation is either delayed or perhaps inhibited , and the cell membrane - cytoskeleton coupling is weakened . the erm proteins function not only as the connector linking the membrane with microfilaments but also are now known to regulate signaling . this is consistent with the erm proteins ' expression that promotes a stronger association between the cell membrane and cytoskeleton . while this model is not intended to represent an exhaustive list of possibly numerous pathways , the differentiation cues may influence the cellular biomechanics through utilizing both ca - dependent and -independent pathways ( see figure 5 ) . soluble factors either bind to the cell surface receptors or diffuse through the cell membrane , and alter gene expressions , modulate [ ca]i oscillation and reorganize cytoskeleton . an elevated [ ca]i level is likely to cause depolymerization of f - actins and therefore an overall decrease in the cell elasticity , thus reinforcing the effects of the soluble factors . , atp release ) , which in turn leads to inhibition of erm proteins ' binding affinity and dissociation of the cytoskeleton from the membrane . the linker proteins seem to play a significant role in modulation of the cell mechanics that is a critical factor in the regulation and even manipulation of stem cells . it is interesting to know that an electrical stimulation alone fails to initiate msc osteodifferentiation but , when combined with the soluble factors , causes a synergistic stem cell differentiation . this brings the cell elastic and structural properties closer to those of fully differentiated osteoblasts , and induces the cell membrane dissociation from cytoskeleton , and consequently decreases the membrane tension to enhance endocytosis and transmembrane trafficking of the soluble factors . when cells are allowed to recover following the electrical exposure in presence of soluble factors
, a further rearrangement of actin and erm proteins will proceed along with expression of osteogenic markers . therefore , a higher physical stimulation might be required to facilitate neurogenic differentiation , which will maximally disrupt the actin cytoskeleton and inhibit erm linkers , suggesting a plausible approach for precisely controlling the physical parameters for selective manipulation of mechanical properties of a particular and pre - selected cell phenotype . taken together , optimal use of the physicochemical cues may lead the researcher to develop strategies for tissue engineering by manipulating cell differentiation , mobility , and cell incorporation into engineered scaffolds , and eventual maturation of tissue substitutes . an in depth understanding of mechanisms that allow regulation of the cell biomechanical and biochemical properties will undoubtedly lead to a more effective development of therapeutics for regenerative medicine . while biologists have long appreciated the important role for soluble factors in the regulation of stem cell differentiation , the mechanical and electrical stimuli are now firmly established to significantly influence the stem cell fate . therefore , the idea of combining multiple differentiation cues is gaining traction and could lead to an active research area of stem cells and perhaps unravel their intricate biological and physical properties . however , completion of the all possible combinatory experiments may be laborious , and it is unlikely that we can determine a set of combinational cues that is widely applicable irrespective of tissue phenotypes . in spite of such difficulties , advances in the molecular , biophysical , and imaging tools
should allow a more detailed understanding of the coupling mechanisms that regulate stem cell differentiation in the near future . establishment of new paradigms in which stem cells can be manipulated physicochemically will undoubtedly expedite the current effort for stem cell - based regenerative medicine by targeting specific combinations of physicochemical cues that optimally exploit the unique biological and biophysical properties of stem cells for the intended differentiation . | [
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] |
alongside the continued , albeit geographically limited , engagement of bilateral cooperation and non - governmental organizations ( ngos ) , it was the commitment of private sector in 2001 to support who that marked a turning point in the fight against human african trypanosomiasis ( hat )
. from that moment ,
control actions by the national sleeping sickness control programmes ( nsscps ) improved across the continent , and a dramatic decrease in the number of gambiense hat cases followed . the annual number of cases reported to who by disease - endemic countries dropped by 73% from 25,865 in 2000 to 7,106 in 2012
.
this observed drop in cases reported was accompanied and made possible by strengthened capacity of nsscps to perform active and passive case - detection , as well as an improvement in reporting
.
the withdrawal of ngos which focused on hat emergencies ( while in the early 2000 31 ngo - run programmes were active , in december 2012 only two were registered ) and the difficulties in recruiting patients for clinical trials to develop new tools for hat control , support the notion that the trend reported over the last decade reflects a real abatement of disease transmission in the field .
furthermore , substantially improved management and reporting of data embodied in the hat atlas initiative
also contribute to the reliability of the available epidemiological information .
nevertheless , because of the occurrence of gambiense hat in remote rural areas and the weakness of health systems , an unspecified number of sleeping sickness cases still escape detection and reporting .
for gambiense hat little is known of the true extent of this underreporting phenomenon , and specific studies are needed to estimate its magnitude and thereby accurately to assess the progress made in curbing transmission globally . at the same time , some insights into the likelihood of underdetection / underreporting can be gained by looking at the distribution and intensity of control and surveillance activities involving both mobile teams and fixed health facilities . arguably , a narrow focus on active case - finding activities alone may give the impression that only a small proportion of the population at - risk of hat is reached by control and surveillance activities . in fact , while recent estimates put the population at risk of gambiense hat at approximately 57 million people
, only an average of 2 million are screened every year by mobile teams ( period 2000 - 2012 ) ( data not shown ) .
active surveillance alone appears inadequate to ensure a satisfactory coverage of populations at - risk of gambiense hat , and consequently it may seem reasonable to question the reported abatement in disease transmission .
however , the role played by fixed health facilities embedded in the health system must be taken into account if a complete picture of the coverage and outcomes of hat control and surveillance is to be drawn . indeed ,
control of gambiense hat has relied , in addition to the impressive and well known active case - finding surveys carried out by mobile teams , also on the quiet contribution of passive screening performed in fixed health structures .
for example , in the period 2000 - 2012 , 93,561 cases of gambiense hat ( 48.9% of the reported total ) were diagnosed by passive case - finding carried out in fixed health facilities ( table
1 ) .
cases of gambiense hat diagnosed by active and passive case - finding ( period : 2000 - 2012 ) " - " , no data reported .
however , the lack of consistent and detailed data on the extent , capacities and coverage of passive case - finding and treatment constrains our ability to appreciate its full relevance in hat control and surveillance .
the paper explores the geographic distribution and the diagnostic and treatment capabilities of fixed health facilities involved in gambiense hat control and surveillance .
it also aims to estimate the at - risk population potentially covered by passive case - finding and treatment by using geographic information systems ( gis ) .
the inventory of this passive surveillance and treatment network provides the dimension of what is available today . at the same time
, it gives insights into the work needed in terms of capacity development and improvement and expansion of the network , aiming at covering the whole population at risk .
the paper focuses on all endemic countries having reported gambiense hat cases or having conducted active screening activities during the period 2000 - 2012 ( i.e. angola , benin , burkina faso , cameroon , central african republic , chad , congo , cte divoire , democratic republic of the congo , equatorial guinea , gabon , ghana , guinea , mali , nigeria , sierra leone , south sudan , togo and uganda ) . capabilities for diagnosis and treatment in non - endemic countries have been reviewed elsewhere
.
rhodesiense hat being characterized by different epidemiological patterns and approaches to disease control is not directly considered in this study .
fixed health facilities active in gambiense hat control and surveillance were identified through direct interviews with the coordinators of the nsscps .
standardized forms were circulated requesting information on the name and location of the health structures , and on their diagnostic and treatment capabilities for hat .
clinical diagnosis ( dxc ) refers to the ability of health centres systematically to raise a clinical suspicion of trypanosomal infection and refer the suspect to the next level for further diagnosis or , if available , to perform it in the same facility .
serological diagnosis ( dxs ) concerns the capability of performing serological tests , thus identifying serological suspects . in this context , the serological test considered is the card agglutination test for trypanosomiasis
parasitological diagnosis ( dxp ) pertains to the competence of the health centre to execute any type of parasitological test in order to confirm trypanosome infection in clinical or serological suspects through direct observation of the parasite in body fluids .
health centres were also classified according to their capability to carry out the stage determination ( dxph ) by examination of cerebrospinal fluid obtained through lumbar puncture .
this includes treatment of first stage infections with pentamidine ( tx1p ) , and treatment of second - stage cases with melarsoprol ( tx2 m ) , eflornithine ( tx2e ) or nifurtimox - eflornithine combination therapy
all data on the hat health centres provided by nsscps were harmonized and assembled in a database .
mapping was subsequently carried out using geo - referencing procedures already described
. physical accessibility to hat diagnosis and treatment was estimated through a cost - distance function .
this type of geographical functions determines the shortest weighted distance ( or least cumulated travel time - cost ) from any location to the nearest destination . in our study ,
travel cost was measured in time units , and the destinations were represented by health facilities having capabilities for hat diagnosis and treatment .
such gis maps as land cover , water bodies , slope and road network are combined to empirically derived travel speeds to estimate a pixel - by - pixel travel time .
the friction grid utilized in this study has a spatial resolution of approximately 1-km ( i.e. 30 arcseconds ) .
importantly , affordability ( or economic cost of travel ) is not considered in this model .
the least time - cost path function enabled a map of accessibility to be generated across the study countries , whereby the shortest travel time to the closest hat diagnostic or treatment facility was estimated for each pixel .
in particular , results are provided for three different travel time - cost thresholds : 1 , 3 and 5 hours .
total population coverage was subsequently stratified by level of gambiense hat risk . to this end , a previously derived risk surface for gambiense hat was used , which was based on spatial smoothing of hat reported cases and human population layers
. from this dataset three broad categories of risk were extracted : high and very high ( 1 hat case per 10 people per annum
p.a . ) , moderate ( 1 hat case per 10 people and < 1 hat case per 10 people p.a . ) , and low and very low ( 1 hat case per 10 people and < 1 hat case per 10 people p.a . )
risk is based on the reported number of gambiense hat cases in the period 2000 - 2009 and on landscan human population layers for the same period
. as a backdrop , the predicted distribution of the palpalis group of tsetse flies is provided .
the palpalis group ( genus : glossina , subgenus : nemorhina ) includes all the major vector species of gambiense hat . for estimating both the total population coverage and the coverage of at - risk population , the dataset landscan 2009
this was the same layer previously used to estimate at - risk population
, and therefore it provided a precise baseline for comparison .
uncertainties and limitations of the available global human population layers , including landscan , have been discussed in a number of papers ( e.g.
) .
the study is based on the analysis of a database obtained through administrative data provided by health authorities in the selected countries .
therefore for this study no institutional review board was sought and the helsinki declaration and ethical guidelines for biomedical research were not applicable .
fixed health facilities active in gambiense hat control and surveillance were identified through direct interviews with the coordinators of the nsscps .
standardized forms were circulated requesting information on the name and location of the health structures , and on their diagnostic and treatment capabilities for hat .
clinical diagnosis ( dxc ) refers to the ability of health centres systematically to raise a clinical suspicion of trypanosomal infection and refer the suspect to the next level for further diagnosis or , if available , to perform it in the same facility .
serological diagnosis ( dxs ) concerns the capability of performing serological tests , thus identifying serological suspects . in this context , the serological test considered is the card agglutination test for trypanosomiasis
parasitological diagnosis ( dxp ) pertains to the competence of the health centre to execute any type of parasitological test in order to confirm trypanosome infection in clinical or serological suspects through direct observation of the parasite in body fluids .
health centres were also classified according to their capability to carry out the stage determination ( dxph ) by examination of cerebrospinal fluid obtained through lumbar puncture .
this includes treatment of first stage infections with pentamidine ( tx1p ) , and treatment of second - stage cases with melarsoprol ( tx2 m ) , eflornithine ( tx2e ) or nifurtimox - eflornithine combination therapy
all data on the hat health centres provided by nsscps were harmonized and assembled in a database .
physical accessibility to hat diagnosis and treatment was estimated through a cost - distance function .
this type of geographical functions determines the shortest weighted distance ( or least cumulated travel time - cost ) from any location to the nearest destination . in our study ,
travel cost was measured in time units , and the destinations were represented by health facilities having capabilities for hat diagnosis and treatment .
such gis maps as land cover , water bodies , slope and road network are combined to empirically derived travel speeds to estimate a pixel - by - pixel travel time .
the friction grid utilized in this study has a spatial resolution of approximately 1-km ( i.e. 30 arcseconds ) .
importantly , affordability ( or economic cost of travel ) is not considered in this model .
the least time - cost path function enabled a map of accessibility to be generated across the study countries , whereby the shortest travel time to the closest hat diagnostic or treatment facility was estimated for each pixel .
in particular , results are provided for three different travel time - cost thresholds : 1 , 3 and 5 hours .
total population coverage was subsequently stratified by level of gambiense hat risk . to this end ,
a previously derived risk surface for gambiense hat was used , which was based on spatial smoothing of hat reported cases and human population layers
. from this dataset
three broad categories of risk were extracted : high and very high ( 1 hat case per 10 people per annum
p.a . ) , moderate ( 1 hat case per 10 people and < 1 hat case per 10 people p.a . ) , and low and very low ( 1 hat case per 10 people and < 1 hat case per 10 people p.a . ) ( figure
1 ) .
risk is based on the reported number of gambiense hat cases in the period 2000 - 2009 and on landscan human population layers for the same period
. as a backdrop ,
the palpalis group ( genus : glossina , subgenus : nemorhina ) includes all the major vector species of gambiense hat . for estimating both the total population coverage and the coverage of at - risk population , the dataset landscan 2009
this was the same layer previously used to estimate at - risk population
, and therefore it provided a precise baseline for comparison .
uncertainties and limitations of the available global human population layers , including landscan , have been discussed in a number of papers ( e.g.
) .
the study is based on the analysis of a database obtained through administrative data provided by health authorities in the selected countries .
therefore for this study no institutional review board was sought and the helsinki declaration and ethical guidelines for biomedical research were not applicable .
information provided by the nsscps revealed the existence of 632 fixed health facilities that are involved in the control and surveillance of gambiense hat in disease - endemic countries .
their diagnostic and treatment capacities are summarized in table
2 , while the complete list of health facilities including the names and geographical coordinates is provided in additional file
1 .
number of fixed health facilities providing diagnosis and treatment for gambiense hat study area : disease - endemic countries having reporting cases or having conducted active screening activities during the period 2000 - 2012 .
dxc : clinical diagnosis ; dxs : serological diagnosis ; dxp : parasitological diagnosis ; dxph : disease staging .
tx1p : treatment of first - stage infection with pentamidine ; tx2 m : treatment of second - stage infection with melarsoprol ; tx2e : treatment of second - stage infection with eflornithine ; tx2n : treatment of second - stage infection with nifurtimox - eflornithine combination therapy ( nect ) ; tx2 : treatment of second stage . * after closing the study , information was received that angola upgraded the tx2e centres to tx2n .
diagnosis is available in 622 facilities , 84.2% of which are found in the democratic republic of the congo ( drc ) .
although all of the 622 centres are able to identify clinical suspects , only 56% of them are able to go further performing serological test .
the number of centres able to carry out parasitological diagnosis ( dxp ) and disease staging ( dxph ) decreases to 49% and 39% respectively , mainly due to the need for complex tools and highly skilled staff .
access to treatment for gambiense hat is slightly more limited ( 495 health centres 81.6% of which in drc ) .
while all of these centres are able to offer treatment for first - stage disease , only 36.4% can offer first line treatment for second - stage disease ( tx2n ) .
this is mainly due to the challenges inherent in the distribution and the administration of nect .
figure
2 shows the geographic distribution of the health structures involved in gambiense hat diagnosis ( a ) and treatment ( b ) . in this figure
separate maps showing the distribution of centres able to perform the various types of diagnosis and treatment are provided in additional file
2 .
distribution maps at the national and focus level will be made available from the who hat web site (
http://www.who.int/trypanosomiasis_african/en/ ) .
geographic distribution of fixed health facilities having capacities for gambiense hat diagnosis ( a ) and treatment ( b ) .
data were collected by who between december 2012 and august 2013 from national sleeping sickness control programmes in the study countries ( i.e. angola , benin , burkina faso , cameroon , central african republic , chad , congo , cte divoire , democratic republic of the congo , equatorial guinea , gabon , ghana , guinea , mali , nigeria , sierra leone , south sudan , togo and uganda ) . the map of physical accessibility to hat diagnostic facilities is shown in figure
3 . in the study countries
, we estimate that 31 , 85 and 154 million people are respectively within 1 , 3 and 5 hours travel of a facility with capacity for hat diagnosis .
travel time ( or physical accessibility ) to health facilities having capabilities for diagnosis of gambiense hat [ hours ] .
travel cost was measured in time units , and the destinations were represented by health facilities having capabilities for hat diagnosis ( figure
2a ) .
focusing on gambiense hat at - risk population , their coverage by diagnostic facilities is summarized in table
3 .
our analysis indicates that 23.4 , 40.2 , and 47.4 million people at risk are respectively within 1 , 3 and 5 hours travel of a facility with capacity for gambiense hat diagnosis .
this corresponds to 41 , 71 , and 83% of the total at - risk population .
for the 10-hour travel threshold , the coverage of at - risk population is 96% ( additional file
3 ) .
people at risk of gambiense hat that are potentially covered by facilities with diagnostic capabilities people at risk are stratified by risk categories as defined in
.
coverage is not homogeneous across the risk categories , with higher risk categories being characterized by comparatively better coverage .
for example , for populations at high and very high risk , coverage is estimated at 48 , 77 and 87% for the travel time thresholds of 1 , 3 and 5 hours respectively .
these percentages decrease for moderate and for low and very low risk categories the health systems deploying better equipped facilities and skilled staff in areas where transmission of the disease is more intense .
full details for a range of travel - times , for all study countries and for all risk categories are provided in additional file
3 .
these country - level data indicate that there are no major differences between countries in terms of the proportion of at - risk population potentially covered by hat diagnostic facilities .
results are summarized in table
4 , while detailed data for a range of travel - times , for all study countries and for all risk categories are provided in additional file
4 .
people at risk of gambiense hat that are potentially covered by facilities with different types of diagnostic capabilities for hat dxc : clinical diagnosis ; dxs : serological diagnosis ; dxp : parasitological diagnosis ; dxph : disease staging .
vh : very high risk ; h : high risk ; m : moderate risk ; l : low risk ; vl : very low risk .
the map of physical accessibility to hat treatment facilities is shown in additional file
5 .
we estimate that in the study countries 27 , 73 and 133 million people are respectively within 1 , 3 and 5 hours travel of a facility with capacity for gambiense hat treatment .
looking at population at risk , the coverage by treatment facilities is summarized in table
5 , while full details for a range of travel - times , for all study countries and for all risk categories are provided in additional file
6 .
our analysis indicates that 21.6 , 38.8 , and 46.5 million people at risk are respectively within 1 , 3 and 5 hours travel of a facility with capacity for gambiense hat treatment .
this corresponds to 38 , 68 , and 82% of the total population at risk .
for the 10-hour travel threshold , the coverage of at - risk population is 95% ( additional file
6 ) .
people at risk of gambiense hat that are potentially covered by facilities with treatment capabilities for hat people at risk are stratified by risk categories as defined in
.
results are summarized in table
6 , while detailed data for a range of travel - times , for all study countries and for all risk categories are provided in additional file
7 .
all facilities involved in gambiense hat treatment have capacity for treatment of the first - stage of the disease .
lower coverage is available for the treatment of the second - stage of the disease .
people at risk of gambiense hat that are potentially covered by facilities with different types of treatment capabilities for hat tx1p : treatment of first - stage infection with pentamidine ; tx2 m : treatment of second - stage infection with melarsoprol ; tx2e : treatment of second - stage infection with eflornithine ; tx2n : treatment of second - stage infection with nifurtimox - eflornithine combination therapy ( nect ) .
vh : very high risk ; h : high risk ; m : moderate risk ; l : low risk ; vl : very low risk .
information provided by the nsscps revealed the existence of 632 fixed health facilities that are involved in the control and surveillance of gambiense hat in disease - endemic countries .
their diagnostic and treatment capacities are summarized in table
2 , while the complete list of health facilities including the names and geographical coordinates is provided in additional file
1 .
number of fixed health facilities providing diagnosis and treatment for gambiense hat study area : disease - endemic countries having reporting cases or having conducted active screening activities during the period 2000 - 2012 .
dxc : clinical diagnosis ; dxs : serological diagnosis ; dxp : parasitological diagnosis ; dxph : disease staging .
tx1p : treatment of first - stage infection with pentamidine ; tx2 m : treatment of second - stage infection with melarsoprol ; tx2e : treatment of second - stage infection with eflornithine ; tx2n : treatment of second - stage infection with nifurtimox - eflornithine combination therapy ( nect ) ; tx2 : treatment of second stage . * after closing the study , information was received that angola upgraded the tx2e centres to tx2n .
diagnosis is available in 622 facilities , 84.2% of which are found in the democratic republic of the congo ( drc ) .
although all of the 622 centres are able to identify clinical suspects , only 56% of them are able to go further performing serological test .
the number of centres able to carry out parasitological diagnosis ( dxp ) and disease staging ( dxph ) decreases to 49% and 39% respectively , mainly due to the need for complex tools and highly skilled staff .
access to treatment for gambiense hat is slightly more limited ( 495 health centres 81.6% of which in drc ) .
while all of these centres are able to offer treatment for first - stage disease , only 36.4% can offer first line treatment for second - stage disease ( tx2n ) .
this is mainly due to the challenges inherent in the distribution and the administration of nect .
figure
2 shows the geographic distribution of the health structures involved in gambiense hat diagnosis ( a ) and treatment ( b ) . in this figure
separate maps showing the distribution of centres able to perform the various types of diagnosis and treatment are provided in additional file
2 .
distribution maps at the national and focus level will be made available from the who hat web site (
http://www.who.int/trypanosomiasis_african/en/ ) .
geographic distribution of fixed health facilities having capacities for gambiense hat diagnosis ( a ) and treatment ( b ) .
data were collected by who between december 2012 and august 2013 from national sleeping sickness control programmes in the study countries ( i.e. angola , benin , burkina faso , cameroon , central african republic , chad , congo , cte divoire , democratic republic of the congo , equatorial guinea , gabon , ghana , guinea , mali , nigeria , sierra leone , south sudan , togo and uganda ) .
the map of physical accessibility to hat diagnostic facilities is shown in figure
3 . in the study countries
, we estimate that 31 , 85 and 154 million people are respectively within 1 , 3 and 5 hours travel of a facility with capacity for hat diagnosis .
travel time ( or physical accessibility ) to health facilities having capabilities for diagnosis of gambiense hat [ hours ] .
travel cost was measured in time units , and the destinations were represented by health facilities having capabilities for hat diagnosis ( figure
2a ) .
focusing on gambiense hat at - risk population , their coverage by diagnostic facilities is summarized in table
3 .
our analysis indicates that 23.4 , 40.2 , and 47.4 million people at risk are respectively within 1 , 3 and 5 hours travel of a facility with capacity for gambiense hat diagnosis .
this corresponds to 41 , 71 , and 83% of the total at - risk population .
for the 10-hour travel threshold , the coverage of at - risk population is 96% ( additional file
3 ) .
people at risk of gambiense hat that are potentially covered by facilities with diagnostic capabilities people at risk are stratified by risk categories as defined in
.
coverage is not homogeneous across the risk categories , with higher risk categories being characterized by comparatively better coverage .
for example , for populations at high and very high risk , coverage is estimated at 48 , 77 and 87% for the travel time thresholds of 1 , 3 and 5 hours respectively .
these percentages decrease for moderate and for low and very low risk categories the health systems deploying better equipped facilities and skilled staff in areas where transmission of the disease is more intense .
full details for a range of travel - times , for all study countries and for all risk categories are provided in additional file
3 .
these country - level data indicate that there are no major differences between countries in terms of the proportion of at - risk population potentially covered by hat diagnostic facilities .
results are summarized in table
4 , while detailed data for a range of travel - times , for all study countries and for all risk categories are provided in additional file
4 .
people at risk of gambiense hat that are potentially covered by facilities with different types of diagnostic capabilities for hat dxc : clinical diagnosis ; dxs : serological diagnosis ; dxp : parasitological diagnosis ; dxph : disease staging .
vh : very high risk ; h : high risk ; m : moderate risk ; l : low risk ; vl : very low risk .
the map of physical accessibility to hat treatment facilities is shown in additional file
5 .
we estimate that in the study countries 27 , 73 and 133 million people are respectively within 1 , 3 and 5 hours travel of a facility with capacity for gambiense hat treatment . looking at population at risk , the coverage by treatment facilities is summarized in table
5 , while full details for a range of travel - times , for all study countries and for all risk categories are provided in additional file
6 .
our analysis indicates that 21.6 , 38.8 , and 46.5 million people at risk are respectively within 1 , 3 and 5 hours travel of a facility with capacity for gambiense hat treatment .
this corresponds to 38 , 68 , and 82% of the total population at risk .
for the 10-hour travel threshold , the coverage of at - risk population is 95% ( additional file
6 ) .
people at risk of gambiense hat that are potentially covered by facilities with treatment capabilities for hat people at risk are stratified by risk categories as defined in
.
results are summarized in table
6 , while detailed data for a range of travel - times , for all study countries and for all risk categories are provided in additional file
7 .
all facilities involved in gambiense hat treatment have capacity for treatment of the first - stage of the disease .
lower coverage is available for the treatment of the second - stage of the disease .
people at risk of gambiense hat that are potentially covered by facilities with different types of treatment capabilities for hat tx1p : treatment of first - stage infection with pentamidine ; tx2 m : treatment of second - stage infection with melarsoprol ; tx2e : treatment of second - stage infection with eflornithine ; tx2n : treatment of second - stage infection with nifurtimox - eflornithine combination therapy ( nect ) .
vh : very high risk ; h : high risk ; m : moderate risk ; l : low risk ; vl : very low risk .
the decreasing trend in the reported number of gambiense hat cases has led to target the disease for elimination in the who road map on neglected tropical disease
. however ,
the low number of people at risk covered by active case - finding surveys is often cited to raise doubts on the reliability of the reported figures . in this study we show that a narrow focus on the intensity of active case - finding is inadequate to fully describe the coverage of hat control and surveillance activities .
gambiense hat is a chronic disease that is characterized by a sufficiently long duration
to allow passive case detection in fixed health facilities to complement the coverage of active case detection . at the same time , the interval between infection and diagnosis in fixed health structure allowed the persistence and the spread of the disease . as a matter of fact , active screening by mobile teams that has been decisive in tackling the worrisome situation of hat at the beginning of this century ,
covered locations are rarely visited more than once a year , and the interval between successive visits is often longer . even on the occasion when a village is visited by a mobile team , coverage is unfortunately far from 100%
.
because of these limitations of active case - finding , the role of the fixed health facilities in gambiense hat transmission areas has been extremely important to fill a part of the screening and coverage gap . indeed , passive screening detected 93,561 cases during the period 2000 - 2012 , which represent approximately half of the total cases reported .
the present analysis reveals the existence of a network of fixed health facilities involved in the passive control and surveillance of gambiense hat .
a census of the diagnosis and treatment capabilities was made for the 632 fixed health facilities identified .
it must be stressed that our survey did not address the quality control of services provided in the fixed health facilities involved in hat control and surveillance , nor did it explore population attendance .
the results illustrate the substantial attempts made by the health system in disease - endemic countries in tackling the disease , although efforts on capacity building and equipment must be undertaken to get this network fully operational . over 80% of the population at - risk lives within 5-hour travel of a fixed health facility offering diagnosis and treatment for gambiense hat .
the results also indicate that coverage for clinical diagnosis is the most widely available , while access to serological and parasitological diagnosis becomes progressively more difficult .
this observation supports the notion that that the current test for serological screening ( catt ) , although very useful for serological mass screening by mobile teams , does not fulfil the requirements for its use in the rural health facilities where gambiense hat is endemic most notably because of the format ( 50 tests per vial ) and the need for cold chain and electric power .
the new individual screening tests ( ist , often called rapid diagnostic tests ) do not require cold chain nor electric power , and they hold the potential to expand the network of fixed health facilities able to detect serological suspects which is presently available only in 55.6% of the facilities able to detect clinical suspects and therefore they could widen and improve access to gambiense hat diagnosis
. concerning parasitological diagnosis ,
the coverage gap can partly be ascribed to the cumbersome nature of and the skilled staff required by the current parasitological diagnostic tests .
first evaluation on clinical samples of new molecular techniques procedures ( i.e. the loop - mediated isothermal amplification lamp ) has shown similar performance to conventional pcr
. however , lamp is still more complicated than the parasitological methods available and requires further studies to assess its accuracy .
therefore it does not fulfil all requirements to replace the laborious process for demonstrating the presence of the parasite in body fluids of people giving positive results in serological screening tests .
at this point in time , the weakness of laboratory capacities in the rural health facilities remains a challenge for parasitological diagnosis of gambiense hat
.
concerning treatment , thanks to the fact that some cases diagnosed and staged in highest centres as first stage can be referred for treatment to lower health centres , 82% of the people at risk of gambiense hat have access to a centre offering treatment for first - stage patients within a 5-hour travel - time , while 69% have access to a health care facility administering the recommended first line treatment for second - stage gambiense hat ( nect ) within the same travel - time threshold .
these figures show that people affected by second - stage gambiense hat have to make more efforts to receive appropriate treatment .
this is mainly because of the complexity of administration and the demanding logistics of current first line treatment for second - stage gambiense hat .
the hopes to overcome these hurdles rest in the ongoing development of new drugs not requiring skilled staff and complex equipment ( i.e. fexinidazole and oxaborol )
. an oral , safe drug , effective for both stages of the disease could greatly improve access to treatment ; at the same time , it would remove the need for staging through lumbar puncture , which is not always well accepted and can only be offered by 38.5% of the facilities involved in the treatment of gambiense hat . in the light of the new challenges to achieve sustainable elimination of gambiense hat , the meeting on hat elimination held in geneva in december 2012
emphasized the importance of maintaining mobile teams in selected areas and in particular epidemiological circumstances , as well as the value of reinforcing the health system and involving it fully in passive control and surveillance .
this message has been voiced by the disease - endemic countries
and in a recent who expert committee on hat
. strengthening and expanding fixed health facilities involved in control and surveillance to reach
support from both international partners and from operational national teams is needed to monitor and evaluate the network , as well as to collect and analyze data obtained and to provide appropriate response .
it is important to note that two factors have contributed to the good performance of passive detection in recent years : the knowledge of the disease among health staff and among affected communities .
this knowledge facilitates diagnosis both at the level of fixed health facilities , but also at the community level , where infected individuals can be identified and advised to visit a hospital for proper diagnosis and care .
retirement of skilled staff and the decreasing number of cases in the community reduce familiarity with the disease and can greatly decrease the effectiveness of passive diagnosis .
a need exists therefore for capacity building of health staff , for less complicated control tools , and for awareness - raising campaigns among populations at risk of gambiense hat
. moreover , low attendance rates in some health care facilities ( the average annual attendance rate of health services in drc is estimated at 0.15 per inhabitant
) are unfortunately not only due to difficulties at the technical level ( i.e. skilled staff and adapted tools ) but are also at the structural level .
indeed , attendance of populations to health facilities does not depend only on physical accessibility , but also on economic and socio - cultural factors and on the perceived benefit of skilled care
.
furthermore , due to the location of the main fixed health structure ( towns ) , passive screening is likely to address more urban than rural people .
therefore , attention must be paid to appreciate the level of attendance of population at risk of hat ( e.g. number of people screen by new ist by year and by village of origin ) , as well as their characteristics ( sex , age , urban / rural , activities , etc . ) in order to evaluate the coverage of fixed health facilities in the case of hat surveillance .
on the other hand , attendance to facilities providing services of diagnosis of hat does not mean that the tests available are applied automatically .
several visits are sometimes necessary before hat diagnostic tools are applied , and awareness of staff in the presence of the disease is required to prescribe their use
.
continuous monitoring and evaluation are therefore needed , with a view to ensuring that an effective surveillance of gambiense hat is in place to sustain the disease elimination process .
the data and coverage estimates herewith presented will be regularly updated , thus continuously providing evidence to inform a rational deployment of control and surveillance efforts .
coverage estimates of population at risk will also provide indicators to monitor the process of elimination of gambiense hat
.
gc supervised the technical aspects related to data management and gis , and conducted the analysis .
pps , jrf , ad and jar collated and screened the data used as input .
pps , jrf , rcm and jgj coordinated and supervised the collaboration between who and fao in the framework of the programme against african trypanosomosis ( paat ) .
all authors have contributed to conceptualizing the manuscript , and commented on and approved the final draft .
fixed health facilities that are active in the control and surveillance of gambiense hat . table s1 .
fixed health facilities that are active in the control and surveillance of gambiense hat in angola , benin , burkina faso , cameroon , central african republic , chad , congo , cte divoire , equatorial guinea , gabon , ghana , guinea , mali , nigeria , sierra leone , south sudan , togo and uganda .
fixed health facilities that are active in the control and surveillance of gambiense hat in the democratic republic of the congo . geographic distribution of fixed health facilities having capacities for diagnosis of gambiense human african trypanosomiasis .
geographic distribution of fixed health facilities having capacities for clinical diagnosis of gambiense hat ( a ) and serological diagnosis ( b ) .
geographic distribution of fixed health facilities having capacities for parasitological diagnosis of gambiense hat ( a ) and stage determination ( b ) .
geographic distribution of fixed health facilities having capacities for treatment of gambiense hat first - stage infections with pentamidine ( a ) and second - stage infection with melarsoprol ( b ) .
geographic distribution of fixed health facilities having capacities for treatment of gambiense hat second - stage infections with eflornithine ( a ) and with nifurtimox - eflornithine combination therapy ( b ) .
diagnosis coverage by fixed health facilities of population at - risk of gambiense human african trypanosomiasis .
diagnosis coverage is provided for a range of travel - times , for all study countries and for all hat - risk categories .
clinical diagnosis coverage is provided for a range of travel - times , for all study countries and for all hat - risk categories .
serological diagnosis coverage is provided for a range of travel - times , for all study countries and for all hat - risk categories .
parasitological diagnosis coverage is provided for a range of travel - times , for all study countries and for all hat - risk categories .
annex_dxph.xlsx : disease staging coverage is provided for a range of travel - times , for all study countries and for all hat - risk categories .
travel time ( or physical accessibility ) to health facilities having capabilities for treatment of gambiense hat [ hours ] .
travel cost was measured in time units , and the destinations were represented by health facilities having capabilities for hat treatment .
treatment coverage by fixed health facilities of population at - risk of gambiense human african trypanosomiasis .
treatment coverage is provided for a range of travel - times , for all study countries and for all hat - risk categories .
annex_tx1p.xlsx : coverage for treatment of first - stage infection with pentamidine is provided for a range of travel - times , for all study countries and for all hat - risk categories .
annex_tx2m.xlsx : coverage for treatment of second - stage infection with melarsoprol is provided for a range of travel - times , for all study countries and for all hat - risk categories .
annex_tx2e.xlsx : coverage for treatment of second - stage infection with eflornithine is provided for a range of travel - times , for all study countries and for all hat - risk categories .
annex_tx2n.xlsx : coverage for treatment of second - stage infection with nifurtimox - eflornithine combination therapy is provided for a range of travel - times , for all study countries and for all hat - risk categories .
the authors would like to acknowledge the coordinators of the national sleeping sickness control programmes that provided data for the analysis : angola , benin , burkina faso , cameroon , central african republic , chad , congo , cte divoire , democratic republic of the congo , equatorial guinea , gabon , ghana , guinea , mali , nigeria , sierra leone , south sudan , togo and uganda .
the activities described in this paper are an initiative of the department of control of neglected tropical diseases - who .
they were implemented through a technical collaboration between who and fao in the framework of paat .
the contribution of fao is supported by the government of italy through the fao trust fund for food security and food safety ( project gtfs / raf/474/ita improving food security in sub - saharan africa by supporting the progressive reduction of tsetse - transmitted trypanosomosis in the framework of the nepad ) . the boundaries and names shown and the designations used on the maps presented in this paper do not imply the expression of any opinion whatsoever on the part of who and fao concerning the legal status of any country , territory , city or area or of its authorities , or concerning the delimitation of its frontiers or boundaries .
the views expressed in this paper are those of the authors and do not necessarily reflect the views of who and fao .
the boundaries and names shown and the designations used on the maps presented in this paper do not imply the expression of any opinion whatsoever on the part of who and fao concerning the legal status of any country , territory , city or area or of its authorities , or concerning the delimitation of its frontiers or boundaries .
the views expressed in this paper are those of the authors and do not necessarily reflect the views of who and fao . | backgroundthe emphasis placed on the activities of mobile teams in the detection of gambiense human african trypanosomiasis ( hat ) can at times obscure the major role played by fixed health facilities in hat control and surveillance .
the lack of consistent and detailed data on the coverage of passive case - finding and treatment further constrains our ability to appreciate the full contribution of the health system to the control of hat.methodsa survey was made of all fixed health facilities that are active in the control and surveillance of gambiense hat .
information on their diagnostic and treatment capabilities was collected , reviewed and harmonized .
health facilities were geo - referenced .
time - cost distance analysis was conducted to estimate physical accessibility and the potential coverage of the population at - risk of gambiense hat.resultsinformation provided by the national sleeping sickness control programmes revealed the existence of 632 fixed health facilities that are active in the control and surveillance of gambiense hat in endemic countries having reported cases or having conducted active screening activities during the period 2000 - 2012 .
different types of diagnosis ( clinical , serological , parasitological and disease staging ) are available from 622 facilities .
treatment with pentamidine for first - stage disease is provided by 495 health facilities , while for second - stage disease various types of treatment are available in 206 health facilities only . over 80% of the population at - risk for gambiense hat lives within 5-hour travel of a fixed health facility offering diagnosis and treatment for the disease.conclusionsfixed health facilities have played a crucial role in the diagnosis , treatment and coverage of at - risk - population for gambiense hat . as the number of reported cases continues to dwindle
, their role will become increasingly important for the prospects of disease elimination .
future updates of the database here presented will regularly provide evidence to inform and monitor a rational deployment of control and surveillance efforts .
support to the development and , if successful , the implementation of new control tools ( e.g. new diagnostics and new drugs ) is crucial , both for strengthening and expanding the existing network of fixed health facilities by improving access to diagnosis and treatment and for securing a sustainable control and surveillance of gambiense hat . | Introduction
Methods
Data collection
Data analysis
Ethical considerations
Results
Fixed health facilities for diagnosis and treatment of gambiense human African trypanosomiasis
Diagnosis coverage by fixed health facilities of population at-risk of gambiense human African trypanosomiasis
Treatment coverage by fixed health facilities of population at-risk of gambiense human African trypanosomiasis
Conclusions
Competing interests
Authors contributions
Supplementary Material
Acknowledgements
Disclaimers | alongside the continued , albeit geographically limited , engagement of bilateral cooperation and non - governmental organizations ( ngos ) , it was the commitment of private sector in 2001 to support who that marked a turning point in the fight against human african trypanosomiasis ( hat )
. from that moment ,
control actions by the national sleeping sickness control programmes ( nsscps ) improved across the continent , and a dramatic decrease in the number of gambiense hat cases followed . nevertheless , because of the occurrence of gambiense hat in remote rural areas and the weakness of health systems , an unspecified number of sleeping sickness cases still escape detection and reporting . at the same time , some insights into the likelihood of underdetection / underreporting can be gained by looking at the distribution and intensity of control and surveillance activities involving both mobile teams and fixed health facilities . arguably , a narrow focus on active case - finding activities alone may give the impression that only a small proportion of the population at - risk of hat is reached by control and surveillance activities . in fact , while recent estimates put the population at risk of gambiense hat at approximately 57 million people
, only an average of 2 million are screened every year by mobile teams ( period 2000 - 2012 ) ( data not shown ) . active surveillance alone appears inadequate to ensure a satisfactory coverage of populations at - risk of gambiense hat , and consequently it may seem reasonable to question the reported abatement in disease transmission . however , the role played by fixed health facilities embedded in the health system must be taken into account if a complete picture of the coverage and outcomes of hat control and surveillance is to be drawn . indeed ,
control of gambiense hat has relied , in addition to the impressive and well known active case - finding surveys carried out by mobile teams , also on the quiet contribution of passive screening performed in fixed health structures . for example , in the period 2000 - 2012 , 93,561 cases of gambiense hat ( 48.9% of the reported total ) were diagnosed by passive case - finding carried out in fixed health facilities ( table
1 ) . cases of gambiense hat diagnosed by active and passive case - finding ( period : 2000 - 2012 ) " - " , no data reported . however , the lack of consistent and detailed data on the extent , capacities and coverage of passive case - finding and treatment constrains our ability to appreciate its full relevance in hat control and surveillance . the paper explores the geographic distribution and the diagnostic and treatment capabilities of fixed health facilities involved in gambiense hat control and surveillance . it also aims to estimate the at - risk population potentially covered by passive case - finding and treatment by using geographic information systems ( gis ) . the paper focuses on all endemic countries having reported gambiense hat cases or having conducted active screening activities during the period 2000 - 2012 ( i.e. fixed health facilities active in gambiense hat control and surveillance were identified through direct interviews with the coordinators of the nsscps . standardized forms were circulated requesting information on the name and location of the health structures , and on their diagnostic and treatment capabilities for hat . in this context , the serological test considered is the card agglutination test for trypanosomiasis
parasitological diagnosis ( dxp ) pertains to the competence of the health centre to execute any type of parasitological test in order to confirm trypanosome infection in clinical or serological suspects through direct observation of the parasite in body fluids . this includes treatment of first stage infections with pentamidine ( tx1p ) , and treatment of second - stage cases with melarsoprol ( tx2 m ) , eflornithine ( tx2e ) or nifurtimox - eflornithine combination therapy
all data on the hat health centres provided by nsscps were harmonized and assembled in a database . risk is based on the reported number of gambiense hat cases in the period 2000 - 2009 and on landscan human population layers for the same period
. for estimating both the total population coverage and the coverage of at - risk population , the dataset landscan 2009
this was the same layer previously used to estimate at - risk population
, and therefore it provided a precise baseline for comparison . fixed health facilities active in gambiense hat control and surveillance were identified through direct interviews with the coordinators of the nsscps . standardized forms were circulated requesting information on the name and location of the health structures , and on their diagnostic and treatment capabilities for hat . in this context , the serological test considered is the card agglutination test for trypanosomiasis
parasitological diagnosis ( dxp ) pertains to the competence of the health centre to execute any type of parasitological test in order to confirm trypanosome infection in clinical or serological suspects through direct observation of the parasite in body fluids . this includes treatment of first stage infections with pentamidine ( tx1p ) , and treatment of second - stage cases with melarsoprol ( tx2 m ) , eflornithine ( tx2e ) or nifurtimox - eflornithine combination therapy
all data on the hat health centres provided by nsscps were harmonized and assembled in a database . risk is based on the reported number of gambiense hat cases in the period 2000 - 2009 and on landscan human population layers for the same period
. for estimating both the total population coverage and the coverage of at - risk population , the dataset landscan 2009
this was the same layer previously used to estimate at - risk population
, and therefore it provided a precise baseline for comparison . information provided by the nsscps revealed the existence of 632 fixed health facilities that are involved in the control and surveillance of gambiense hat in disease - endemic countries . their diagnostic and treatment capacities are summarized in table
2 , while the complete list of health facilities including the names and geographical coordinates is provided in additional file
1 . number of fixed health facilities providing diagnosis and treatment for gambiense hat study area : disease - endemic countries having reporting cases or having conducted active screening activities during the period 2000 - 2012 . diagnosis is available in 622 facilities , 84.2% of which are found in the democratic republic of the congo ( drc ) . the number of centres able to carry out parasitological diagnosis ( dxp ) and disease staging ( dxph ) decreases to 49% and 39% respectively , mainly due to the need for complex tools and highly skilled staff . access to treatment for gambiense hat is slightly more limited ( 495 health centres 81.6% of which in drc ) . while all of these centres are able to offer treatment for first - stage disease , only 36.4% can offer first line treatment for second - stage disease ( tx2n ) . figure
2 shows the geographic distribution of the health structures involved in gambiense hat diagnosis ( a ) and treatment ( b ) . in this figure
separate maps showing the distribution of centres able to perform the various types of diagnosis and treatment are provided in additional file
2 . geographic distribution of fixed health facilities having capacities for gambiense hat diagnosis ( a ) and treatment ( b ) . data were collected by who between december 2012 and august 2013 from national sleeping sickness control programmes in the study countries ( i.e. for the 10-hour travel threshold , the coverage of at - risk population is 96% ( additional file
3 ) . people at risk of gambiense hat that are potentially covered by facilities with different types of diagnostic capabilities for hat dxc : clinical diagnosis ; dxs : serological diagnosis ; dxp : parasitological diagnosis ; dxph : disease staging . we estimate that in the study countries 27 , 73 and 133 million people are respectively within 1 , 3 and 5 hours travel of a facility with capacity for gambiense hat treatment . looking at population at risk , the coverage by treatment facilities is summarized in table
5 , while full details for a range of travel - times , for all study countries and for all risk categories are provided in additional file
6 . for the 10-hour travel threshold , the coverage of at - risk population is 95% ( additional file
6 ) . people at risk of gambiense hat that are potentially covered by facilities with treatment capabilities for hat people at risk are stratified by risk categories as defined in
. all facilities involved in gambiense hat treatment have capacity for treatment of the first - stage of the disease . lower coverage is available for the treatment of the second - stage of the disease . people at risk of gambiense hat that are potentially covered by facilities with different types of treatment capabilities for hat tx1p : treatment of first - stage infection with pentamidine ; tx2 m : treatment of second - stage infection with melarsoprol ; tx2e : treatment of second - stage infection with eflornithine ; tx2n : treatment of second - stage infection with nifurtimox - eflornithine combination therapy ( nect ) . information provided by the nsscps revealed the existence of 632 fixed health facilities that are involved in the control and surveillance of gambiense hat in disease - endemic countries . their diagnostic and treatment capacities are summarized in table
2 , while the complete list of health facilities including the names and geographical coordinates is provided in additional file
1 . number of fixed health facilities providing diagnosis and treatment for gambiense hat study area : disease - endemic countries having reporting cases or having conducted active screening activities during the period 2000 - 2012 . the number of centres able to carry out parasitological diagnosis ( dxp ) and disease staging ( dxph ) decreases to 49% and 39% respectively , mainly due to the need for complex tools and highly skilled staff . access to treatment for gambiense hat is slightly more limited ( 495 health centres 81.6% of which in drc ) . while all of these centres are able to offer treatment for first - stage disease , only 36.4% can offer first line treatment for second - stage disease ( tx2n ) . figure
2 shows the geographic distribution of the health structures involved in gambiense hat diagnosis ( a ) and treatment ( b ) . in this figure
separate maps showing the distribution of centres able to perform the various types of diagnosis and treatment are provided in additional file
2 . geographic distribution of fixed health facilities having capacities for gambiense hat diagnosis ( a ) and treatment ( b ) . data were collected by who between december 2012 and august 2013 from national sleeping sickness control programmes in the study countries ( i.e. for the 10-hour travel threshold , the coverage of at - risk population is 96% ( additional file
3 ) . these percentages decrease for moderate and for low and very low risk categories the health systems deploying better equipped facilities and skilled staff in areas where transmission of the disease is more intense . people at risk of gambiense hat that are potentially covered by facilities with different types of diagnostic capabilities for hat dxc : clinical diagnosis ; dxs : serological diagnosis ; dxp : parasitological diagnosis ; dxph : disease staging . we estimate that in the study countries 27 , 73 and 133 million people are respectively within 1 , 3 and 5 hours travel of a facility with capacity for gambiense hat treatment . for the 10-hour travel threshold , the coverage of at - risk population is 95% ( additional file
6 ) . all facilities involved in gambiense hat treatment have capacity for treatment of the first - stage of the disease . people at risk of gambiense hat that are potentially covered by facilities with different types of treatment capabilities for hat tx1p : treatment of first - stage infection with pentamidine ; tx2 m : treatment of second - stage infection with melarsoprol ; tx2e : treatment of second - stage infection with eflornithine ; tx2n : treatment of second - stage infection with nifurtimox - eflornithine combination therapy ( nect ) . however ,
the low number of people at risk covered by active case - finding surveys is often cited to raise doubts on the reliability of the reported figures . in this study we show that a narrow focus on the intensity of active case - finding is inadequate to fully describe the coverage of hat control and surveillance activities . gambiense hat is a chronic disease that is characterized by a sufficiently long duration
to allow passive case detection in fixed health facilities to complement the coverage of active case detection . at the same time , the interval between infection and diagnosis in fixed health structure allowed the persistence and the spread of the disease . because of these limitations of active case - finding , the role of the fixed health facilities in gambiense hat transmission areas has been extremely important to fill a part of the screening and coverage gap . indeed , passive screening detected 93,561 cases during the period 2000 - 2012 , which represent approximately half of the total cases reported . the present analysis reveals the existence of a network of fixed health facilities involved in the passive control and surveillance of gambiense hat . a census of the diagnosis and treatment capabilities was made for the 632 fixed health facilities identified . it must be stressed that our survey did not address the quality control of services provided in the fixed health facilities involved in hat control and surveillance , nor did it explore population attendance . the results illustrate the substantial attempts made by the health system in disease - endemic countries in tackling the disease , although efforts on capacity building and equipment must be undertaken to get this network fully operational . over 80% of the population at - risk lives within 5-hour travel of a fixed health facility offering diagnosis and treatment for gambiense hat . this observation supports the notion that that the current test for serological screening ( catt ) , although very useful for serological mass screening by mobile teams , does not fulfil the requirements for its use in the rural health facilities where gambiense hat is endemic most notably because of the format ( 50 tests per vial ) and the need for cold chain and electric power . the new individual screening tests ( ist , often called rapid diagnostic tests ) do not require cold chain nor electric power , and they hold the potential to expand the network of fixed health facilities able to detect serological suspects which is presently available only in 55.6% of the facilities able to detect clinical suspects and therefore they could widen and improve access to gambiense hat diagnosis
. concerning parasitological diagnosis ,
the coverage gap can partly be ascribed to the cumbersome nature of and the skilled staff required by the current parasitological diagnostic tests . concerning treatment , thanks to the fact that some cases diagnosed and staged in highest centres as first stage can be referred for treatment to lower health centres , 82% of the people at risk of gambiense hat have access to a centre offering treatment for first - stage patients within a 5-hour travel - time , while 69% have access to a health care facility administering the recommended first line treatment for second - stage gambiense hat ( nect ) within the same travel - time threshold . this is mainly because of the complexity of administration and the demanding logistics of current first line treatment for second - stage gambiense hat . an oral , safe drug , effective for both stages of the disease could greatly improve access to treatment ; at the same time , it would remove the need for staging through lumbar puncture , which is not always well accepted and can only be offered by 38.5% of the facilities involved in the treatment of gambiense hat . in the light of the new challenges to achieve sustainable elimination of gambiense hat , the meeting on hat elimination held in geneva in december 2012
emphasized the importance of maintaining mobile teams in selected areas and in particular epidemiological circumstances , as well as the value of reinforcing the health system and involving it fully in passive control and surveillance . strengthening and expanding fixed health facilities involved in control and surveillance to reach
support from both international partners and from operational national teams is needed to monitor and evaluate the network , as well as to collect and analyze data obtained and to provide appropriate response . retirement of skilled staff and the decreasing number of cases in the community reduce familiarity with the disease and can greatly decrease the effectiveness of passive diagnosis . a need exists therefore for capacity building of health staff , for less complicated control tools , and for awareness - raising campaigns among populations at risk of gambiense hat
. therefore , attention must be paid to appreciate the level of attendance of population at risk of hat ( e.g. in order to evaluate the coverage of fixed health facilities in the case of hat surveillance . continuous monitoring and evaluation are therefore needed , with a view to ensuring that an effective surveillance of gambiense hat is in place to sustain the disease elimination process . the data and coverage estimates herewith presented will be regularly updated , thus continuously providing evidence to inform a rational deployment of control and surveillance efforts . fixed health facilities that are active in the control and surveillance of gambiense hat . fixed health facilities that are active in the control and surveillance of gambiense hat in angola , benin , burkina faso , cameroon , central african republic , chad , congo , cte divoire , equatorial guinea , gabon , ghana , guinea , mali , nigeria , sierra leone , south sudan , togo and uganda . fixed health facilities that are active in the control and surveillance of gambiense hat in the democratic republic of the congo . geographic distribution of fixed health facilities having capacities for diagnosis of gambiense human african trypanosomiasis . geographic distribution of fixed health facilities having capacities for clinical diagnosis of gambiense hat ( a ) and serological diagnosis ( b ) . geographic distribution of fixed health facilities having capacities for parasitological diagnosis of gambiense hat ( a ) and stage determination ( b ) . geographic distribution of fixed health facilities having capacities for treatment of gambiense hat first - stage infections with pentamidine ( a ) and second - stage infection with melarsoprol ( b ) . geographic distribution of fixed health facilities having capacities for treatment of gambiense hat second - stage infections with eflornithine ( a ) and with nifurtimox - eflornithine combination therapy ( b ) . diagnosis coverage by fixed health facilities of population at - risk of gambiense human african trypanosomiasis . treatment coverage by fixed health facilities of population at - risk of gambiense human african trypanosomiasis . annex_tx1p.xlsx : coverage for treatment of first - stage infection with pentamidine is provided for a range of travel - times , for all study countries and for all hat - risk categories . annex_tx2m.xlsx : coverage for treatment of second - stage infection with melarsoprol is provided for a range of travel - times , for all study countries and for all hat - risk categories . annex_tx2e.xlsx : coverage for treatment of second - stage infection with eflornithine is provided for a range of travel - times , for all study countries and for all hat - risk categories . annex_tx2n.xlsx : coverage for treatment of second - stage infection with nifurtimox - eflornithine combination therapy is provided for a range of travel - times , for all study countries and for all hat - risk categories . the authors would like to acknowledge the coordinators of the national sleeping sickness control programmes that provided data for the analysis : angola , benin , burkina faso , cameroon , central african republic , chad , congo , cte divoire , democratic republic of the congo , equatorial guinea , gabon , ghana , guinea , mali , nigeria , sierra leone , south sudan , togo and uganda . | [
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] |
meniscal injuries located in the low or nonvascularized zones ( red - white and white - white zones ) are often treated with partial or total meniscectomy .
although meniscectomy is a relatively simple and quick surgery with good immediate postoperative clinical outcomes , the long - term results are poor .
these poor outcomes are not surprising since many studies have demonstrated the importance of the meniscus for the knee function .
it has also been accepted that surgeons should preserve as much meniscal tissue as possible . both complete and partial
, it is now suggested that meniscal tears should be treated by meniscal repair instead of meniscectomy whenever possible .
they include use of a fibrin clot , platelet - rich plasma , pro - inflammatory cytokines , and application of growth factors .
further progress in meniscus healing has been made possible by the innovative use of cells and scaffolds
. improved methods to isolate and concentrate mesenchymal stem cells from autologous sources ( e.g. , bone marrow or adipose tissue ) allows the evaluation of the role of cell - based techniques to augment healing .
furthermore , the development of advanced scaffold materials and associated techniques has contributed to creating an intracapsular biological environment for improved meniscus healing .
notably , jakobi and jakob tested 30 patients with the treatment of meniscal tears by suturing and wrapping the repair in a collagen matrix .
the results obtained were encouraging , but the surgical technique , an inside - out suturing , presents some challenges since it can be time - consuming and requires an additional surgical approach . in a goat animal study ,
jlke et al . showed that a transient , short - term guided tissue regeneration of avascular meniscal tears occurs when a collagen membrane is used .
the addition of expanded autologous chondrocytes supports more sustainable long - term tear healing in the goat model .
inspired by those advances in the treatment of injured menisci , in 2010 a fully arthroscopic technique has been developed to treat combined ( possessing horizontal and radial or longitudinal component ) and complex ( tear extended through all vascular zones or / and composed with 2 or more morphological tear pattern ) meniscal tears .
conceptually , the surgical technique , is a modification of the historical henning s and jakob procedure , included suturing and wrapping the menisci in a collagen matrix , followed by the injection of liquid bone marrow collected from the tibial proximal epiphysis , into the area of the meniscal lesion .
a detailed description of the surgical method had been published by piontek et al . in 2012 . by using this surgical technique in patients sustaining complex and/or combined meniscal injuries ,
meniscus resection could be avoided in these tear patterns cases that otherwise would have been treated with meniscetomy .
we hypothesized that complex and combined meniscal tears occurring in white - white and white - red zones can safely and successfully be treated arthroscopically by meniscal suturing and wrapping in the collagen matrix , instead of being partially or totally resected .
the purpose of this work is to present a 2-year follow - up of clinical and magnetic resonance imaging ( mri ) results from a prospective consecutive case series of patients treated with a newly developed , fully arthroscopic technique of collagen matrix - based meniscus repair ( ammr ) and the injection of bone marrow aspirate into the area of the meniscal lesion .
this clinical study has been approved by the institutional review board committee of the medical university of poznan , poland , and has been performed according to the ethical standards laid down in the 1964 declaration of helsinki .
inclusion criteria for the procedure comprised ( 1 ) full - thickness , combined meniscal tear greater than 20 mm in length , ( 2 ) horizontal and radial tear , ( 3 ) tear location reaching more than 6 mm from the meniscocapsular junction including the avascular zone , or ( 4 ) both degenerative and non - degenerative meniscus ( i.e. , horizontal and radial tears , involving the white - white and red - white zones , as well as extensive tears of the bucket - handle type ) .
an example of meniscus injury treated as a part of this study is shown in figure 1 and as a schematic drawing in figure 2 .
patients were excluded from the procedure if ( 1 ) full - thickness combined meniscus tear was larger than 85% of the meniscus body or ( 2 ) meniscus tear with fibrillation extended in all area of tear which was not able to be stabilized with a suture ( meniscus lesion beyond repair ) .
time to surgery was defined as a time elapsed between injury or patient decision to seek orthopaedic treatment to the ammr surgical intervention .
the length of the meniscal tear was measured during arthroscopic procedure by using arthroscopic ruler .
degenerative meniscus was in most cases defined as a adipose ( fat ) degeneration or significant fibrillations not exceeding 85% body of the meniscus .
the primary outcome of the study defined as a result of a clinical of the knee joint and meniscus related symptoms before and after treatment ( at 2-year follow - up ) .
the safety of the procedure was assessed based on pass - fail criteria s and adverse events related to the procedure or medical device used ( chondro - gide collagen matrix ) .
the clinical results were assessed and evaluated by the number of different scoring systems based on patient reported outcomes and clinical findings .
the following information were recorded at each assessment point : international knee documentation committee ( ikdc ) 2000 subjective score , ikdc 2000 clinical evaluation score , lysholm score , and barrett clinical criteria of meniscal healing .
based on original barrett criteria score , we have developed a converted method ( described in details in appendix a ) to allow for statistical analysis .
the aim of this calculation is to convert the four clinically meaningful signs ( pain , effusion , clicking / locking , mcmurray test ) , which are coded binary ( 0 = absence and 1 = presence ) into a numerical notation .
the final values may vary from 0 to 15 and each value represents the discreet stage of the knee joint with respect to the initial barrett s evaluation criteria . in an attempt to lower the confounding influence of the concomitant knee diseases in the patients included in the study , the analysis of the subgroup of patients with isolated meniscus injury was performed .
magnetic resonance imaging scans were acquired with a 1.5-tesla whole - body scanner using a commercial circumferential knee coil .
imaging sequences included , axial t1-weighted spin - echo ( se : 700/11 [ tr ms / te ms ] , 20 cm field of view ( fov ) , 5 mm/1 mm [ slice thickness / interslice gap ] , 256 192 matrix , frequency encoding [ fe ] anterior - posterior , one excitation ) , coronal t1-weighted se ( 600/11 , 16 cm fov , 4 mm/0.5 mm , 256 192 , fe superior - inferior , 2 excitations averaged ) , sagittal t1-weighted se ( 600/11 , 16 cm fov , 4 mm/0.5 mm , 256 192 , fe anterior - posterior , 2 excitations averaged ) , sagittal t2-weighted fast spin echo ( fse : 2500/90 ; echo train length ( etl ) of 8 ; 14 cm fov , 4 mm/0 mm , 256 192 , fe superior - inferior , 2 excitations averaged ) with fat suppression ( frequency - selective presaturation ) , and sagittal fat - suppressed t1-weighted 3-dimensional ( 3d ) spoiled gradient echo ( fs-3dspgr : 58/6 , 40 flip angle , 14 cm fov , 256 128 matrix , 60 contiguous 2-mm slices covering all articular cartilage plates in the knee , fe , superior - inferior , one excitation , frequency - selective fat saturation , superior - inferior saturation bands to minimize pulsation artifacts ) .
all images were transferred to the osirix v.5.8.5 dicom viewer software ( pixmeo sarl , bern , switzerland ) .
images were scored with respect to modified whole organ mri score ( worms ) criteria for medial and lateral meniscal integrity .
image assessments were performed independently by 2 orthopedic surgeons ( tp , kc - g ) .
the anterior horn , meniscal body segment , and posterior horn of the medial and lateral menisci were scored separately on the scale from 0 to 4 based on both the sagittal and coronal images : 0 = intact ; 1 = nonhomogeneous signal but not a meniscal tear ( in original worms scale minor radial tear or parrot - beak tear ) ; 2 = nondisplaced tear or prior surgical repair ; 3 = displaced tear or partial resection ; 4 = complete maceration / destruction or complete resection .
a cumulative grade for each meniscus was determined using the following scheme : 0 = all 0 ; 1 = at least one 1 , but no > 1 ; 2 = 2 in only one region ; 3 = 2 in more than one region ; 4 = 3 in one or more regions ; 5 = 4 in only one region ; 6 = 4 in more than one region .
these modifications were introduced in order to accurately describe mri scans . in this study , the following pass - fail criteria were used : failure if at the follow - up process patient underwent partial / complete meniscectomy or knee replacement after ammr procedure pass was defined as an overall grade of a or b at final follow - up with clinical ikdc 2000 assessment .
pass was also defined if the patient exhibited no pain at rest or with activity and a negative mcmurray s test .
patients who did not met the aforementioned criteria were characterized as poor with regard to their latest clinical follow - up . additionally , a worms cumulative grade 2 and more classified patients in the poor mri results group .
the chondro - gide collagen matrix ( geistlich pharma ag , wolhusen , switzerland ) was used for meniscus wrapping .
this is a non cross - linked collagen type i / iii matrix , with a 2-layer ( bilayer ) structure .
the porous layer ( placed facing the defect ) allows the in - growth of cells and a newly formed tissue .
the compact layer acts as a scaffold to prevent cells being flushed out of the meniscal site defect .
a diagnostic knee arthroscopy was performed to identify other pathologies , such as ligament or cartilage lesions .
all cartilage and ligament lesions were repaired during surgery . the extent and type of meniscal lesion was evaluated to determine whether or not the meniscal lesion meets the study inclusion criteria .
lesions were reduced and meniscal fragments were fixed with a meniscal fixing device ( fast - fix , smith & nephew , andover , ma , usa ) . the matrix , usually of the size 30 20 mm , was prepared by addition of nonabsorbable suture loops ( ethibond 2 , ethicon inc ) , passing through the surface of the matrix on both sides . the matrix was then inserted into an applicator . using direct arthroscopic vision ,
the loops of threads running through the meniscal posterior horn and the meniscal body were passed with a special suture shuttle ( accupass , smith & nephew , andover , ma , usa ) at the level of the anterior border of the tear .
ethibond 2 , matrix - passing sutures , were inserted to the meniscal posterior horn and to the anterior part of the lesion .
the matrix was inserted into the knee joint with smooth surface facing cartilaginous surfaces of the tibia and femur and with porous part directed to the meniscal surface . after assuring that the matrix adhered to the meniscus from the tibial side the scaffold
, the meniscus was wrapped by the collagen matrix on both sides , and the matrix was fixed into the meniscus in a stable way .
additionally , 1 to 4 ( 3 on average , depending on the extension of the tear ) fast - fix sutures were inserted into the meniscus wrapped with the matrix for a better stabilization of the meniscal tear and for an increased tightness between the meniscus and the matrix .
an example of suture placement and meniscus wrapping with collagen matrix is shown in figure 3 and as a schematic drawing in figure 4 .
the bone marrow aspirate was obtained from the tibial proximal epiphysis of the operated knee .
the bone marrow aspirate was injected between the chondro - gide matrix and the meniscus , under direct arthroscopic control and carried out in
an extensive surgical description of the technique has been published by piontek et al . in 2012 .
patients began postoperative treatment from toe touch crutch walking and a range of motion exercises on the first postoperative day , for a period of 4 weeks . over the next 2 to 4 weeks
detailed rehabilitation guidelines used in our center have been presented in appendix b. statistical evaluation included descriptive statistics to compare the pre- and postoperative indexed values .
the comparison between preoperative ( time 0 ) and postoperative ( 24 months follow - up ) values of ikdc 2000 clinical , ikdc subjective , and lysholm separately had been calculated by nonparametric wilcoxon test .
the statistical significance of values between the preoperative and 2-year follow - up values of ikdc 2000 clinical , ikdc 2000 subjective , and lysholm scores with respect to the worms mri cumulative score were performed using kruskal - wallis test .
spearman s rank coefficient test was used to calculate the correlation coefficients between ikdc 200 , ikdc subjective , and lysholm score preoperatively and at 2-year follow - up and against worms mri cumulative score .
computations were done with statistica data analysis software system version 10 ( statsoft inc , 2011 ) .
inclusion criteria for the procedure comprised ( 1 ) full - thickness , combined meniscal tear greater than 20 mm in length , ( 2 ) horizontal and radial tear , ( 3 ) tear location reaching more than 6 mm from the meniscocapsular junction including the avascular zone , or ( 4 ) both degenerative and non - degenerative meniscus ( i.e. , horizontal and radial tears , involving the white - white and red - white zones , as well as extensive tears of the bucket - handle type ) .
an example of meniscus injury treated as a part of this study is shown in figure 1 and as a schematic drawing in figure 2 .
patients were excluded from the procedure if ( 1 ) full - thickness combined meniscus tear was larger than 85% of the meniscus body or ( 2 ) meniscus tear with fibrillation extended in all area of tear which was not able to be stabilized with a suture ( meniscus lesion beyond repair ) .
time to surgery was defined as a time elapsed between injury or patient decision to seek orthopaedic treatment to the ammr surgical intervention .
the length of the meniscal tear was measured during arthroscopic procedure by using arthroscopic ruler .
degenerative meniscus was in most cases defined as a adipose ( fat ) degeneration or significant fibrillations not exceeding 85% body of the meniscus .
the primary outcome of the study defined as a result of a clinical of the knee joint and meniscus related symptoms before and after treatment ( at 2-year follow - up ) .
the safety of the procedure was assessed based on pass - fail criteria s and adverse events related to the procedure or medical device used ( chondro - gide collagen matrix ) .
the clinical results were assessed and evaluated by the number of different scoring systems based on patient reported outcomes and clinical findings .
the following information were recorded at each assessment point : international knee documentation committee ( ikdc ) 2000 subjective score , ikdc 2000 clinical evaluation score , lysholm score , and barrett clinical criteria of meniscal healing .
based on original barrett criteria score , we have developed a converted method ( described in details in appendix a ) to allow for statistical analysis .
the aim of this calculation is to convert the four clinically meaningful signs ( pain , effusion , clicking / locking , mcmurray test ) , which are coded binary ( 0 = absence and 1 = presence ) into a numerical notation .
the final values may vary from 0 to 15 and each value represents the discreet stage of the knee joint with respect to the initial barrett s evaluation criteria . in an attempt to lower the confounding influence of the concomitant knee diseases in the patients included in the study ,
magnetic resonance imaging scans were acquired with a 1.5-tesla whole - body scanner using a commercial circumferential knee coil .
imaging sequences included , axial t1-weighted spin - echo ( se : 700/11 [ tr ms / te ms ] , 20 cm field of view ( fov ) , 5 mm/1 mm [ slice thickness / interslice gap ] , 256 192 matrix , frequency encoding [ fe ] anterior - posterior , one excitation ) , coronal t1-weighted se ( 600/11 , 16 cm fov , 4 mm/0.5 mm , 256 192 , fe superior - inferior , 2 excitations averaged ) , sagittal t1-weighted se ( 600/11 , 16 cm fov , 4 mm/0.5 mm , 256 192 , fe anterior - posterior , 2 excitations averaged ) , sagittal t2-weighted fast spin echo ( fse : 2500/90 ; echo train length ( etl ) of 8 ; 14 cm fov , 4 mm/0 mm , 256 192 , fe superior - inferior , 2 excitations averaged ) with fat suppression ( frequency - selective presaturation ) , and sagittal fat - suppressed t1-weighted 3-dimensional ( 3d ) spoiled gradient echo ( fs-3dspgr : 58/6 , 40 flip angle , 14 cm fov , 256 128 matrix , 60 contiguous 2-mm slices covering all articular cartilage plates in the knee , fe , superior - inferior , one excitation , frequency - selective fat saturation , superior - inferior saturation bands to minimize pulsation artifacts ) .
all images were transferred to the osirix v.5.8.5 dicom viewer software ( pixmeo sarl , bern , switzerland ) .
images were scored with respect to modified whole organ mri score ( worms ) criteria for medial and lateral meniscal integrity .
image assessments were performed independently by 2 orthopedic surgeons ( tp , kc - g ) .
the anterior horn , meniscal body segment , and posterior horn of the medial and lateral menisci were scored separately on the scale from 0 to 4 based on both the sagittal and coronal images : 0 = intact ; 1 = nonhomogeneous signal but not a meniscal tear ( in original worms scale minor radial tear or parrot - beak tear ) ; 2 = nondisplaced tear or prior surgical repair ; 3 = displaced tear or partial resection ; 4 = complete maceration / destruction or complete resection .
a cumulative grade for each meniscus was determined using the following scheme : 0 = all 0 ; 1 = at least one 1 , but no > 1 ; 2 = 2 in only one region ; 3 = 2 in more than one region ; 4 = 3 in one or more regions ; 5 = 4 in only one region ; 6 = 4 in more than one region .
these modifications were introduced in order to accurately describe mri scans . in this study , the following pass - fail criteria were used : failure if at the follow - up process patient underwent partial / complete meniscectomy or knee replacement after ammr procedure pass was defined as an overall grade of a or b at final follow - up with clinical ikdc 2000 assessment .
pass was also defined if the patient exhibited no pain at rest or with activity and a negative mcmurray s test .
patients who did not met the aforementioned criteria were characterized as poor with regard to their latest clinical follow - up . additionally ,
a worms cumulative grade 2 and more classified patients in the poor mri results group .
the chondro - gide collagen matrix ( geistlich pharma ag , wolhusen , switzerland ) was used for meniscus wrapping .
this is a non cross - linked collagen type i / iii matrix , with a 2-layer ( bilayer ) structure .
the porous layer ( placed facing the defect ) allows the in - growth of cells and a newly formed tissue .
the compact layer acts as a scaffold to prevent cells being flushed out of the meniscal site defect .
a diagnostic knee arthroscopy was performed to identify other pathologies , such as ligament or cartilage lesions .
all cartilage and ligament lesions were repaired during surgery . the extent and type of meniscal lesion was evaluated to determine whether or not the meniscal lesion meets the study inclusion criteria .
lesions were reduced and meniscal fragments were fixed with a meniscal fixing device ( fast - fix , smith & nephew , andover , ma , usa ) . the matrix , usually of the size 30 20 mm , was prepared by addition of nonabsorbable suture loops ( ethibond 2 , ethicon inc ) , passing through the surface of the matrix on both sides . the matrix was then inserted into an applicator . using direct arthroscopic vision ,
the loops of threads running through the meniscal posterior horn and the meniscal body were passed with a special suture shuttle ( accupass , smith & nephew , andover , ma , usa ) at the level of the anterior border of the tear .
ethibond 2 , matrix - passing sutures , were inserted to the meniscal posterior horn and to the anterior part of the lesion .
the matrix was inserted into the knee joint with smooth surface facing cartilaginous surfaces of the tibia and femur and with porous part directed to the meniscal surface . after assuring that the matrix adhered to the meniscus from the tibial side the scaffold
was fixed to the meniscus , with arthroscopic simple knotted sutures . as a result ,
the meniscus was wrapped by the collagen matrix on both sides , and the matrix was fixed into the meniscus in a stable way .
additionally , 1 to 4 ( 3 on average , depending on the extension of the tear ) fast - fix sutures were inserted into the meniscus wrapped with the matrix for a better stabilization of the meniscal tear and for an increased tightness between the meniscus and the matrix .
an example of suture placement and meniscus wrapping with collagen matrix is shown in figure 3 and as a schematic drawing in figure 4 .
the bone marrow aspirate was obtained from the tibial proximal epiphysis of the operated knee .
the bone marrow aspirate was injected between the chondro - gide matrix and the meniscus , under direct arthroscopic control and carried out in
an extensive surgical description of the technique has been published by piontek et al . in 2012 .
patients began postoperative treatment from toe touch crutch walking and a range of motion exercises on the first postoperative day , for a period of 4 weeks . over the next 2 to 4 weeks
the comparison between preoperative ( time 0 ) and postoperative ( 24 months follow - up ) values of ikdc 2000 clinical , ikdc subjective , and lysholm separately had been calculated by nonparametric wilcoxon test .
the statistical significance of values between the preoperative and 2-year follow - up values of ikdc 2000 clinical , ikdc 2000 subjective , and lysholm scores with respect to the worms mri cumulative score were performed using kruskal - wallis test .
spearman s rank coefficient test was used to calculate the correlation coefficients between ikdc 200 , ikdc subjective , and lysholm score preoperatively and at 2-year follow - up and against worms mri cumulative score .
computations were done with statistica data analysis software system version 10 ( statsoft inc , 2011 ) .
the comprehensive data , including demographics , pre- and postoperative data of the patients participating in the study , are summarized in table 1 .
abbreviations : bmi , body mass index ; f , female ; fu , follow - up ; ikdc , international knee documentation committee ; m , male ; n / a , not aapplicable ; tts , time to surgery ; barrett scores , binary value for pain , effusion , clicking / locking , mcmurray test ; barrett values , converted barrett scores ( att.1 ) .
mri cummulative based on worms modified criterias . between april 2010 and november 2011 , 53 consecutive patients ( 40 male and 13 female , age 18 - 59 years ) meeting inclusion criteria were included in the study and treated with the ammr method . in total , 32 right and
meniscal lesions were associated with other knee joint surgeries for concomitant lesions in 32 cases , and are reported in table 2 . concomitant knee joint surgical treatment provided at the time of indexed procedure .
abbreviations : acl , anterior cruciate ligament ; amic , autologous matrix - induced chondrogenesis ; mfc , medial femoral condyle .
the overall study participants distribution is depicted on consort case series diagram in figure 5 .
two - year follow - up period was achieved in 50 cases , 3 ( 5.7% ) patients being lost to follow - up . in addition , 2 patients were completely excluded from the evaluation due to the incomplete pre- or postoperative data .
two patients ( 4.2% ) underwent partial meniscectomy and according to the pass - fail criteria were classified as failures . based on pass - fail criteria used in this study
this value represents 95.8% of the cases entered the prospective evaluation and 86.8% of the intended to treat cases .
ikdc 2000 subjective and lysholm scores pre and postop comparison ( table 3 ) indicated a statistically significant improvement ( p = 0.0001 , wilcoxon test ) .
the subjective ikdc and lysholm scores preoperative and 24 months postoperative follow - up results .
abbreviations : ci , confidence interval ; fu , follow - up ; ikdc , international knee documentation committee .
the analysis of the ikdc clinical scores after 2 years ( table 4 ) demonstrated that 96% patients achieved a or b scores , compared with none of the patients present in either category preoperatively .
a statistically significant improvement in ikdc 2000 clinical assessment was also observed between preoperative and 2-year follow - up ( p = 0.0001 , wilcoxon test ) .
the clinical assessment based on barrett criteria ( calculated using the method described in appendix a ) is presented in table 5 , panels a and b. calculated values demonstrate a tendency for the patients to score low ( good clinical outcome ) at the 24-month follow - up .
significant improvement for barrett s clinical score assessment between pre and postoperative values was observed ( p = 0.0001 , wilcoxon test ) .
no patient was presented with positive mcmurray test . in the subgroup of isolated meniscal injuries ,
the ikdc 2000 subjective and lysholm scores were slightly lower preoperatively than in the overall study population , but the results were in the range of the general study population after 2 years ( tables 6 and 7 ) .
an improvement in ikdc 2000 subjective and lysholm scores in this subgroup of patients was observed on average and in patient by patient assessments ( data not included in this article )
. results of ikdc 2000 subjective scores for the patients with isolated meniscal injuries . results of lysholm scores for patients with isolated meniscal injuries . in 11 patients , an isolated horizontal cleavage tear of the menisci were observed ( average age 41 years , range 20 - 59 years ) . in this group of patients , an average improvement of subjective ikdc ( from 47 to 72 ) and lysholm ( from 79 to 89 ) outcomes were observed .
none of those 11 cases had undergone any additional surgical knee treatment and had their meniscus saved .
the worst results were obtained among patients suffering from cartilage damage ( grade iv ) irrespective of age .
the remaining patients with additional damage to the knee ( mainly posttraumatic ) , obtained very good and excellent results .
no significant difference in the rate of meniscal repair failure was noted for anterior cruciate ligament
intact , compared with anterior cruciate ligament deficient knees . in the 2 failures , a second - look arthroscopy was performed . in 1 case ,
the meniscal healing in arthroscopic assessment was found but due to persisting pain symptoms , a partial meniscectomy was performed 12 months after initial treatment .
postoperatively the patient still complaint about the pain localized in the region of the medial tibial condyle .
after appropriate treatment , the patient reported pain - free knee and full knee joint function .
the second patient did not comply with the physiotherapy protocol and returned to full physical activity 3 months postoperatively , despite the doctor s recommendations . as a result
, he developed a second injury of the treated meniscus leading to partial meniscectomy 6 months after indexed procedure .
analysis of the follow - up mri images revealed 85% of good meniscus outcomes based on the worms classification ( cumulative score 1 ) ( table 1 ) . in the mri examination at 2 years
postoperatively , 76% of all operated menisci showed meniscal abnormalities corresponding to a nonhomogeneous signal without meniscal tear and were classified as grade 1 according to worms classification .
eleven percent of menisci had been classified as grade 0 , with a fully regenerating meniscal cartilage with homogenous mri signal .
the worms cumulative grade 2 was established in 6 out of 45 patients ( 13% ) and they were classified as a poor mri results .
the analysis of the mri results and clinical findings at 2 years fu demonstrated that in this case series the cumulative worms results did not correlate with either clinical ( clinical ikdc 2000 and barrett score ) or subjective scores ( ikdc 2000 subjective and lysholm score ) ( table 8) .
notably , in 29 ( 62% ) of the cases , mri revealed the presence of a cyst located in the vicinity of the t - fix anchor .
the presence of the cyst had no correlation with clinical ( ikdc 2000 clinical , p = 0.47 ; and barrett criteria ) or cumulative worms results .
additionally , no correlation was found between the number of used t - fix anchors and presence or absence of the cyst ( p = 0.22 ) and with cumulative worms results ( r = 0.08 ; p = 0.62 , spearman s rank coefficient test , kruskal - wallis test ) .
figure 6a d illustrates the mri history of one representative case of a 50-year - old man who had undergone medial meniscus repair with ammr technique and cartilage regeneration of medial femoral condyle with arthroscopic amic procedure in a one - stage knee arthroscopy .
an example of sagittal proton density weighted fat - saturated image magnetic resonance iamging ( mri ) of a 50-year - old man who had undergone medial meniscus regeneration with arthroscopic technique of collagen matrix - based meniscus repair ( ammr ) and cartilage regeneration of medial femoral condyle with arthroscopic autologous matrix - induced chondrogenesis ( amic ) procedure in one - stage knee arthroscopy .
abnormal signal of the medial meniscus ( thick arrow ) , bone marrow edema ( asterisk ) , a parameniscal cyst ( thin arrow ) , and abnormal signal from regenerated cartilage area ( dotted line arrow ) .
. abnormal signal extending to the superior surface of the body of the medial meniscus ( thick arrow ) .
normal signal of the medial meniscus ( thick arrow ) , a parameniscal small cysts containing fast - fix anchors ( thin arrow ) , bone marrow edema ( asterisk ) , and abnormal signal from regenerated cartilage area ( dotted line arrow ) .
normal signal of the medial meniscus with scar tissue ( thick arrow ) , fast - fix anchors with small cysts ( thin arrow ) , normal signal from bone marrow ( asterisk ) and normal signal from the regenerated cartilage area ( dotted line arrow ) . in our series , 13 serious adverse events ( including 2 failures ) were registered . however
, none of these events were related to either the indexed procedure or material used .
abbreviations : acl , anterior cruciate ligament ; ammr , arthroscopic technique of collagen matrix - based meniscus repair ; mpfl , medial patellofemoral ligament .
the data obtained demonstrate that combined and complex meniscal tears located in the white - white and white - red zones can be treated arthroscopically safely and with clinical success treated by meniscal suturing and wrapping in the collagen matrix .
all patients participating in this study would have had their menisci partially or totally resected otherwise .
the present results , demonstrate that the specially developed all - inside technique of suture wrapping of the lesion with a collagen membrane and in situ administration of blood bone marrow aspirate through an arthroscopic technique , is feasible .
it presents , in our opinion , an alternative to total or partial meniscectomies in cases of combined tears located in the red - white and white - white zones .
as previously suggested , the collagen scaffold may facilitate healing by providing an appropriate biological environment for cells and bone marrow blood to facilitate the process of natural healing and regeneration .
the primary outcome results of our 2-year follow - up data indicate not only possibility for meniscus preservation but also increase in the ikdc subjective score from 44 to 79 and in the lysholm score from 66 to 86 on average preoperatively and at 2-year follow - up , respectively .
ikdc 2000 clinical and barrett scores were also improved at 24 months postsurgery . in the subgroup of 11 cases of horizontal tear
we were able to save the meniscus in all cases with 82% of good to excellent clinical results , and 64% of very good and excellent subjective score assessment . in our case series
it provides good access to the operating site , is simple and was preferred by the operating surgeon .
there is , however , no reason to restrict the surgical technique to the one approach only .
the goal is to fix the torn meniscus and wrap it in to the collagen membrane .
collagen membrane use to protect the cartilage healing after micro fracture is an established procedure since almost 10 years .
recently , jlke et al . described encouraging results of collagen membrane use for short - term guided tissue regeneration of avascular meniscal tears on the goat model , especially if the autologous chondrocytes were applied .
autologous bone marrow aspirate can be obtained in a relatively simple and inexpensive method , and is known as a source of stem cells as well as growth factors . although alternative sources of stem cells are described in the literature , there is currently no evidence of superiority one over the other source or method of preparation . after reviewing the clinical results of the series
, we found no reason to limit the method to the patients presented with absent signs of an advanced arthrosis of the knee , neither to the patients of the young age .
based on our experience great care needs to be taken in patients with grade iv cartilage lesions which scored lowest in our case series .
additional research on a larger population may help in finalizing the indications for the proposed method of treatment .
while there are number of publications indicating mri as the imaging modality of choice for diagnosis of primary meniscal injuries , to date there is no suitable scoring system offering the assessment classification and allowing for an objective analysis of the mri data after meniscus repair .
conventional mr accuracy for detection of meniscal recurrent tears status post - surgery has been previously reported as 66% to 82% . in the absence of established mri scoring system for assessment of meniscal repair
, we have chosen the worms score as the classification best suited for the purpose of this study .
we found a higher prevalence of increased signal in the posterior horn of the treated meniscus in 35 cases ( 73% ) in which degeneration and tears are most frequently found , and the differentiation of a high signal intensity from a meniscal tear at this site is of great clinical importance .
the reason and meaning of this finding remains unclear , but it may be due to the scar formation and increased vascularization after regeneration . in 7 ( 15% ) cases ,
interestingly , we had observed a homogenous signal in 5 ( 10% ) cases , which is uncommon after meniscus suturing without biological healing enhancing methods . we also had observed changes in shape of meniscus ; however ,
this finding has been reported as sensitive but not specific for the diagnosis of recurrent tears . similarly to popescu et al .
, no correlation between mri outcomes , healing outcomes and clinical scores , barrett s criteria , patient s satisfaction , or the return to previous level of activity was found in the study . on the other hand ,
of these , 65 % had healed completely and 19 % had healed incompletely , leaving a failure of 16% .
have recently compared meniscal treatment techniques , and found that strict clinical evaluation resulted in lower estimates of the healing rate compared with mri or second - look arthroscopy .
similarly to miao and pujol , we are of the opinion that a thorough clinical evaluation , including medical history and physical examination , seems to remain the gold standard in short - term follow - up , and this may be supplemented with imaging studies when needed .
all - inside suture materials , such as fast - fix , are now widely available .
the occurrence of meniscal cyst is rather frequently related to all known suture techniques and materials . in the present study , anchor cysts
were noted in 29 ( 62% ) menisci in follow - up mri evaluation , without significant or clinically relevant correlation with clinical outcome .
type of lesion , type of surgery , timing of biological healing , and the patient s symptoms determine the various types of rehabilitation protocol available for a full recovery .
specific physiotherapy treatment applicable after ammr procedure has been designed and used for the patients of this study ( see appendix b ) .
the results presented in this study were collected on a consecutive case series from a single center .
the alternative of treatment for the participants of current study would be total or partial meniscetomy . from the literature and clinical experience
it is known that such treatment leads to biomechanical suboptimal condition and increases the risk of development of the arthritic changes in the knee joint .
possibility of control group containing simple suturing of menisci was also contemplated ; however , the clinical experience of menisci sutured in white - white zone were not satisfactory .
it has proven to be ethically unjustified to propose the clinical study with the control group , which will evidently not be benefitting from the treatment and such proposal was in fact not considered by our institutional review board .
the goal of this study was to evaluate the safety of the procedure and early clinical outcome of the preserved menisci treated with the collagen wrapping technique . since majority of patients in the study had additional lesions , including cartilage defects and anterior cruciate ligament deficiency .
it can not be excluded that those concomitatnt defects affected the final clicnial outcome due to potential differences in the biology of the intrasynovial fluid .
such a study design may be confounded by selection bias , which limits statements on the causality of correlations observed .
the inclusion criteria based on the extent and type of meniscal lesion were assessed and operated by one surgeon .
video and photographic documentation have been however obtained for each case documenting meniscal lesion and the justification for using the collagen wrapping surgical technique according to the selection criteria of the study design . for the recruitment for this study ,
the study objective was to assess the clinical state after applying the new technology regardless of the axis of the leg .
is a well - established tool used for many years for research and clinical purposes .
the numerical representation of the barrett score , described in detail in appendix a was simple conversion of the single variables into the one summarized value .
safety of the procedure was evaluated based on pass - fail criteria and adverse events recording .
none of the recorded adverse events were related to the procedure or material used in the procedure .
both patients have been successfully treated and achieved full recovery and full range of motion in the knee joint .
based on the published data the frequency of arthrofibrosis of the knee joint after arthroscopic interventions is reported between 8% and 10% .
we agree that an accumulation of irrigation fluid passing through a popliteal cyst into the superficial flexor compartment can be potential couseor at least should be suspected . in summary ,
even with an optimal perioperative management the subsequent compartment syndrome due to knee arthroscopy can not be completely avoided .
the results of this study can be considered for salvage treatment of complex meniscal tears in patients who otherwise would have undergone total or partial meniscectomies . the 2-year follow - up
is generally considered as a minimum time span to assess the results of the orthopedic intervention in the knee joint .
in fact , the current understanding is that meniscal healing is reported to be achieved at 6 months .
the surgical technique applied in this study will be further validated through an extended follow - up for the next 3 years .
a clinical results at the 2-year follow - up of the patients operated by ammr technique due to the meniscal lesions localized in the white - white and white - red zones demonstrated that presented surgical technique can offer a safe and promising additional alternative for surgeons who are willing to make an effort to save the meniscus in the patients otherwise scheduled for meniscal removal . | objectiveto collect and analyze the 2-year follow - up clinical and mri results of patients treated with an arthroscopic technique of collagen membrane - based meniscus repair.design53 consecutive patients with combined ( horizontal and radial or longitudinal component ) and complex meniscal tears ( tear extended through avascular zones or / and composed with two or more morphological tear pattern ) were treated with an all - inside arthroscopic suture of meniscus and wrapping with a collagen membrane ( chondro - gide ) technique with bone marrow blood injection .
the ikdc 2000 subjective score , ikdc 2000 clinical evaluation score , lysholm score and barret clinical criteria of meniscal healing were recorded .
all patients were examinated by mri 2 years postoperatively , using modified worms criteria for meniscal integrity.resultsthe 2 year follow - up was achieved in 50 cases .
of these , 2 patients were excluded from the evaluation due to incomplete data and 2 patients underwent partial meniscectomy and were classified as failures . in 46 patients ( 86.8% of the intended to treat cases ) , a statistically significant improvement in ikdc 2000 subjective , lysholm scores and ikdc 2000 clinical assessment between preoperative and the 2-year follow - up time points were obsereved .
barret criteria demonstrated an improved clinical outcome between pre- and post - operative values .
mri revealed a non - homogeneous signal without meniscal tear ( worms grade 1 ) in 76% of the operated menisci ( 13% worms grade 2).conclusionsthe
2-year follow - up data demonstrate that this technique is safe and can offer an additional tool to save the meniscus in the patients otherwise scheduled for meniscal removal .
level of evidence iv | Introduction
Materials and Methods
Study Design
Collagen Matrix
Surgical Technique
Postoperative Physiotherapy Protocol
Statistical Evaluation
Results
Discussion
Conclusions | , bone marrow or adipose tissue ) allows the evaluation of the role of cell - based techniques to augment healing . notably , jakobi and jakob tested 30 patients with the treatment of meniscal tears by suturing and wrapping the repair in a collagen matrix . inspired by those advances in the treatment of injured menisci , in 2010 a fully arthroscopic technique has been developed to treat combined ( possessing horizontal and radial or longitudinal component ) and complex ( tear extended through all vascular zones or / and composed with 2 or more morphological tear pattern ) meniscal tears . conceptually , the surgical technique , is a modification of the historical henning s and jakob procedure , included suturing and wrapping the menisci in a collagen matrix , followed by the injection of liquid bone marrow collected from the tibial proximal epiphysis , into the area of the meniscal lesion . the purpose of this work is to present a 2-year follow - up of clinical and magnetic resonance imaging ( mri ) results from a prospective consecutive case series of patients treated with a newly developed , fully arthroscopic technique of collagen matrix - based meniscus repair ( ammr ) and the injection of bone marrow aspirate into the area of the meniscal lesion . inclusion criteria for the procedure comprised ( 1 ) full - thickness , combined meniscal tear greater than 20 mm in length , ( 2 ) horizontal and radial tear , ( 3 ) tear location reaching more than 6 mm from the meniscocapsular junction including the avascular zone , or ( 4 ) both degenerative and non - degenerative meniscus ( i.e. , horizontal and radial tears , involving the white - white and red - white zones , as well as extensive tears of the bucket - handle type ) . patients were excluded from the procedure if ( 1 ) full - thickness combined meniscus tear was larger than 85% of the meniscus body or ( 2 ) meniscus tear with fibrillation extended in all area of tear which was not able to be stabilized with a suture ( meniscus lesion beyond repair ) . the primary outcome of the study defined as a result of a clinical of the knee joint and meniscus related symptoms before and after treatment ( at 2-year follow - up ) . the safety of the procedure was assessed based on pass - fail criteria s and adverse events related to the procedure or medical device used ( chondro - gide collagen matrix ) . the following information were recorded at each assessment point : international knee documentation committee ( ikdc ) 2000 subjective score , ikdc 2000 clinical evaluation score , lysholm score , and barrett clinical criteria of meniscal healing . in an attempt to lower the confounding influence of the concomitant knee diseases in the patients included in the study , the analysis of the subgroup of patients with isolated meniscus injury was performed . imaging sequences included , axial t1-weighted spin - echo ( se : 700/11 [ tr ms / te ms ] , 20 cm field of view ( fov ) , 5 mm/1 mm [ slice thickness / interslice gap ] , 256 192 matrix , frequency encoding [ fe ] anterior - posterior , one excitation ) , coronal t1-weighted se ( 600/11 , 16 cm fov , 4 mm/0.5 mm , 256 192 , fe superior - inferior , 2 excitations averaged ) , sagittal t1-weighted se ( 600/11 , 16 cm fov , 4 mm/0.5 mm , 256 192 , fe anterior - posterior , 2 excitations averaged ) , sagittal t2-weighted fast spin echo ( fse : 2500/90 ; echo train length ( etl ) of 8 ; 14 cm fov , 4 mm/0 mm , 256 192 , fe superior - inferior , 2 excitations averaged ) with fat suppression ( frequency - selective presaturation ) , and sagittal fat - suppressed t1-weighted 3-dimensional ( 3d ) spoiled gradient echo ( fs-3dspgr : 58/6 , 40 flip angle , 14 cm fov , 256 128 matrix , 60 contiguous 2-mm slices covering all articular cartilage plates in the knee , fe , superior - inferior , one excitation , frequency - selective fat saturation , superior - inferior saturation bands to minimize pulsation artifacts ) . the anterior horn , meniscal body segment , and posterior horn of the medial and lateral menisci were scored separately on the scale from 0 to 4 based on both the sagittal and coronal images : 0 = intact ; 1 = nonhomogeneous signal but not a meniscal tear ( in original worms scale minor radial tear or parrot - beak tear ) ; 2 = nondisplaced tear or prior surgical repair ; 3 = displaced tear or partial resection ; 4 = complete maceration / destruction or complete resection . in this study , the following pass - fail criteria were used : failure if at the follow - up process patient underwent partial / complete meniscectomy or knee replacement after ammr procedure pass was defined as an overall grade of a or b at final follow - up with clinical ikdc 2000 assessment . additionally , a worms cumulative grade 2 and more classified patients in the poor mri results group . using direct arthroscopic vision ,
the loops of threads running through the meniscal posterior horn and the meniscal body were passed with a special suture shuttle ( accupass , smith & nephew , andover , ma , usa ) at the level of the anterior border of the tear . after assuring that the matrix adhered to the meniscus from the tibial side the scaffold
, the meniscus was wrapped by the collagen matrix on both sides , and the matrix was fixed into the meniscus in a stable way . additionally , 1 to 4 ( 3 on average , depending on the extension of the tear ) fast - fix sutures were inserted into the meniscus wrapped with the matrix for a better stabilization of the meniscal tear and for an increased tightness between the meniscus and the matrix . the bone marrow aspirate was obtained from the tibial proximal epiphysis of the operated knee . the bone marrow aspirate was injected between the chondro - gide matrix and the meniscus , under direct arthroscopic control and carried out in
an extensive surgical description of the technique has been published by piontek et al . the comparison between preoperative ( time 0 ) and postoperative ( 24 months follow - up ) values of ikdc 2000 clinical , ikdc subjective , and lysholm separately had been calculated by nonparametric wilcoxon test . the statistical significance of values between the preoperative and 2-year follow - up values of ikdc 2000 clinical , ikdc 2000 subjective , and lysholm scores with respect to the worms mri cumulative score were performed using kruskal - wallis test . spearman s rank coefficient test was used to calculate the correlation coefficients between ikdc 200 , ikdc subjective , and lysholm score preoperatively and at 2-year follow - up and against worms mri cumulative score . inclusion criteria for the procedure comprised ( 1 ) full - thickness , combined meniscal tear greater than 20 mm in length , ( 2 ) horizontal and radial tear , ( 3 ) tear location reaching more than 6 mm from the meniscocapsular junction including the avascular zone , or ( 4 ) both degenerative and non - degenerative meniscus ( i.e. , horizontal and radial tears , involving the white - white and red - white zones , as well as extensive tears of the bucket - handle type ) . patients were excluded from the procedure if ( 1 ) full - thickness combined meniscus tear was larger than 85% of the meniscus body or ( 2 ) meniscus tear with fibrillation extended in all area of tear which was not able to be stabilized with a suture ( meniscus lesion beyond repair ) . the primary outcome of the study defined as a result of a clinical of the knee joint and meniscus related symptoms before and after treatment ( at 2-year follow - up ) . the safety of the procedure was assessed based on pass - fail criteria s and adverse events related to the procedure or medical device used ( chondro - gide collagen matrix ) . the following information were recorded at each assessment point : international knee documentation committee ( ikdc ) 2000 subjective score , ikdc 2000 clinical evaluation score , lysholm score , and barrett clinical criteria of meniscal healing . in an attempt to lower the confounding influence of the concomitant knee diseases in the patients included in the study ,
magnetic resonance imaging scans were acquired with a 1.5-tesla whole - body scanner using a commercial circumferential knee coil . images were scored with respect to modified whole organ mri score ( worms ) criteria for medial and lateral meniscal integrity . the anterior horn , meniscal body segment , and posterior horn of the medial and lateral menisci were scored separately on the scale from 0 to 4 based on both the sagittal and coronal images : 0 = intact ; 1 = nonhomogeneous signal but not a meniscal tear ( in original worms scale minor radial tear or parrot - beak tear ) ; 2 = nondisplaced tear or prior surgical repair ; 3 = displaced tear or partial resection ; 4 = complete maceration / destruction or complete resection . in this study , the following pass - fail criteria were used : failure if at the follow - up process patient underwent partial / complete meniscectomy or knee replacement after ammr procedure pass was defined as an overall grade of a or b at final follow - up with clinical ikdc 2000 assessment . additionally ,
a worms cumulative grade 2 and more classified patients in the poor mri results group . using direct arthroscopic vision ,
the loops of threads running through the meniscal posterior horn and the meniscal body were passed with a special suture shuttle ( accupass , smith & nephew , andover , ma , usa ) at the level of the anterior border of the tear . as a result ,
the meniscus was wrapped by the collagen matrix on both sides , and the matrix was fixed into the meniscus in a stable way . additionally , 1 to 4 ( 3 on average , depending on the extension of the tear ) fast - fix sutures were inserted into the meniscus wrapped with the matrix for a better stabilization of the meniscal tear and for an increased tightness between the meniscus and the matrix . the bone marrow aspirate was obtained from the tibial proximal epiphysis of the operated knee . the bone marrow aspirate was injected between the chondro - gide matrix and the meniscus , under direct arthroscopic control and carried out in
an extensive surgical description of the technique has been published by piontek et al . over the next 2 to 4 weeks
the comparison between preoperative ( time 0 ) and postoperative ( 24 months follow - up ) values of ikdc 2000 clinical , ikdc subjective , and lysholm separately had been calculated by nonparametric wilcoxon test . the statistical significance of values between the preoperative and 2-year follow - up values of ikdc 2000 clinical , ikdc 2000 subjective , and lysholm scores with respect to the worms mri cumulative score were performed using kruskal - wallis test . spearman s rank coefficient test was used to calculate the correlation coefficients between ikdc 200 , ikdc subjective , and lysholm score preoperatively and at 2-year follow - up and against worms mri cumulative score . the comprehensive data , including demographics , pre- and postoperative data of the patients participating in the study , are summarized in table 1 . abbreviations : bmi , body mass index ; f , female ; fu , follow - up ; ikdc , international knee documentation committee ; m , male ; n / a , not aapplicable ; tts , time to surgery ; barrett scores , binary value for pain , effusion , clicking / locking , mcmurray test ; barrett values , converted barrett scores ( att.1 ) . between april 2010 and november 2011 , 53 consecutive patients ( 40 male and 13 female , age 18 - 59 years ) meeting inclusion criteria were included in the study and treated with the ammr method . two - year follow - up period was achieved in 50 cases , 3 ( 5.7% ) patients being lost to follow - up . in addition , 2 patients were completely excluded from the evaluation due to the incomplete pre- or postoperative data . two patients ( 4.2% ) underwent partial meniscectomy and according to the pass - fail criteria were classified as failures . based on pass - fail criteria used in this study
this value represents 95.8% of the cases entered the prospective evaluation and 86.8% of the intended to treat cases . ikdc 2000 subjective and lysholm scores pre and postop comparison ( table 3 ) indicated a statistically significant improvement ( p = 0.0001 , wilcoxon test ) . the subjective ikdc and lysholm scores preoperative and 24 months postoperative follow - up results . the analysis of the ikdc clinical scores after 2 years ( table 4 ) demonstrated that 96% patients achieved a or b scores , compared with none of the patients present in either category preoperatively . a statistically significant improvement in ikdc 2000 clinical assessment was also observed between preoperative and 2-year follow - up ( p = 0.0001 , wilcoxon test ) . the clinical assessment based on barrett criteria ( calculated using the method described in appendix a ) is presented in table 5 , panels a and b. calculated values demonstrate a tendency for the patients to score low ( good clinical outcome ) at the 24-month follow - up . in the subgroup of isolated meniscal injuries ,
the ikdc 2000 subjective and lysholm scores were slightly lower preoperatively than in the overall study population , but the results were in the range of the general study population after 2 years ( tables 6 and 7 ) . an improvement in ikdc 2000 subjective and lysholm scores in this subgroup of patients was observed on average and in patient by patient assessments ( data not included in this article )
. results of ikdc 2000 subjective scores for the patients with isolated meniscal injuries . results of lysholm scores for patients with isolated meniscal injuries . in 1 case ,
the meniscal healing in arthroscopic assessment was found but due to persisting pain symptoms , a partial meniscectomy was performed 12 months after initial treatment . analysis of the follow - up mri images revealed 85% of good meniscus outcomes based on the worms classification ( cumulative score 1 ) ( table 1 ) . in the mri examination at 2 years
postoperatively , 76% of all operated menisci showed meniscal abnormalities corresponding to a nonhomogeneous signal without meniscal tear and were classified as grade 1 according to worms classification . the worms cumulative grade 2 was established in 6 out of 45 patients ( 13% ) and they were classified as a poor mri results . the analysis of the mri results and clinical findings at 2 years fu demonstrated that in this case series the cumulative worms results did not correlate with either clinical ( clinical ikdc 2000 and barrett score ) or subjective scores ( ikdc 2000 subjective and lysholm score ) ( table 8) . notably , in 29 ( 62% ) of the cases , mri revealed the presence of a cyst located in the vicinity of the t - fix anchor . the presence of the cyst had no correlation with clinical ( ikdc 2000 clinical , p = 0.47 ; and barrett criteria ) or cumulative worms results . an example of sagittal proton density weighted fat - saturated image magnetic resonance iamging ( mri ) of a 50-year - old man who had undergone medial meniscus regeneration with arthroscopic technique of collagen matrix - based meniscus repair ( ammr ) and cartilage regeneration of medial femoral condyle with arthroscopic autologous matrix - induced chondrogenesis ( amic ) procedure in one - stage knee arthroscopy . abnormal signal of the medial meniscus ( thick arrow ) , bone marrow edema ( asterisk ) , a parameniscal cyst ( thin arrow ) , and abnormal signal from regenerated cartilage area ( dotted line arrow ) . normal signal of the medial meniscus ( thick arrow ) , a parameniscal small cysts containing fast - fix anchors ( thin arrow ) , bone marrow edema ( asterisk ) , and abnormal signal from regenerated cartilage area ( dotted line arrow ) . normal signal of the medial meniscus with scar tissue ( thick arrow ) , fast - fix anchors with small cysts ( thin arrow ) , normal signal from bone marrow ( asterisk ) and normal signal from the regenerated cartilage area ( dotted line arrow ) . abbreviations : acl , anterior cruciate ligament ; ammr , arthroscopic technique of collagen matrix - based meniscus repair ; mpfl , medial patellofemoral ligament . the data obtained demonstrate that combined and complex meniscal tears located in the white - white and white - red zones can be treated arthroscopically safely and with clinical success treated by meniscal suturing and wrapping in the collagen matrix . the present results , demonstrate that the specially developed all - inside technique of suture wrapping of the lesion with a collagen membrane and in situ administration of blood bone marrow aspirate through an arthroscopic technique , is feasible . the primary outcome results of our 2-year follow - up data indicate not only possibility for meniscus preservation but also increase in the ikdc subjective score from 44 to 79 and in the lysholm score from 66 to 86 on average preoperatively and at 2-year follow - up , respectively . ikdc 2000 clinical and barrett scores were also improved at 24 months postsurgery . in the subgroup of 11 cases of horizontal tear
we were able to save the meniscus in all cases with 82% of good to excellent clinical results , and 64% of very good and excellent subjective score assessment . described encouraging results of collagen membrane use for short - term guided tissue regeneration of avascular meniscal tears on the goat model , especially if the autologous chondrocytes were applied . we found a higher prevalence of increased signal in the posterior horn of the treated meniscus in 35 cases ( 73% ) in which degeneration and tears are most frequently found , and the differentiation of a high signal intensity from a meniscal tear at this site is of great clinical importance . similarly to miao and pujol , we are of the opinion that a thorough clinical evaluation , including medical history and physical examination , seems to remain the gold standard in short - term follow - up , and this may be supplemented with imaging studies when needed . in the present study , anchor cysts
were noted in 29 ( 62% ) menisci in follow - up mri evaluation , without significant or clinically relevant correlation with clinical outcome . from the literature and clinical experience
it is known that such treatment leads to biomechanical suboptimal condition and increases the risk of development of the arthritic changes in the knee joint . the goal of this study was to evaluate the safety of the procedure and early clinical outcome of the preserved menisci treated with the collagen wrapping technique . it can not be excluded that those concomitatnt defects affected the final clicnial outcome due to potential differences in the biology of the intrasynovial fluid . video and photographic documentation have been however obtained for each case documenting meniscal lesion and the justification for using the collagen wrapping surgical technique according to the selection criteria of the study design . the results of this study can be considered for salvage treatment of complex meniscal tears in patients who otherwise would have undergone total or partial meniscectomies . the 2-year follow - up
is generally considered as a minimum time span to assess the results of the orthopedic intervention in the knee joint . a clinical results at the 2-year follow - up of the patients operated by ammr technique due to the meniscal lesions localized in the white - white and white - red zones demonstrated that presented surgical technique can offer a safe and promising additional alternative for surgeons who are willing to make an effort to save the meniscus in the patients otherwise scheduled for meniscal removal . | [
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in 2013 , there were 251,454 deaths in the united states stemming from medical error , which made medical error the third leading cause of death .
while published data are lacking from china , it is an important problem to be addressed .
simulation , as a valid and safe adjunct modality in healthcare education , is widely embraced by academic medical centers around the world .
it provides a no - risk platform for learners , not only to acquaint themselves with and develop new skills , but also to refine and perfect their skills to achieve expertise and mastery .
when integrated into medical education , simulation not only enables the trainees to bypass the early error - prone period and reduce the length of the learning curve , but also secures the safety of patients .
simulation is being used to train and assess all of the domains that are linked to performance in healthcare , including technical and nontechnical skills for both individuals and healthcare teams .
simulation as a solution for training and assessment is a worldwide phenomenon that is beginning to evolve in china , the world 's most populous country .
an assessment of the state and need for simulation - based education in china has not been published in the literature .
the purpose of this study was to assess the current status of the use of simulation - based training in beijing , identify the availability and adequacy of simulation resources , assess financial capacity , investigate where simulation is being used , and explore the barriers to further development .
the study included hospitals in beijing accredited by the standardized residency training ( srt ) program .
the list of the potential qualified hospitals was obtained from the beijing srt management system website .
hospitals accredited for at least 1 of the core specialties / disciplines during 2015 were selected for the study .
these specialties / disciplines included anesthesiology , emergency medicine , internal medicine , obstetrics - gynecology ( ob / gyn ) , pediatric internal medicine , pediatric surgery , and surgery .
hospitals qualified only for the srt in clinical pathology , dermatology , family medicine , laboratory medicine , medical imaging , neurology , oncology , ophthalmology , otorhinolaryngology , pharmacy , psychiatry , rehabilitation medicine , or stomatology were excluded in this study .
participants were queried as to the status of their organizations from january 1 , 2015 , to december 31 , 2015 .
survey invitations with website links were sent to the srt management departments by e - mail or instant message from may through june of 2016 .
survey question methodology included single - choice and multiple - choice questions ( one or more answer ) and semantic differential scale questions .
demographic classification of trainees as surgeons or physicians in practice ( attending or above ) , residents ( or postgraduate residents ) , fellows , medical students ( or interns ) , nurses , and allied health professionals was performed .
we assessed the availability of simulation equipment , curricula , and identification of the targeted learners .
the categories of simulation equipment in this survey included standardized patients , full - scale mannequins , ex vivo animal tissue models , live animals , human cadavers , synthetic task trainers ( or bench - top models ) , screen - based virtual reality ( vr ) workstations and online simulation modules . to examine the curricula , the survey divided simulation - based educational activities into several areas , such as learning medical knowledge , practice - based skill training , skill competency evaluation , doctor - patient communication , leadership and teamwork , critical thinking and decision - making , patient safety , health advocacy , medical ethics , curriculum validation , and follow - up of learners .
the survey also assessed infrastructure support , such as the affiliations of their simulation department , accreditation status , financial capacity , and the availability of specialized simulation - trained staff .
finally , it queried their attitudes , needs and perceived barriers in the development of simulation programs .
survey question methodology included single - choice and multiple - choice questions ( one or more answer ) and semantic differential scale questions .
demographic classification of trainees as surgeons or physicians in practice ( attending or above ) , residents ( or postgraduate residents ) , fellows , medical students ( or interns ) , nurses , and allied health professionals was performed .
we assessed the availability of simulation equipment , curricula , and identification of the targeted learners .
the categories of simulation equipment in this survey included standardized patients , full - scale mannequins , ex vivo animal tissue models , live animals , human cadavers , synthetic task trainers ( or bench - top models ) , screen - based virtual reality ( vr ) workstations and online simulation modules . to examine the curricula , the survey divided simulation - based educational activities into several areas , such as learning medical knowledge , practice - based skill training , skill competency evaluation , doctor - patient communication , leadership and teamwork , critical thinking and decision - making , patient safety , health advocacy , medical ethics , curriculum validation , and follow - up of learners .
the survey also assessed infrastructure support , such as the affiliations of their simulation department , accreditation status , financial capacity , and the availability of specialized simulation - trained staff .
finally , it queried their attitudes , needs and perceived barriers in the development of simulation programs .
thirty hospitals were invited to participate in the survey , and 15 of them ( 50% response rate ) replied and completed the questionnaire .
of these 15 hospitals , the qualification rate for srt in each specialty were internal medicine 93.33% ( 14/15 ) , surgery 73.33% ( 11/15 ) , anesthesiology 60% ( 9/15 ) , ob - gyn 60% ( 9/15 ) , emergency medicine 53.33% ( 8/15 ) , pediatric internal medicine 13.33% ( 2/15 ) , and pediatric surgery 6.67% ( 1/15 ) .
task trainers ( 15/15 ) , full - scale mannequins ( 14/15 ) , standardized patients ( 12/15 ) , and vr workstations ( 11/15 ) were the top 4 common types of simulation modalities available for use , followed by ex vivo animal tissue models ( 8/15 ) , live animal models ( 7/15 ) , and an online simulation module ( 1/15 ) .
the most commonly available task trainers ( or bench - top models ) in these institutions were cardiopulmonary resuscitation torso trainers ( 15/15 ) , incision and suturing models ( 14/15 ) , and heart and lung sound - recognition models ( 12/15 ) , followed by genitourinary models ( 11/15 ) , airway management models ( 11/15 ) , vascular access models ( 10/15 ) , lumbar puncture models ( 8/15 ) , and nasogastric tube models ( 8/15 ) .
of the 14 hospitals that owned the full - scale mannequin , most had basic adult mannequins without physiology ( 12/14 ) ; 8/14 had mannequins with physiology .
four of them had pediatric mannequins without physiology , and only 1 owned pediatric mannequins with physiology ; 1 had basic infant mannequins and 1 owned infant mannequins with physiology .
the top 2 available vr simulators in these 9 hospitals were for laparoscopic skills ( 9/11 ) , and gastrointestinal ( gi ) endoscopy skills ( 5/11 ) , followed by urologic endoscopy skills ( 4/11 ) , arthroscopic skills ( 3/11 ) , bronchoscopic skills ( 3/11 ) , and endovascular skills ( 2/11 ) . among the given specialties for srt , the availability of simulation courses was 100% ( 2/2 ) for pediatric internal medicine , 100% ( 1/1 ) for pediatric surgery , 90% ( 10/11 ) for surgery , 78.57% ( 11/14 ) for internal medicine , 77.78% ( 7/9 ) for anesthesiology , 75% ( 6/8 ) for emergency medicine , and 33.33% ( 3/9 ) for ob / gyn ( figure 1 ) .
some institutes had simulation curricula , even though they were not qualified for srt in the given disciplines .
there were 2 hospitals without any available training course in any of the listed specialties .
qualified hospitals for str and availabilities of simulation curriculum in the given specialties . of the 13 institutions with available simulation curricula ,
12 had simulations focused on proficiency - based skill training , 11 had medical knowledge learning , and 10 had skill competency assessment .
simulation was cited less for patient safety ( 6/13 ) , leadership and teamwork ( 5/13 ) , trainee follow - up ( 5/13 ) , doctor
patient communication ( 4/13 ) , critical thinking and decision - making ( 3/13 ) , and curriculum validation ( 3/13 ) , and even less frequently for medical ethics ( 1/13 ) .
all of the 13 hospitals with available simulation courses targeted residents or postgraduate residents as their trainees .
most of them targeted medical students or interns ( 12/13 ) , fellows ( 9/13 ) , practicing surgeons or physicians ( 8/13 ) , and nurses ( 7/13 ) .
allied health professionals ( 1/13 ) and high school students ( 1/13 ) were the target learning audience in 1 hospital .
simulation - based curricula or courses were not compulsory in the srt program in beijing ; hence , the hospitals do not need an accredited simulation department to be accredited for the srt program .
only one of these 14 simulation department was accredited by the ministry of health , the rest of them were not accredited by any organization or society .
thirteen of the 15 departments were affiliated with their hospitals ; the remaining 2 were affiliated with universities or colleges .
the simulation facilities in the majority of them ( 14/15 ) were centralized in a specific department within their institutions . only 1 of them was a separate entity from the clinical departments .
the majority ( 14/15 ) indicated that they were financed by their hospitals or clinical departments .
some of them also reported financial support from medical universities or colleges ( 5/15 ) .
few of them had economic support from grants or foundations ( 2/15 ) , governments ( 2/15 ) , or for - profit entities ( 1/15 ) .
none of them received funding from philanthropy . concerning human resources for simulation programs ( figure 2 ) , most of the 14 hospitals had full - time ( 10/15 ) and part - time ( 11/15 ) clinical directors , full - time ( 8/15 ) and part - time ( 9/15 ) administrators , and part - time instructors ( 13/15 ) . the full - time instructors ( 3/15 ) , part - time researchers ( 2/15 ) , and full - time ( 2/15 ) and part - time ( 4/15 ) information technology ( it ) specialists and technicians were available in some organizations .
however , there was no full - time researcher for simulation in any of the hospitals surveyed .
the center of figure 2 represents 0 , and the percentages increase by 20% as the pentagon progress outward with the outline representing 100% .
a 5-point likert scale was used to assess participants ' attitudes toward the sufficiency of simulation resources at their institutions .
one point represented extremely insufficient while 5 points represented extremely sufficient .
the mean score was 2.93 , and none of the participants thought the simulation resources were extremely sufficient or insufficient .
one point indicated totally unnecessary , while 5 points represented highly necessary .
the participants ' attitudes toward the given factors that may affect the development of simulation programs were demonstrated in figure 3 .
the respondents were asked to point out the barriers in their simulation programs ( figure 4 ) .
the top 2 barriers were the shortage of sustainable financial resources ( 12/15 ) and advocacy from their institutional authorities ( 7/15 ) .
some of them indicated the insufficiency of research programs ( 6/15 ) , curricula ( 6/15 ) , clinical instructors with protected time ( 6/15 ) , dedicated it specialists and technicians ( 4/15 ) , trainees ' need ( 3/15 ) , and endorsement by local healthcare authorities ( 2/15 ) .
the center of figure 4 represents 0 , and the percentages increase by 20% as the nonagon progresses outward , with the outline representing 100% .
task trainers ( 15/15 ) , full - scale mannequins ( 14/15 ) , standardized patients ( 12/15 ) , and vr workstations ( 11/15 ) were the top 4 common types of simulation modalities available for use , followed by ex vivo animal tissue models ( 8/15 ) , live animal models ( 7/15 ) , and an online simulation module ( 1/15 ) .
the most commonly available task trainers ( or bench - top models ) in these institutions were cardiopulmonary resuscitation torso trainers ( 15/15 ) , incision and suturing models ( 14/15 ) , and heart and lung sound - recognition models ( 12/15 ) , followed by genitourinary models ( 11/15 ) , airway management models ( 11/15 ) , vascular access models ( 10/15 ) , lumbar puncture models ( 8/15 ) , and nasogastric tube models ( 8/15 ) .
of the 14 hospitals that owned the full - scale mannequin , most had basic adult mannequins without physiology ( 12/14 ) ; 8/14 had mannequins with physiology .
four of them had pediatric mannequins without physiology , and only 1 owned pediatric mannequins with physiology ; 1 had basic infant mannequins and 1 owned infant mannequins with physiology .
the top 2 available vr simulators in these 9 hospitals were for laparoscopic skills ( 9/11 ) , and gastrointestinal ( gi ) endoscopy skills ( 5/11 ) , followed by urologic endoscopy skills ( 4/11 ) , arthroscopic skills ( 3/11 ) , bronchoscopic skills ( 3/11 ) , and endovascular skills ( 2/11 ) . among the given specialties for srt , the availability of simulation courses was 100% ( 2/2 ) for pediatric internal medicine , 100% ( 1/1 ) for pediatric surgery , 90% ( 10/11 ) for surgery , 78.57% ( 11/14 ) for internal medicine , 77.78% ( 7/9 ) for anesthesiology , 75% ( 6/8 ) for emergency medicine , and 33.33% ( 3/9 ) for ob / gyn ( figure 1 ) .
some institutes had simulation curricula , even though they were not qualified for srt in the given disciplines . there were 2 hospitals without any available training course in any of the listed specialties .
qualified hospitals for str and availabilities of simulation curriculum in the given specialties . of the 13 institutions with available simulation curricula ,
12 had simulations focused on proficiency - based skill training , 11 had medical knowledge learning , and 10 had skill competency assessment .
simulation was cited less for patient safety ( 6/13 ) , leadership and teamwork ( 5/13 ) , trainee follow - up ( 5/13 ) , doctor
patient communication ( 4/13 ) , critical thinking and decision - making ( 3/13 ) , and curriculum validation ( 3/13 ) , and even less frequently for medical ethics ( 1/13 ) .
all of the 13 hospitals with available simulation courses targeted residents or postgraduate residents as their trainees .
most of them targeted medical students or interns ( 12/13 ) , fellows ( 9/13 ) , practicing surgeons or physicians ( 8/13 ) , and nurses ( 7/13 ) .
allied health professionals ( 1/13 ) and high school students ( 1/13 ) were the target learning audience in 1 hospital .
simulation - based curricula or courses were not compulsory in the srt program in beijing ; hence , the hospitals do not need an accredited simulation department to be accredited for the srt program .
only one of these 14 simulation department was accredited by the ministry of health , the rest of them were not accredited by any organization or society .
thirteen of the 15 departments were affiliated with their hospitals ; the remaining 2 were affiliated with universities or colleges .
the simulation facilities in the majority of them ( 14/15 ) were centralized in a specific department within their institutions . only 1 of them was a separate entity from the clinical departments .
the majority ( 14/15 ) indicated that they were financed by their hospitals or clinical departments .
some of them also reported financial support from medical universities or colleges ( 5/15 ) .
few of them had economic support from grants or foundations ( 2/15 ) , governments ( 2/15 ) , or for - profit entities ( 1/15 ) .
none of them received funding from philanthropy . concerning human resources for simulation programs ( figure 2 )
, most of the 14 hospitals had full - time ( 10/15 ) and part - time ( 11/15 ) clinical directors , full - time ( 8/15 ) and part - time ( 9/15 ) administrators , and part - time instructors ( 13/15 ) . the full - time instructors ( 3/15 ) , part - time researchers ( 2/15 ) , and full - time ( 2/15 ) and part - time ( 4/15 )
however , there was no full - time researcher for simulation in any of the hospitals surveyed .
the center of figure 2 represents 0 , and the percentages increase by 20% as the pentagon progress outward with the outline representing 100% .
a 5-point likert scale was used to assess participants ' attitudes toward the sufficiency of simulation resources at their institutions .
one point represented extremely insufficient while 5 points represented extremely sufficient . the mean score was 2.93 , and none of the participants thought the simulation resources were extremely sufficient or insufficient .
one point indicated totally unnecessary , while 5 points represented highly necessary . the mean score was 4.07 .
the participants ' attitudes toward the given factors that may affect the development of simulation programs were demonstrated in figure 3 .
the respondents were asked to point out the barriers in their simulation programs ( figure 4 ) .
the top 2 barriers were the shortage of sustainable financial resources ( 12/15 ) and advocacy from their institutional authorities ( 7/15 ) .
some of them indicated the insufficiency of research programs ( 6/15 ) , curricula ( 6/15 ) , clinical instructors with protected time ( 6/15 ) , dedicated it specialists and technicians ( 4/15 ) , trainees ' need ( 3/15 ) , and endorsement by local healthcare authorities ( 2/15 ) .
the center of figure 4 represents 0 , and the percentages increase by 20% as the nonagon progresses outward , with the outline representing 100% .
simulation among the high - ranking srt hospitals in beijing is clearly important , underresourced , and in demand .
all of the 15 hospitals included in the study are tertiary hospitals , which are the top - ranking hospitals on the chinese mainland .
simulation is being used worldwide for both technical and nontechnical skills and is emerging as an important modality in china .
issues specific to china include an increasingly strained relationship between doctors and patients , in which patients do not trust their doctors and even refuse to be operated on by resident surgeons .
hands - on time in each specialty and the enormously discrepant distribution of case volumes all ultimately contribute to the relatively unequal or insufficient exposure for learners in medical practice .
simulation has a profound ability to provide significant advantages as a platform for repetitive practice in a no - risk environment away from patients .
the specialties included in this study are the ones where simulation - based training had been broadly used internationally . the most common simulation equipment in the responding hospitals were task trainers , basic or advanced full - scale mannequins , standardized patients , and vr simulators .
the quantities of simulators , curricula , and trainees were not asked for in the survey , as most of the hospitals in beijing did not have an independent simulation department .
the availability of simulators may not reflect the exact frequency of use or activities of an institution , but these types of simulators are universally applicable in primary specialties , and they could meet the common needs of novices in a clinical rotation if embedded within a well - designed simulation curriculum .
most of the respondents ( 11/15 ) agreed or strongly agreed that curriculum is one of the influence factors in simulation education and training , and 40% ( 6/15 ) of them indicated that the shortage of curriculum was one of the barriers in their simulation program . to fully take advantage the benefit of simulation
, proficiency - based training should not be a single method of education , but instead , must be integrated into a comprehensive curriculum .
the equipment available and purchased will directly follow the curricular needs . according to this survey , in beijing
, the focus for application of simulation is primarily for competency - based technical skills training , medical knowledge learning , and competency assessment . despite evidence that 70% of medical errors occur within the communication domain ,
fewer than half of the programs included patient safety and nontechnical skills , such as leadership , teamwork , communication , decision - making , and critical thinking .
this finding was in contrast to the united states and europe , where simulation is frequently used to address these competencies .
healthcare is a team sport , and to achieve a good patient outcome , technical ability must be complemented by excellent , nontechnical skills across the entire team taking care of the patient . to fully integrate patient safety into the curriculum , simulation programs should work closely with risk management , patient safety committees , and hospital administrations to pointedly address problems of patient safety .
the main targeted trainees in these hospitals were residents ( or postgraduate residents ) and medical students ( or interns ) .
novices are always the target audience in simulation training , because premature exposure to patients often results in increased complication rates related to suboptimal technical skills .
simulation training can help novices to maximize their practice time and ensure that they get as much technical exposure as possible . as nontechnical skills have not been found to necessarily correlate with experience , more advanced trainees and practitioners may also benefit from simulation training
. either novices or physicians who want to develop new skills or maintain their skills could be the eligible trainees in a comprehensive simulation course .
only 23.08% ( 3/13 ) of the institutions reported validation activities in their simulation courses , and only 1 had published articles related to simulation in 2015 .
first , for a simulator to be used to assess competence , it must be evaluated vigorously and objectively to determine its reliability and validity .
second , one of the most significant incentives of simulation in medical education is that simulation can improve patients ' outcomes by minimizing preventable mistakes , and research or academic activities are therefore also necessary for advocating the superiority of simulation - based medical education .
hence , a qualified simulation curriculum should be well designed and validated ; it should be composed of both technical and nontechnical skills training .
with such a course , the real value of simulation would be appreciated . as most of their simulation departments were centralized and affiliated with their hospitals ,
the financing mainly came from their hospitals or clinical departments . according to this study ,
one of the most frequent barriers they encountered in their development was a lack of sustainable funding sources ( 12/15 ) , and most of the respondents ( 14/15 ) indicated that the funding was one of the factors that may influence the growth of simulation programs .
simulation education and training in healthcare could improve patients ' safety , and most respondents ( 13/15 ) held a positive attitude toward the necessity of simulation in healthcare education .
however , few of them ( 3/15 ) thought that simulation training resources were sufficient at their organizations . given the massive investment up front , the on - going consumption , and the time needed for validating the curriculum , simulation may fail to demonstrate the return on investment in the short term .
the return is difficult to monetize , which may be the reason that simulation commonly fails to acquire adequate support from the authorities at their hospitals .
however , taking into consideration that one of the benefits of simulation is to reduce the cost of the complications and malpractice suits of preventable mistakes , the savings could pay for an entire program .
for beijing , coordination and cooperation among simulation centers seems to be a plausible solution for optimizing the allocation of resources to serve its large population of healthcare workers and teams .
basic questions around simulation - based education can not be answered without strong collaboration among simulation centers for increased funding support , faculty training , generating guidelines , and changing policies .
they could also launch multicenter validation or research to demonstrate the benefits of simulation for introduction of new technology , entry into training programs , training , and even credentialing .
showing links to improved patient outcomes may ultimately compensate for the governments ' or authorities ' expenditure by reduction of preventable medical errors in the long run .
the need for curricula , research program , and cooperation with other centers were also demonstrated in this study ( figure 4 ) .
as indicated in this survey , 46.67% participants felt there to be insufficient support from their institutions : 86.67% thought such advocacy from their institutions could influence their simulation programs .
most of them had both full - time and part - time clinical directors and administrators . however , as most of the instructors are part - time in their simulation departments , the deficiency of instructors with protected time was cited as a major obstacle ; 93.33% of them believed that dedicated teachers could positively influence their simulation programs . in china
, the clinical directors and instructors drive the research , so the part - time status may be a contributor to the lack of emphasis on research .
the leaders of simulation programs should promote their simulation - based medical curricula through carefully designed studies linking training to patient outcomes and improving the healthcare team 's competency and efficiency . as validating evidence accumulates
, it is evident that there is a need for careful development of training facilities , training the
trainers and administrators , financial funding , and curricular design ( including theory and practical teaching ) , and research to continue to build a long - term , sustainable , effective program .
because the questionnaires were distributed through e - mail or instant message and completed online , the expectation of the time spend on completing the survey , and the complication of the items was confining to some extent .
therefore , it was not feasible to investigate quantitative data on the online survey , such as the number of trainees and simulation equipment in each category , the quantitative degree of integration of simulation into their medical education , the priority of teaching content areas in simulation activities , the contact hours ( the hours spent on training ) for different types trainees , and the frequency of use of simulation facilities .
we could not guarantee all the respondents were the director or administrator of their simulation departments , which could introduce bias into the result . the information provided by the respondents
the most common simulation equipment in the responding hospitals were task trainers , basic or advanced full - scale mannequins , standardized patients , and vr simulators .
the quantities of simulators , curricula , and trainees were not asked for in the survey , as most of the hospitals in beijing did not have an independent simulation department .
the availability of simulators may not reflect the exact frequency of use or activities of an institution , but these types of simulators are universally applicable in primary specialties , and they could meet the common needs of novices in a clinical rotation if embedded within a well - designed simulation curriculum .
most of the respondents ( 11/15 ) agreed or strongly agreed that curriculum is one of the influence factors in simulation education and training , and 40% ( 6/15 ) of them indicated that the shortage of curriculum was one of the barriers in their simulation program . to fully take advantage the benefit of simulation
, proficiency - based training should not be a single method of education , but instead , must be integrated into a comprehensive curriculum .
the equipment available and purchased will directly follow the curricular needs . according to this survey , in beijing
, the focus for application of simulation is primarily for competency - based technical skills training , medical knowledge learning , and competency assessment . despite evidence that 70% of medical errors occur within the communication domain , fewer than half of the programs included patient safety and nontechnical skills , such as leadership , teamwork , communication , decision - making , and critical thinking .
this finding was in contrast to the united states and europe , where simulation is frequently used to address these competencies .
healthcare is a team sport , and to achieve a good patient outcome , technical ability must be complemented by excellent , nontechnical skills across the entire team taking care of the patient . to fully integrate patient safety into the curriculum
, simulation programs should work closely with risk management , patient safety committees , and hospital administrations to pointedly address problems of patient safety .
the main targeted trainees in these hospitals were residents ( or postgraduate residents ) and medical students ( or interns ) .
novices are always the target audience in simulation training , because premature exposure to patients often results in increased complication rates related to suboptimal technical skills .
simulation training can help novices to maximize their practice time and ensure that they get as much technical exposure as possible . as nontechnical skills have not been found to necessarily correlate with experience
either novices or physicians who want to develop new skills or maintain their skills could be the eligible trainees in a comprehensive simulation course .
only 23.08% ( 3/13 ) of the institutions reported validation activities in their simulation courses , and only 1 had published articles related to simulation in 2015 .
first , for a simulator to be used to assess competence , it must be evaluated vigorously and objectively to determine its reliability and validity .
second , one of the most significant incentives of simulation in medical education is that simulation can improve patients ' outcomes by minimizing preventable mistakes , and research or academic activities are therefore also necessary for advocating the superiority of simulation - based medical education .
hence , a qualified simulation curriculum should be well designed and validated ; it should be composed of both technical and nontechnical skills training .
as most of their simulation departments were centralized and affiliated with their hospitals , the financing mainly came from their hospitals or clinical departments . according to this study ,
one of the most frequent barriers they encountered in their development was a lack of sustainable funding sources ( 12/15 ) , and most of the respondents ( 14/15 ) indicated that the funding was one of the factors that may influence the growth of simulation programs .
simulation education and training in healthcare could improve patients ' safety , and most respondents ( 13/15 ) held a positive attitude toward the necessity of simulation in healthcare education .
however , few of them ( 3/15 ) thought that simulation training resources were sufficient at their organizations . given the massive investment up front , the on - going consumption , and the time needed for validating the curriculum , simulation may fail to demonstrate the return on investment in the short term .
the return is difficult to monetize , which may be the reason that simulation commonly fails to acquire adequate support from the authorities at their hospitals .
however , taking into consideration that one of the benefits of simulation is to reduce the cost of the complications and malpractice suits of preventable mistakes , the savings could pay for an entire program .
for beijing , coordination and cooperation among simulation centers seems to be a plausible solution for optimizing the allocation of resources to serve its large population of healthcare workers and teams .
basic questions around simulation - based education can not be answered without strong collaboration among simulation centers for increased funding support , faculty training , generating guidelines , and changing policies .
they could also launch multicenter validation or research to demonstrate the benefits of simulation for introduction of new technology , entry into training programs , training , and even credentialing .
showing links to improved patient outcomes may ultimately compensate for the governments ' or authorities ' expenditure by reduction of preventable medical errors in the long run .
the need for curricula , research program , and cooperation with other centers were also demonstrated in this study ( figure 4 ) .
as indicated in this survey , 46.67% participants felt there to be insufficient support from their institutions : 86.67% thought such advocacy from their institutions could influence their simulation programs .
most of them had both full - time and part - time clinical directors and administrators . however , as most of the instructors are part - time in their simulation departments , the deficiency of instructors with protected time was cited as a major obstacle ; 93.33% of them believed that dedicated teachers could positively influence their simulation programs . in china ,
the clinical directors and instructors drive the research , so the part - time status may be a contributor to the lack of emphasis on research .
the leaders of simulation programs should promote their simulation - based medical curricula through carefully designed studies linking training to patient outcomes and improving the healthcare team 's competency and efficiency . as validating evidence accumulates
it is evident that there is a need for careful development of training facilities , training the
trainers and administrators , financial funding , and curricular design ( including theory and practical teaching ) , and research to continue to build a long - term , sustainable , effective program .
because the questionnaires were distributed through e - mail or instant message and completed online , the expectation of the time spend on completing the survey , and the complication of the items was confining to some extent .
therefore , it was not feasible to investigate quantitative data on the online survey , such as the number of trainees and simulation equipment in each category , the quantitative degree of integration of simulation into their medical education , the priority of teaching content areas in simulation activities , the contact hours ( the hours spent on training ) for different types trainees , and the frequency of use of simulation facilities .
we could not guarantee all the respondents were the director or administrator of their simulation departments , which could introduce bias into the result . the information provided by the respondents
the results of this survey indicated that most of the participating hospitals had simulation facilities .
the primary targeted audiences were residents ( or postgraduate residents ) and medical students ( or interns ) .
internal medicine , surgery , and anesthesiology were the 3 most common specialties where simulation training was used .
the teaching content domains should be refined , and there should be a greater emphasis on nontechnical skills .
the simulation education and training in most if them had a shortage of research , sustainable financial resources , and endorsement from their authorities .
the building of a consortium and leadership specializing in the science of simulation would help bridge the gaps . | background and objectives : in 2013 , medical error was the third leading cause of death in the united states.1 in china , as in the case with the united states , training and assessment are developing as a strategy to reduce the occurrence of such errors .
the objective of this study was to assess the current state of the use of simulation - based training in beijing and to explore the barriers to further development.methods:this study included hospitals in beijing accredited by the standardized residency training ( srt ) program .
the questionnaire was designed online and distributed to the srt management departments by e - mail or instant message.results:thirty hospitals were invited to participate in this survey , and 15 responses were completed and met the inclusion criteria .
task trainers ( 15/15 ) , full - scale mannequins ( 14/15 ) , standardized patients ( 12/15 ) , and virtual reality workstations ( 11/15 ) were the most common types of simulation modalities available for use . among the given specialties for srt , the availability of simulation courses was 2/2 for pediatric internal medicine , 1/1 for pediatric surgery , 10/11 for surgery , 11/14 for internal medicine , 7/9 for anesthesiology , 6/8 for emergency medicine , and 3/9 for obstetrics / gynecology .
of the 13 institutions with available simulation curricula , 12/13 had simulation focused on proficiency - based skill training , 11/13 had medical knowledge learning , 10/13 had skill competency assessment .
the main targeted trainees in these hospitals were residents ( or postgraduate residents ) and medical students ( or interns ) .
the top 2 barriers were the shortage of sustainable financial resources ( 12/15 ) and advocacy from their institutional authorities ( 7/15).conclusion : it is evident that there is a need for more development of training facilities , and for training the
trainers and administrators . financial funding , curricular design , and research seem to be crucial for building a long - term , sustainable , effective program . | INTRODUCTION
MATERIALS AND METHODS
Survey
RESULTS
Simulator, Curriculum, and Learner
Infrastructure Support
Barriers
DISCUSSION
Simulator, Curriculum, and Learner
Infrastructure Support and Barriers
A Beijing Simulation Alliance?
Limitations
CONCLUSION | in 2013 , there were 251,454 deaths in the united states stemming from medical error , which made medical error the third leading cause of death . simulation as a solution for training and assessment is a worldwide phenomenon that is beginning to evolve in china , the world 's most populous country . an assessment of the state and need for simulation - based education in china has not been published in the literature . the purpose of this study was to assess the current status of the use of simulation - based training in beijing , identify the availability and adequacy of simulation resources , assess financial capacity , investigate where simulation is being used , and explore the barriers to further development . the study included hospitals in beijing accredited by the standardized residency training ( srt ) program . these specialties / disciplines included anesthesiology , emergency medicine , internal medicine , obstetrics - gynecology ( ob / gyn ) , pediatric internal medicine , pediatric surgery , and surgery . hospitals qualified only for the srt in clinical pathology , dermatology , family medicine , laboratory medicine , medical imaging , neurology , oncology , ophthalmology , otorhinolaryngology , pharmacy , psychiatry , rehabilitation medicine , or stomatology were excluded in this study . survey invitations with website links were sent to the srt management departments by e - mail or instant message from may through june of 2016 . demographic classification of trainees as surgeons or physicians in practice ( attending or above ) , residents ( or postgraduate residents ) , fellows , medical students ( or interns ) , nurses , and allied health professionals was performed . we assessed the availability of simulation equipment , curricula , and identification of the targeted learners . the categories of simulation equipment in this survey included standardized patients , full - scale mannequins , ex vivo animal tissue models , live animals , human cadavers , synthetic task trainers ( or bench - top models ) , screen - based virtual reality ( vr ) workstations and online simulation modules . to examine the curricula , the survey divided simulation - based educational activities into several areas , such as learning medical knowledge , practice - based skill training , skill competency evaluation , doctor - patient communication , leadership and teamwork , critical thinking and decision - making , patient safety , health advocacy , medical ethics , curriculum validation , and follow - up of learners . the survey also assessed infrastructure support , such as the affiliations of their simulation department , accreditation status , financial capacity , and the availability of specialized simulation - trained staff . demographic classification of trainees as surgeons or physicians in practice ( attending or above ) , residents ( or postgraduate residents ) , fellows , medical students ( or interns ) , nurses , and allied health professionals was performed . we assessed the availability of simulation equipment , curricula , and identification of the targeted learners . the categories of simulation equipment in this survey included standardized patients , full - scale mannequins , ex vivo animal tissue models , live animals , human cadavers , synthetic task trainers ( or bench - top models ) , screen - based virtual reality ( vr ) workstations and online simulation modules . to examine the curricula , the survey divided simulation - based educational activities into several areas , such as learning medical knowledge , practice - based skill training , skill competency evaluation , doctor - patient communication , leadership and teamwork , critical thinking and decision - making , patient safety , health advocacy , medical ethics , curriculum validation , and follow - up of learners . the survey also assessed infrastructure support , such as the affiliations of their simulation department , accreditation status , financial capacity , and the availability of specialized simulation - trained staff . thirty hospitals were invited to participate in the survey , and 15 of them ( 50% response rate ) replied and completed the questionnaire . of these 15 hospitals , the qualification rate for srt in each specialty were internal medicine 93.33% ( 14/15 ) , surgery 73.33% ( 11/15 ) , anesthesiology 60% ( 9/15 ) , ob - gyn 60% ( 9/15 ) , emergency medicine 53.33% ( 8/15 ) , pediatric internal medicine 13.33% ( 2/15 ) , and pediatric surgery 6.67% ( 1/15 ) . task trainers ( 15/15 ) , full - scale mannequins ( 14/15 ) , standardized patients ( 12/15 ) , and vr workstations ( 11/15 ) were the top 4 common types of simulation modalities available for use , followed by ex vivo animal tissue models ( 8/15 ) , live animal models ( 7/15 ) , and an online simulation module ( 1/15 ) . the most commonly available task trainers ( or bench - top models ) in these institutions were cardiopulmonary resuscitation torso trainers ( 15/15 ) , incision and suturing models ( 14/15 ) , and heart and lung sound - recognition models ( 12/15 ) , followed by genitourinary models ( 11/15 ) , airway management models ( 11/15 ) , vascular access models ( 10/15 ) , lumbar puncture models ( 8/15 ) , and nasogastric tube models ( 8/15 ) . the top 2 available vr simulators in these 9 hospitals were for laparoscopic skills ( 9/11 ) , and gastrointestinal ( gi ) endoscopy skills ( 5/11 ) , followed by urologic endoscopy skills ( 4/11 ) , arthroscopic skills ( 3/11 ) , bronchoscopic skills ( 3/11 ) , and endovascular skills ( 2/11 ) . among the given specialties for srt , the availability of simulation courses was 100% ( 2/2 ) for pediatric internal medicine , 100% ( 1/1 ) for pediatric surgery , 90% ( 10/11 ) for surgery , 78.57% ( 11/14 ) for internal medicine , 77.78% ( 7/9 ) for anesthesiology , 75% ( 6/8 ) for emergency medicine , and 33.33% ( 3/9 ) for ob / gyn ( figure 1 ) . some institutes had simulation curricula , even though they were not qualified for srt in the given disciplines . qualified hospitals for str and availabilities of simulation curriculum in the given specialties . of the 13 institutions with available simulation curricula ,
12 had simulations focused on proficiency - based skill training , 11 had medical knowledge learning , and 10 had skill competency assessment . all of the 13 hospitals with available simulation courses targeted residents or postgraduate residents as their trainees . most of them targeted medical students or interns ( 12/13 ) , fellows ( 9/13 ) , practicing surgeons or physicians ( 8/13 ) , and nurses ( 7/13 ) . allied health professionals ( 1/13 ) and high school students ( 1/13 ) were the target learning audience in 1 hospital . simulation - based curricula or courses were not compulsory in the srt program in beijing ; hence , the hospitals do not need an accredited simulation department to be accredited for the srt program . concerning human resources for simulation programs ( figure 2 ) , most of the 14 hospitals had full - time ( 10/15 ) and part - time ( 11/15 ) clinical directors , full - time ( 8/15 ) and part - time ( 9/15 ) administrators , and part - time instructors ( 13/15 ) . the full - time instructors ( 3/15 ) , part - time researchers ( 2/15 ) , and full - time ( 2/15 ) and part - time ( 4/15 ) information technology ( it ) specialists and technicians were available in some organizations . the top 2 barriers were the shortage of sustainable financial resources ( 12/15 ) and advocacy from their institutional authorities ( 7/15 ) . task trainers ( 15/15 ) , full - scale mannequins ( 14/15 ) , standardized patients ( 12/15 ) , and vr workstations ( 11/15 ) were the top 4 common types of simulation modalities available for use , followed by ex vivo animal tissue models ( 8/15 ) , live animal models ( 7/15 ) , and an online simulation module ( 1/15 ) . the most commonly available task trainers ( or bench - top models ) in these institutions were cardiopulmonary resuscitation torso trainers ( 15/15 ) , incision and suturing models ( 14/15 ) , and heart and lung sound - recognition models ( 12/15 ) , followed by genitourinary models ( 11/15 ) , airway management models ( 11/15 ) , vascular access models ( 10/15 ) , lumbar puncture models ( 8/15 ) , and nasogastric tube models ( 8/15 ) . the top 2 available vr simulators in these 9 hospitals were for laparoscopic skills ( 9/11 ) , and gastrointestinal ( gi ) endoscopy skills ( 5/11 ) , followed by urologic endoscopy skills ( 4/11 ) , arthroscopic skills ( 3/11 ) , bronchoscopic skills ( 3/11 ) , and endovascular skills ( 2/11 ) . among the given specialties for srt , the availability of simulation courses was 100% ( 2/2 ) for pediatric internal medicine , 100% ( 1/1 ) for pediatric surgery , 90% ( 10/11 ) for surgery , 78.57% ( 11/14 ) for internal medicine , 77.78% ( 7/9 ) for anesthesiology , 75% ( 6/8 ) for emergency medicine , and 33.33% ( 3/9 ) for ob / gyn ( figure 1 ) . some institutes had simulation curricula , even though they were not qualified for srt in the given disciplines . qualified hospitals for str and availabilities of simulation curriculum in the given specialties . of the 13 institutions with available simulation curricula ,
12 had simulations focused on proficiency - based skill training , 11 had medical knowledge learning , and 10 had skill competency assessment . all of the 13 hospitals with available simulation courses targeted residents or postgraduate residents as their trainees . most of them targeted medical students or interns ( 12/13 ) , fellows ( 9/13 ) , practicing surgeons or physicians ( 8/13 ) , and nurses ( 7/13 ) . allied health professionals ( 1/13 ) and high school students ( 1/13 ) were the target learning audience in 1 hospital . simulation - based curricula or courses were not compulsory in the srt program in beijing ; hence , the hospitals do not need an accredited simulation department to be accredited for the srt program . the simulation facilities in the majority of them ( 14/15 ) were centralized in a specific department within their institutions . concerning human resources for simulation programs ( figure 2 )
, most of the 14 hospitals had full - time ( 10/15 ) and part - time ( 11/15 ) clinical directors , full - time ( 8/15 ) and part - time ( 9/15 ) administrators , and part - time instructors ( 13/15 ) . the full - time instructors ( 3/15 ) , part - time researchers ( 2/15 ) , and full - time ( 2/15 ) and part - time ( 4/15 )
however , there was no full - time researcher for simulation in any of the hospitals surveyed . the top 2 barriers were the shortage of sustainable financial resources ( 12/15 ) and advocacy from their institutional authorities ( 7/15 ) . simulation among the high - ranking srt hospitals in beijing is clearly important , underresourced , and in demand . the specialties included in this study are the ones where simulation - based training had been broadly used internationally . the most common simulation equipment in the responding hospitals were task trainers , basic or advanced full - scale mannequins , standardized patients , and vr simulators . the quantities of simulators , curricula , and trainees were not asked for in the survey , as most of the hospitals in beijing did not have an independent simulation department . the availability of simulators may not reflect the exact frequency of use or activities of an institution , but these types of simulators are universally applicable in primary specialties , and they could meet the common needs of novices in a clinical rotation if embedded within a well - designed simulation curriculum . most of the respondents ( 11/15 ) agreed or strongly agreed that curriculum is one of the influence factors in simulation education and training , and 40% ( 6/15 ) of them indicated that the shortage of curriculum was one of the barriers in their simulation program . to fully take advantage the benefit of simulation
, proficiency - based training should not be a single method of education , but instead , must be integrated into a comprehensive curriculum . according to this survey , in beijing
, the focus for application of simulation is primarily for competency - based technical skills training , medical knowledge learning , and competency assessment . healthcare is a team sport , and to achieve a good patient outcome , technical ability must be complemented by excellent , nontechnical skills across the entire team taking care of the patient . the main targeted trainees in these hospitals were residents ( or postgraduate residents ) and medical students ( or interns ) . second , one of the most significant incentives of simulation in medical education is that simulation can improve patients ' outcomes by minimizing preventable mistakes , and research or academic activities are therefore also necessary for advocating the superiority of simulation - based medical education . according to this study ,
one of the most frequent barriers they encountered in their development was a lack of sustainable funding sources ( 12/15 ) , and most of the respondents ( 14/15 ) indicated that the funding was one of the factors that may influence the growth of simulation programs . however , taking into consideration that one of the benefits of simulation is to reduce the cost of the complications and malpractice suits of preventable mistakes , the savings could pay for an entire program . they could also launch multicenter validation or research to demonstrate the benefits of simulation for introduction of new technology , entry into training programs , training , and even credentialing . the need for curricula , research program , and cooperation with other centers were also demonstrated in this study ( figure 4 ) . as indicated in this survey , 46.67% participants felt there to be insufficient support from their institutions : 86.67% thought such advocacy from their institutions could influence their simulation programs . however , as most of the instructors are part - time in their simulation departments , the deficiency of instructors with protected time was cited as a major obstacle ; 93.33% of them believed that dedicated teachers could positively influence their simulation programs . in china
, the clinical directors and instructors drive the research , so the part - time status may be a contributor to the lack of emphasis on research . as validating evidence accumulates
, it is evident that there is a need for careful development of training facilities , training the
trainers and administrators , financial funding , and curricular design ( including theory and practical teaching ) , and research to continue to build a long - term , sustainable , effective program . because the questionnaires were distributed through e - mail or instant message and completed online , the expectation of the time spend on completing the survey , and the complication of the items was confining to some extent . therefore , it was not feasible to investigate quantitative data on the online survey , such as the number of trainees and simulation equipment in each category , the quantitative degree of integration of simulation into their medical education , the priority of teaching content areas in simulation activities , the contact hours ( the hours spent on training ) for different types trainees , and the frequency of use of simulation facilities . the information provided by the respondents
the most common simulation equipment in the responding hospitals were task trainers , basic or advanced full - scale mannequins , standardized patients , and vr simulators . the quantities of simulators , curricula , and trainees were not asked for in the survey , as most of the hospitals in beijing did not have an independent simulation department . the availability of simulators may not reflect the exact frequency of use or activities of an institution , but these types of simulators are universally applicable in primary specialties , and they could meet the common needs of novices in a clinical rotation if embedded within a well - designed simulation curriculum . most of the respondents ( 11/15 ) agreed or strongly agreed that curriculum is one of the influence factors in simulation education and training , and 40% ( 6/15 ) of them indicated that the shortage of curriculum was one of the barriers in their simulation program . to fully take advantage the benefit of simulation
, proficiency - based training should not be a single method of education , but instead , must be integrated into a comprehensive curriculum . according to this survey , in beijing
, the focus for application of simulation is primarily for competency - based technical skills training , medical knowledge learning , and competency assessment . healthcare is a team sport , and to achieve a good patient outcome , technical ability must be complemented by excellent , nontechnical skills across the entire team taking care of the patient . the main targeted trainees in these hospitals were residents ( or postgraduate residents ) and medical students ( or interns ) . second , one of the most significant incentives of simulation in medical education is that simulation can improve patients ' outcomes by minimizing preventable mistakes , and research or academic activities are therefore also necessary for advocating the superiority of simulation - based medical education . according to this study ,
one of the most frequent barriers they encountered in their development was a lack of sustainable funding sources ( 12/15 ) , and most of the respondents ( 14/15 ) indicated that the funding was one of the factors that may influence the growth of simulation programs . however , taking into consideration that one of the benefits of simulation is to reduce the cost of the complications and malpractice suits of preventable mistakes , the savings could pay for an entire program . basic questions around simulation - based education can not be answered without strong collaboration among simulation centers for increased funding support , faculty training , generating guidelines , and changing policies . the need for curricula , research program , and cooperation with other centers were also demonstrated in this study ( figure 4 ) . as indicated in this survey , 46.67% participants felt there to be insufficient support from their institutions : 86.67% thought such advocacy from their institutions could influence their simulation programs . however , as most of the instructors are part - time in their simulation departments , the deficiency of instructors with protected time was cited as a major obstacle ; 93.33% of them believed that dedicated teachers could positively influence their simulation programs . in china ,
the clinical directors and instructors drive the research , so the part - time status may be a contributor to the lack of emphasis on research . as validating evidence accumulates
it is evident that there is a need for careful development of training facilities , training the
trainers and administrators , financial funding , and curricular design ( including theory and practical teaching ) , and research to continue to build a long - term , sustainable , effective program . because the questionnaires were distributed through e - mail or instant message and completed online , the expectation of the time spend on completing the survey , and the complication of the items was confining to some extent . therefore , it was not feasible to investigate quantitative data on the online survey , such as the number of trainees and simulation equipment in each category , the quantitative degree of integration of simulation into their medical education , the priority of teaching content areas in simulation activities , the contact hours ( the hours spent on training ) for different types trainees , and the frequency of use of simulation facilities . the information provided by the respondents
the results of this survey indicated that most of the participating hospitals had simulation facilities . the primary targeted audiences were residents ( or postgraduate residents ) and medical students ( or interns ) . internal medicine , surgery , and anesthesiology were the 3 most common specialties where simulation training was used . the simulation education and training in most if them had a shortage of research , sustainable financial resources , and endorsement from their authorities . | [
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the microelectronics industries have refined the fabrication methods to make ever smaller devices , but these methods will soon reach their fundamental limits . a promising alternative way for the fabrication of nanometer functional systems is to grow self - organized atoms and molecules on well - defined surface templates with periodic structures .
this idea is of great interest not only for its promising applications in technology but also for a fundamental study [ 1 - 7 ] .
when the mechanisms controlling the self - organized phenomena are fully disclosed , the self - organized growth processes can be steered to create a wide range of surface nanostructures from metallic , semiconducting and molecular materials .
theoretically , the energetic driving forces for self - organization have been explained by various mechanisms , such as overlapping electric , magnetic , and bulk elastic strain fields .
however , many of these systems are too complex to predict a new self - organized structure with sufficient accuracy .
besides , as far as we know , there are only a few first - principles studies on the self - organization template because the experimentally observed super cells are too large to perform such a time - consuming calculation , such as cu bilayer on pt(111 ) and ag bilayer on pt(111 ) with ( 25 25 ) super cells .
recently , the ordered arrays of clusters on metallic substrate are reported experimentally . as the substrate of the ordered clusters , the self - organized template plays a vital role in the growth process of the ordered clusters arrays .
indeed , the detail properties of these self - organization phenomena need thoroughly theoretical research , such as the structure property of the first adsorbing layer under the second layer in metal - bilayer / metal(111 ) system , because they can not be observed directly in experiment . our previous works focused on the behavior of identical metallic clusters arrays on the clean metallic or clean semiconductor substrate .
now , we turn to the self - organized metallic substrate with metal - bilayer / metal(111 ) structure , which can be used as a template . in this paper , we apply two methods to perform energy minimization respectively on a series of complex pb / cu(111 ) interfaces with [ ( n 1 ) ( n 1)]/(n n ) ( n = 2 , 3 , , 12 ) super cells to search a stable template and study its atomic and electronic structures .
the two methods are molecular dynamics ( md ) method based on chen mbius inversion interfacial potential [ 14 - 18 ] and self - consistent first - principles method . some experiments described a method to create almost monodisperse , equally spaced nanostructures through the self - organization of a fcc metal film on fcc metal ( 111 ) substrates with a periodic strain - relief template .
the nanostructures are stable , which are partly due to that the fcc metal ( 111 ) surface is a very stable surface with a very low surface energy .
recently , there are increasing experiments studying the system of pb atoms deposited on cu(111 ) surface using the low - energy electron microscope ( leem ) and the scanning tunneling microscopy ( stm ) .
but as far as we know , very little theoretical study has been performed on the structure of pb bilayer on cu(111 ) surface .
the lattice constant misfit ratio ( 37.1% ) between pb and cu is very large , so the achievable periodicity of the pb / cu(111 ) template can be very short , if the system is stable .
thus , it will be easy to perform further theoretical study on the stable template with a very high density periodic structure , which may reveal important information for potential applications , e.g. in the high - density memory , catalysis or developing nanostructured device technology . in this paper
, we identify a stable template of pb bilayer on cu(111 ) surface with a periodic nanometer structure and reveal the atomic and electronic structure properties of the template .
in order to study the stability of the pb / cu(111 ) interface structures with misfit , the proper interfacial potentials are developed with chen mbius lattice inversion method [ 14 - 18 ] , and then the potentials are applied to relax the interface structures with energy minimization method in cerius2 software package .
the interface structures are modeled in super cells of [ ( n 1 ) ( n 1)]/(n n ) pb / cu(111 ) ( n = 2 , 3 , , 12 ) to take the misfit into account . the ( n 1 ) ( n 1 ) indicates the lateral super cell size of the pb bilayer , and the ( n n ) is for the super cell of cu(111 ) substrate .
the periodic length of the stable pb / cu(111 ) system is expected to be around the lease common multiple of the lattice constants of pb and cu . the [ ( n 1 ) ( n 1)]/(n n ) super cells with the increasing value of n will be easy to meet the lease common multiple condition .
the cu(111 ) surface is modeled by repeated slabs with five cu layers separated by a vacuum region equivalent to twelve cu layers . each metal layer in the super cell
contains n ncu atoms that form a ( n n ) surface cell .
such a super cell can well simulate the system of pb bilayer on the cu(111 ) surface , including a good description of the interaction between the two sides of the interface .
all the cu atoms are initially located at their bulk positions with the equilibrium lattice constant 3.61 . upon pb bilayer adsorptions , all the atoms in the unit cell except for the central cu layer
the pb atoms in the pb bilayer are also initially located at their bulk positions with the equilibrium lattice constant 4.95 .
then , for the stable structures that are found with energy minimization method based on the interfacial potentials , the first - principles calculations are carried out on these stable structures , which is based on a density functional theory implemented in a projector augmented wave ( paw ) representation [ 21 - 23 ] .
correlation effect is treated with the generalized gradient approximation ( gga ) .the plane wave kinetic energy cutoff employed is 25.73 ry , and the monkhorst
the total energy convergences with respect to the energy cutoff and the number of k points have been tested .
optimizations of the atomic structures are done by the conjugate - gradient technique , using the calculated hellmann
all the atomic geometries are fully relaxed , except the fixed center cu layer , till the forces on all relaxed atoms are less than 0.01 ev / .
the interface system is totally different from bulk system , and then the potentials for bulk system are not applicable for the interfacial system . it is necessary to develop proper potentials for the interfacial system .
the potentials cu cu and pb pb are extracted using the method in .
lemberg function : the values of the parameters for the interaction potentials cu pb , cu cu and pb pb are shown in table 1 .
using these potentials , the lattice constants of bulk cu and pb are calculated , respectively , to be 3.61 and 4.95 , which consist well with the values of 3.61 in ref . and 4.949 in ref . .
these results indicate that the interaction potentials cu cu and pb pb are accurate to describe the interatomic interactions of cu and pb , respectively .
figure 1 shows the average energies per atom for the [ ( n 1 ) ( n 1)]/(n
n ) pb / cu(111 ) ( n = 2 , 3 , , 12 ) structures , which are calculated using the energy minimization calculation based on the interfacial potential .
the average energy means that the total energy of each relaxed structure is divided by the total number of atoms in the same structure .
when n varies from 5 to 12 , the pb bilayer does not remain in the bilayer structure but reorganize into three or even more layers and become disordered due to the increasing misfit strain .
therefore , these n 5 structures can not be used as stable templates to support ordered arrays of atoms or clusters .
however , when n < 5 , the relaxed structures retain the well - ordered two pb layers on the cu(111 ) substrate .
these facts mean that n = 5 ( i.e. 4 4/5 5 ) is a critical case .
when n = 4 ( i.e. 3 3/4 4 ) , each layer of the pb bilayer completely covers the cu surface at 0.56 ml ( here , 1 ml is defined as one pb atom per surface cu atom ) with the lattice constant close to 4/3 that of the cu .
the average energy of the n = 3 is slightly lower than that of n = 4 , so the ( 2 2)/(3 3 ) pb bilayer / cu ( 111 ) is more stable than the structure of n = 4 , while both structures of n = 4 and n = 3 are stable .
when n = 2 , the pb surface cell will be 1 1 , it is not suitable to be used as a template for selective adsorptions of atoms or clusters .
pb for the pb bilayer / cu(111 ) structure the average energies for the [ ( n 1 ) ( n 1)]/(n n ) pb bilayer / cu(111 ) ( n = 2,3 , , 12 ) structures .
the dark(big ) and red(small ) balls are for pb and cu atoms , respectively now , we carry out a comprehensive study on the atomic and electronic properties of the stable ( 2 2)/(3 3 ) and ( 3 3)/(4 4 ) pb / cu(111 ) structures with first - principles calculations . for the ( 2 2)/(3 3 ) structure , fig .
2a shows the top view of the fully relaxed structure . in each super cell ( marked with the green bigger parallelogram ) , the four pb atoms of the second pb layer form a relatively close packed semi - cluster structure . here
, we define the semi - cluster as a kind of structure exhibiting the cluster character only in 2-d plane ( in the ( 111 ) surface plane for this paper ) .
one is the area within semi - cluster ; the other is the area between neighboring semi - clusters .
the two areas have different character , which will lead to selective adsorption for atoms or clusters . for the second pb layer ,
the nearest distance of the pb atoms is 3.60 ( but 3.75 for the first pb layer ) that is shorter than that of 4.06 ( 3.90 ) between neighboring semi - clusters by 0.46 ( 0.15 ) .
the semi - cluster is induced by misfit strains between the pb and cu atoms at the interface .
this fact indicates that within the semi - cluster , the pb atoms are closer and have stronger interaction than that between the neighboring semi - clusters .
these atoms in ( 2 2)/(3 3 ) pb / cu(111 ) structure are shifted into one cu(111 ) ( 1 1 ) surface cell , but retain their relative positions .
the a , b and c points denote the cu atoms in the central , upper and top cu layers of the slab respectively , which are the standard fcc sites .
one of the blue triangles that correspond to the first pb layer locates at the upper corner that is equivalent to a site that belongs to fcc site relative to the top cu layer . the other one blue triangle is near the
another two blue triangles are near the borderline to form the transition area that can release the misfit strains .
these facts indicate : ( 1 ) there is a strong interaction between the first pb layer and the cu substrate , which shortens the distance between the neighboring pb semi - clusters in the first pb layer ; ( 2 ) the interaction between the cu substrate and the first pb layer is different from the interaction between the first and second pb layers .
finally , these different interactions arrange the atoms in first pb layer in a balance state . atop view of the relaxed ( 2 2)/(3 3 ) pb / cu(111 ) structure , and
b the relative positions of pb and cu atoms in cu(111 ) 1 1 surface cell for the same structure as ( a ) . the black dot , blue triangle and red square are for the atoms of cu substrate , first pb layer and second pb layer , respectively . the distances ( ) between some pb atoms
are also shown figure 3a shows the top view of the fully relaxed structure of the ( 3 3)/(4 4 ) pb / cu(111 ) structure .
contrast to the ( 2 2)/(3 3 ) pb / cu(111 ) structure , the fcc area and hcp area for the first pb layer in ( 3 3)/(4 4 ) structure can be clearly seen in fig . 3a . in this case , the fcc and hcp sites mean that the pb atom of the first layer locates near the hollow site of the regular and inverted triangles made by three cu surface atoms , respectively .
there is a very clear borderline ( green dashed ) between the fcc and hcp areas .
the three pb atoms on the straight borderline locate near the bridge sites of the cu(111 ) surface , and these three pb atoms form the transition area between the fcc and hcp areas to release the misfit strains at the interface . compared with the ( 2 2)/(3 3 ) pb / cu(111 ) structures , the separation of the pb atoms in the first layer are almost equal ( around 3.41 0.04 ) in the ( 3 3)/(4 4 ) pb / cu(111 ) structure of fig .
the configuration of these pb atoms is similar to those in the first pb layer . because the differences in the inter - atomic distances for the pb atoms in second pb layer are small ( less than 0.1 ) , the surface of the pb bilayer can be regarded as the clean pb(111 ) surface in some degree for the adsorption atoms . this fact is due to that the pb atoms fully cover the cu(111 ) surface and then they are closely packed . in other words , for the new adsorption atoms , pb atoms in the second layer are almost identical . as a result ,
each hollow site in the pb second layer will have equal chance to hold the deposited atoms , and the same thing would happen to each top and bridge site .
hence , the ( 3 3)/(4 4 ) pb / cu(111 ) structure is not a suitable candidate to be used as a template . atop view of the relaxed ( 3 3)/(4 4 ) pb / cu(111 ) structure , and
b the relative positions of pb and cu atoms in cu(111 ) 1 1 surface cell for the same structure as ( a ) . the black dot , blue triangle and red square are for the atoms of cu substrate , first pb layer and second pb layer , respectively .
the distances ( ) between some pb atoms are also shown figure 4 shows the differences of electron charge densities for the ( 2 2)/(3 3 ) pb / cu(111 ) structure , in order to see the nature of interaction between the pb bilayer and the cu(111 ) substrate , and the interaction between pb atoms in pb bilayer .
the difference of electron charge density helps to visualize the characteristics of bonding and is defined as the differences between the pb / cu(111 ) structure and the superposition of atomic electron charge densities , i.e.,in fig .
4a , there are obvious covalent bondings between pb atoms in first pb layer and the cu atoms at cu(111 ) surface , which indicate the strong interactions between them . in the second pb layer ,
such a picture reveals that the direct interactions between these pb atoms are alternatively strong and weak .
figure 4b shows a very clear picture of the difference of the electron charge density in the second pb layer .
4b ) forms the area with a high charge density and is surrounded by the zero charge density area .
the semi - clusters are separated by the dipole repulsion caused by elastic interactions due to the strain - relief of the lattice misfit in the interface .
the atoms that will be deposited on the surface of ( 2 2)/(3 3 ) pb / cu(111 ) structure will have different interactions with the two different areas .
such mechanisms will help to realize the selective adsorption of atoms on the surface of pb bilayer / cu(111 ) .
so the periodic ( 2 2)/(3 3 ) pb / cu(111 ) structure will be a promising candidate for new self - organized template . furthermore , the periodic length of this ( 2 2)/(3 3 ) pb bilayer / cu(111 ) structure is 7.67 , which is very short for self - organized template .
therefore , this structure may be a unique template for growing ordered quantum dots with a very high density .
afor the ( 2 2)/(3 3 ) pb / cu(111 ) structure , and the plotted plane is vertical to the cu(111 ) surface and along the longer diagonal of the parallelogram pattern ( in fig .
2a ) ; b the same as a , but the plotted plane is parallel to the cu(111 ) surface and crossing the pb atoms in the second layer .
solid lines ( red area indicates highest value ) and dashed lines(blue area indicates lowest value ) correspond to > 0 and < 0 , respectively .
the white area corresponds to = 0 as for the ( 3 3)/(4 4 ) pb / cu(111 ) structure , the differences of electron charge densities are shown in fig .
5 . the alternatively high and zero electronic charge density areas , which are found in ( 2 2)/(3 3 ) pb / cu(111 ) structures , do not appear in ( 3 3)/(4 4 ) structure .
5a , 5b ) are hardly affected by the cu substrate , and very similar to clean pb ( 111 ) surface with ( 1 1 ) periodical cell .
the electron charge density areas between neighboring pb atoms along the pb layer are almost same , because the pb atoms are close packed along the ( 111 ) surface due to that the pb over layer completely covers the cu(111 ) surface in the ( 3 3)/(4 4 ) structure . the ( 3 3)/(4 4 ) pb / cu(111 ) , which shows almost ( 1 1 ) surface electronic structure , is not suitable to be used as a template .
afor the ( 3 3)/(4 4 ) pb / cu(111 ) structure , and the plotted plane is vertical to the cu(111 ) surface and along the longer diagonal of the parallelogram pattern ( in fig .
3a ) ; b the same as a , but the plotted plane is parallel to the cu(111 ) surface and crossing the pb atoms in the second layer .
solid lines ( red area indicates highest value ) and dashed lines(blue area indicates lowest value ) correspond to > 0 and < 0 , respectively .
the white area corresponds to = 0 as a test , we perform the first - principles calculations for the ge atom adsorption on the ( 2 2)/(3 3 ) pb / cu(111 ) template .
the structures with ge atoms adsorbing on the top and bridge sites of pb bilayer are unstable .
there are eight possible hollow sites for adsorption within one surface supercell of this stable template ( fig .
6b , the total energies of eight adsorption systems show that the sites s3 ( representing the site 3 in fig .
this fact implies that experimentally the ge atoms will prefer to adsorb on the s3 , s4 and s6 sites .
figure 6c shows that the binding energies of these three adsorption sites on ( 2 2)/(3 3 ) pb / cu(111 ) are higher than the other sites .
the lower total energies and higher binding energies of the s3 , s4 and s6 adsorption sites will realize the selective adsorption of ge atoms on ( 2 2)/(3 3 ) pb / cu(111 ) .
it confirms that the ( 2 2)/(3 3 ) pb / cu(111 ) is a promising candidate to be used as a periodic template to support ordered arrays of atoms . during an actual deposition process ,
if one ge atom lands on the s1 , s2 , s5 , s7 or s8 , under a moderate temperature , the ge atom could get enough energy to overcome a diffuse barrier and fall into the neighboring sites of lower total energy , for instance transferring from s5 to s4 or s6 . furthermore , it is necessary that the deposited ge atoms should cover the surface of ( 2 2)/(3 3 ) pb / cu(111 ) in a range from 0.11 to 0.33 ml .
the right coverage of ge atoms will contribute to the realization of an ordered arrangement of ge atoms on the ( 2 2)/(3 3 ) pb / cu(111 ) surface .
therefore , under an appropriate growth temperature and with careful control of the deposition rate , the adsorption of ordered ge atoms on the surface of ( 2 2)/(3 3 ) pb / cu(111 ) should be possible .
a the eight important sites of ge atom adsorption on ( 2 2)/(3 3 ) pb / cu(111 ) surface , and the green parallelogram shows one super cell ; b the total energies and c the binding energies of the eight important adsorption systems of ge atom on ( 2 2)/(3 3 ) pb / cu(111 ) surface
in summary , we find a stable periodic strain - relief template by using energy minimizations with the md calculations and the self - consistent first - principles calculations .
the ( 2 2)/(3 3 ) and ( 3 3)/(4 4 ) pb bilayer / cu(111 ) are stable structures among the [ ( n 1 ) ( n 1)]/(n n ) pb bilayer / cu(111 ) ( n = 2 , 3 , , 12 ) structures , which are calculated with md simulations based on chen mbius inversion potential . however , first - principles calculations show that the ( 3 3)/(4 4 ) pb bilayer / cu(111 ) is fully covered by pb atoms with almost ( 1 1 ) atomic and electronic structures on the surface of pb bilayer , and thus it is not suitable to be used as a periodic template . for the ( 2 2)/(3 3 ) pb bilayer / cu(111 ) stable structure , the ordered pb
semi - clusters are found to show a high - density array on the first pb atoms layer , and the high and zero electron charge density areas periodically locate in the second pb layer .
these different areas with different atomic and electronic structure will lead to the selective adsorption of atoms on the surface of ( 2 2)/(3 3 ) pb bilayer / cu(111 ) structure .
the further calculations on the eight adsorptions sites of ge atoms on ( 2 2)/(3 3 ) pb bilayer / cu(111 ) structure also confirm this conclusion .
this stable periodic template can be realized under certain experimental conditions . as a result ,
the strain - relief periodic ( 2 2)/(3 3 ) pb bilayer / cu(111 ) structure is a promising candidate for the new self - organized template to assemble ordered quantum dots on it with a very high density , and may be one of the new platforms for studying next - generation microelectronics .
our method may be useful for the search of other stable templates for quantum structure arrays , that is , superlattice of nanostructures with size and period much smaller than the wavelength of an electron .
the work at tsinghua university was supported by the nature science foundation of china ( nsfc , no .
50531050 ) , the 973 project ( no.2006cb605100 ) , and china postdoctoral science foundation funded project ( no . 20090450426 ) .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . | using the energy minimization calculations based on an interfacial potential and a first - principles total energy method , respectively , we show that ( 2 2)/(3 3 ) pb / cu(111 ) system is a stable structure among all the [ ( n 1 ) ( n 1)]/(n n ) pb / cu(111 ) ( n = 2 , 3 , , 12 ) structures .
the electronic structure calculations indicate that self - organized ordered pb semi - clusters arrays are formed on the first pb monolayer of ( 2 2)/(3 3 ) pb / cu(111 ) , which is due to a strain - release effect induced by the inherent misfits .
the pb semi - clusters structure can generate selective adsorption of atoms of semiconductor materials ( e.g. , ge ) around the semi - clusters , therefore , can be used as a template for the growth of nanoscale structures with a very short periodic length ( 7.67 ) . | Introduction
Calculation Method
Results and Discussions
Conclusions
Acknowledgments
Open Access | a promising alternative way for the fabrication of nanometer functional systems is to grow self - organized atoms and molecules on well - defined surface templates with periodic structures . when the mechanisms controlling the self - organized phenomena are fully disclosed , the self - organized growth processes can be steered to create a wide range of surface nanostructures from metallic , semiconducting and molecular materials . besides , as far as we know , there are only a few first - principles studies on the self - organization template because the experimentally observed super cells are too large to perform such a time - consuming calculation , such as cu bilayer on pt(111 ) and ag bilayer on pt(111 ) with ( 25 25 ) super cells . as the substrate of the ordered clusters , the self - organized template plays a vital role in the growth process of the ordered clusters arrays . indeed , the detail properties of these self - organization phenomena need thoroughly theoretical research , such as the structure property of the first adsorbing layer under the second layer in metal - bilayer / metal(111 ) system , because they can not be observed directly in experiment . now , we turn to the self - organized metallic substrate with metal - bilayer / metal(111 ) structure , which can be used as a template . in this paper , we apply two methods to perform energy minimization respectively on a series of complex pb / cu(111 ) interfaces with [ ( n 1 ) ( n 1)]/(n n ) ( n = 2 , 3 , , 12 ) super cells to search a stable template and study its atomic and electronic structures . the two methods are molecular dynamics ( md ) method based on chen mbius inversion interfacial potential [ 14 - 18 ] and self - consistent first - principles method . some experiments described a method to create almost monodisperse , equally spaced nanostructures through the self - organization of a fcc metal film on fcc metal ( 111 ) substrates with a periodic strain - relief template . the nanostructures are stable , which are partly due to that the fcc metal ( 111 ) surface is a very stable surface with a very low surface energy . recently , there are increasing experiments studying the system of pb atoms deposited on cu(111 ) surface using the low - energy electron microscope ( leem ) and the scanning tunneling microscopy ( stm ) . but as far as we know , very little theoretical study has been performed on the structure of pb bilayer on cu(111 ) surface . the lattice constant misfit ratio ( 37.1% ) between pb and cu is very large , so the achievable periodicity of the pb / cu(111 ) template can be very short , if the system is stable . thus , it will be easy to perform further theoretical study on the stable template with a very high density periodic structure , which may reveal important information for potential applications , e.g. in this paper
, we identify a stable template of pb bilayer on cu(111 ) surface with a periodic nanometer structure and reveal the atomic and electronic structure properties of the template . in order to study the stability of the pb / cu(111 ) interface structures with misfit , the proper interfacial potentials are developed with chen mbius lattice inversion method [ 14 - 18 ] , and then the potentials are applied to relax the interface structures with energy minimization method in cerius2 software package . the interface structures are modeled in super cells of [ ( n 1 ) ( n 1)]/(n n ) pb / cu(111 ) ( n = 2 , 3 , , 12 ) to take the misfit into account . the ( n 1 ) ( n 1 ) indicates the lateral super cell size of the pb bilayer , and the ( n n ) is for the super cell of cu(111 ) substrate . the periodic length of the stable pb / cu(111 ) system is expected to be around the lease common multiple of the lattice constants of pb and cu . the [ ( n 1 ) ( n 1)]/(n n ) super cells with the increasing value of n will be easy to meet the lease common multiple condition . such a super cell can well simulate the system of pb bilayer on the cu(111 ) surface , including a good description of the interaction between the two sides of the interface . upon pb bilayer adsorptions , all the atoms in the unit cell except for the central cu layer
the pb atoms in the pb bilayer are also initially located at their bulk positions with the equilibrium lattice constant 4.95 . then , for the stable structures that are found with energy minimization method based on the interfacial potentials , the first - principles calculations are carried out on these stable structures , which is based on a density functional theory implemented in a projector augmented wave ( paw ) representation [ 21 - 23 ] . correlation effect is treated with the generalized gradient approximation ( gga ) .the plane wave kinetic energy cutoff employed is 25.73 ry , and the monkhorst
the total energy convergences with respect to the energy cutoff and the number of k points have been tested . optimizations of the atomic structures are done by the conjugate - gradient technique , using the calculated hellmann
all the atomic geometries are fully relaxed , except the fixed center cu layer , till the forces on all relaxed atoms are less than 0.01 ev / . the interface system is totally different from bulk system , and then the potentials for bulk system are not applicable for the interfacial system . using these potentials , the lattice constants of bulk cu and pb are calculated , respectively , to be 3.61 and 4.95 , which consist well with the values of 3.61 in ref . these results indicate that the interaction potentials cu cu and pb pb are accurate to describe the interatomic interactions of cu and pb , respectively . figure 1 shows the average energies per atom for the [ ( n 1 ) ( n 1)]/(n
n ) pb / cu(111 ) ( n = 2 , 3 , , 12 ) structures , which are calculated using the energy minimization calculation based on the interfacial potential . the average energy means that the total energy of each relaxed structure is divided by the total number of atoms in the same structure . therefore , these n 5 structures can not be used as stable templates to support ordered arrays of atoms or clusters . the average energy of the n = 3 is slightly lower than that of n = 4 , so the ( 2 2)/(3 3 ) pb bilayer / cu ( 111 ) is more stable than the structure of n = 4 , while both structures of n = 4 and n = 3 are stable . when n = 2 , the pb surface cell will be 1 1 , it is not suitable to be used as a template for selective adsorptions of atoms or clusters . pb for the pb bilayer / cu(111 ) structure the average energies for the [ ( n 1 ) ( n 1)]/(n n ) pb bilayer / cu(111 ) ( n = 2,3 , , 12 ) structures . the dark(big ) and red(small ) balls are for pb and cu atoms , respectively now , we carry out a comprehensive study on the atomic and electronic properties of the stable ( 2 2)/(3 3 ) and ( 3 3)/(4 4 ) pb / cu(111 ) structures with first - principles calculations . for the ( 2 2)/(3 3 ) structure , fig . in each super cell ( marked with the green bigger parallelogram ) , the four pb atoms of the second pb layer form a relatively close packed semi - cluster structure . here
, we define the semi - cluster as a kind of structure exhibiting the cluster character only in 2-d plane ( in the ( 111 ) surface plane for this paper ) . for the second pb layer ,
the nearest distance of the pb atoms is 3.60 ( but 3.75 for the first pb layer ) that is shorter than that of 4.06 ( 3.90 ) between neighboring semi - clusters by 0.46 ( 0.15 ) . the semi - cluster is induced by misfit strains between the pb and cu atoms at the interface . this fact indicates that within the semi - cluster , the pb atoms are closer and have stronger interaction than that between the neighboring semi - clusters . these atoms in ( 2 2)/(3 3 ) pb / cu(111 ) structure are shifted into one cu(111 ) ( 1 1 ) surface cell , but retain their relative positions . the a , b and c points denote the cu atoms in the central , upper and top cu layers of the slab respectively , which are the standard fcc sites . one of the blue triangles that correspond to the first pb layer locates at the upper corner that is equivalent to a site that belongs to fcc site relative to the top cu layer . these facts indicate : ( 1 ) there is a strong interaction between the first pb layer and the cu substrate , which shortens the distance between the neighboring pb semi - clusters in the first pb layer ; ( 2 ) the interaction between the cu substrate and the first pb layer is different from the interaction between the first and second pb layers . atop view of the relaxed ( 2 2)/(3 3 ) pb / cu(111 ) structure , and
b the relative positions of pb and cu atoms in cu(111 ) 1 1 surface cell for the same structure as ( a ) . the black dot , blue triangle and red square are for the atoms of cu substrate , first pb layer and second pb layer , respectively . the distances ( ) between some pb atoms
are also shown figure 3a shows the top view of the fully relaxed structure of the ( 3 3)/(4 4 ) pb / cu(111 ) structure . contrast to the ( 2 2)/(3 3 ) pb / cu(111 ) structure , the fcc area and hcp area for the first pb layer in ( 3 3)/(4 4 ) structure can be clearly seen in fig . in this case , the fcc and hcp sites mean that the pb atom of the first layer locates near the hollow site of the regular and inverted triangles made by three cu surface atoms , respectively . there is a very clear borderline ( green dashed ) between the fcc and hcp areas . compared with the ( 2 2)/(3 3 ) pb / cu(111 ) structures , the separation of the pb atoms in the first layer are almost equal ( around 3.41 0.04 ) in the ( 3 3)/(4 4 ) pb / cu(111 ) structure of fig . the configuration of these pb atoms is similar to those in the first pb layer . because the differences in the inter - atomic distances for the pb atoms in second pb layer are small ( less than 0.1 ) , the surface of the pb bilayer can be regarded as the clean pb(111 ) surface in some degree for the adsorption atoms . this fact is due to that the pb atoms fully cover the cu(111 ) surface and then they are closely packed . hence , the ( 3 3)/(4 4 ) pb / cu(111 ) structure is not a suitable candidate to be used as a template . atop view of the relaxed ( 3 3)/(4 4 ) pb / cu(111 ) structure , and
b the relative positions of pb and cu atoms in cu(111 ) 1 1 surface cell for the same structure as ( a ) . the black dot , blue triangle and red square are for the atoms of cu substrate , first pb layer and second pb layer , respectively . the distances ( ) between some pb atoms are also shown figure 4 shows the differences of electron charge densities for the ( 2 2)/(3 3 ) pb / cu(111 ) structure , in order to see the nature of interaction between the pb bilayer and the cu(111 ) substrate , and the interaction between pb atoms in pb bilayer . the difference of electron charge density helps to visualize the characteristics of bonding and is defined as the differences between the pb / cu(111 ) structure and the superposition of atomic electron charge densities , i.e.,in fig . 4a , there are obvious covalent bondings between pb atoms in first pb layer and the cu atoms at cu(111 ) surface , which indicate the strong interactions between them . 4b ) forms the area with a high charge density and is surrounded by the zero charge density area . the semi - clusters are separated by the dipole repulsion caused by elastic interactions due to the strain - relief of the lattice misfit in the interface . the atoms that will be deposited on the surface of ( 2 2)/(3 3 ) pb / cu(111 ) structure will have different interactions with the two different areas . such mechanisms will help to realize the selective adsorption of atoms on the surface of pb bilayer / cu(111 ) . so the periodic ( 2 2)/(3 3 ) pb / cu(111 ) structure will be a promising candidate for new self - organized template . furthermore , the periodic length of this ( 2 2)/(3 3 ) pb bilayer / cu(111 ) structure is 7.67 , which is very short for self - organized template . therefore , this structure may be a unique template for growing ordered quantum dots with a very high density . afor the ( 2 2)/(3 3 ) pb / cu(111 ) structure , and the plotted plane is vertical to the cu(111 ) surface and along the longer diagonal of the parallelogram pattern ( in fig . 2a ) ; b the same as a , but the plotted plane is parallel to the cu(111 ) surface and crossing the pb atoms in the second layer . the white area corresponds to = 0 as for the ( 3 3)/(4 4 ) pb / cu(111 ) structure , the differences of electron charge densities are shown in fig . the alternatively high and zero electronic charge density areas , which are found in ( 2 2)/(3 3 ) pb / cu(111 ) structures , do not appear in ( 3 3)/(4 4 ) structure . the electron charge density areas between neighboring pb atoms along the pb layer are almost same , because the pb atoms are close packed along the ( 111 ) surface due to that the pb over layer completely covers the cu(111 ) surface in the ( 3 3)/(4 4 ) structure . the ( 3 3)/(4 4 ) pb / cu(111 ) , which shows almost ( 1 1 ) surface electronic structure , is not suitable to be used as a template . afor the ( 3 3)/(4 4 ) pb / cu(111 ) structure , and the plotted plane is vertical to the cu(111 ) surface and along the longer diagonal of the parallelogram pattern ( in fig . 3a ) ; b the same as a , but the plotted plane is parallel to the cu(111 ) surface and crossing the pb atoms in the second layer . the white area corresponds to = 0 as a test , we perform the first - principles calculations for the ge atom adsorption on the ( 2 2)/(3 3 ) pb / cu(111 ) template . the structures with ge atoms adsorbing on the top and bridge sites of pb bilayer are unstable . figure 6c shows that the binding energies of these three adsorption sites on ( 2 2)/(3 3 ) pb / cu(111 ) are higher than the other sites . the lower total energies and higher binding energies of the s3 , s4 and s6 adsorption sites will realize the selective adsorption of ge atoms on ( 2 2)/(3 3 ) pb / cu(111 ) . it confirms that the ( 2 2)/(3 3 ) pb / cu(111 ) is a promising candidate to be used as a periodic template to support ordered arrays of atoms . furthermore , it is necessary that the deposited ge atoms should cover the surface of ( 2 2)/(3 3 ) pb / cu(111 ) in a range from 0.11 to 0.33 ml . the right coverage of ge atoms will contribute to the realization of an ordered arrangement of ge atoms on the ( 2 2)/(3 3 ) pb / cu(111 ) surface . therefore , under an appropriate growth temperature and with careful control of the deposition rate , the adsorption of ordered ge atoms on the surface of ( 2 2)/(3 3 ) pb / cu(111 ) should be possible . a the eight important sites of ge atom adsorption on ( 2 2)/(3 3 ) pb / cu(111 ) surface , and the green parallelogram shows one super cell ; b the total energies and c the binding energies of the eight important adsorption systems of ge atom on ( 2 2)/(3 3 ) pb / cu(111 ) surface
in summary , we find a stable periodic strain - relief template by using energy minimizations with the md calculations and the self - consistent first - principles calculations . the ( 2 2)/(3 3 ) and ( 3 3)/(4 4 ) pb bilayer / cu(111 ) are stable structures among the [ ( n 1 ) ( n 1)]/(n n ) pb bilayer / cu(111 ) ( n = 2 , 3 , , 12 ) structures , which are calculated with md simulations based on chen mbius inversion potential . however , first - principles calculations show that the ( 3 3)/(4 4 ) pb bilayer / cu(111 ) is fully covered by pb atoms with almost ( 1 1 ) atomic and electronic structures on the surface of pb bilayer , and thus it is not suitable to be used as a periodic template . for the ( 2 2)/(3 3 ) pb bilayer / cu(111 ) stable structure , the ordered pb
semi - clusters are found to show a high - density array on the first pb atoms layer , and the high and zero electron charge density areas periodically locate in the second pb layer . these different areas with different atomic and electronic structure will lead to the selective adsorption of atoms on the surface of ( 2 2)/(3 3 ) pb bilayer / cu(111 ) structure . the further calculations on the eight adsorptions sites of ge atoms on ( 2 2)/(3 3 ) pb bilayer / cu(111 ) structure also confirm this conclusion . as a result ,
the strain - relief periodic ( 2 2)/(3 3 ) pb bilayer / cu(111 ) structure is a promising candidate for the new self - organized template to assemble ordered quantum dots on it with a very high density , and may be one of the new platforms for studying next - generation microelectronics . | [
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enteric diseases constitute a major problem in growing pigs and have a large economic impact on pig production .
however , clinical signs such as diarrhoea and poor growth may be caused by a wide range of microorganisms such as parasites , bacteria , and viruses .
hence , to implement adequate treatment and prophylactic strategies it is important to identify the causative organism and the necessity of a reliable diagnosis is obvious .
the diagnostic methods required should be sensitive and specific and ideally also cheap , fast , robust , readily available , and easy to handle .
all these requirements can rarely be fulfilled and there is an ongoing need for further improvement of already existing methods and the development of new techniques .
the bacterium lawsonia intracellularis is a major cause of enteritis in weaned pigs and is demonstrated in up to 94% of the herds .
necropsy is used to establish a relationship between clinical signs and the presence of representative lesions but to specifically identify the causative agent ; other techniques must be added [ 3 , 4 ] .
further , serological methods are cheap , fast , and easy to handle and are used to scan large numbers of samples . however , a causal relationship will not be established and false reactions may be difficult to interpret [ 57 ] .
identification of specific dna by polymerase chain reaction ( pcr ) and immunohistochemistry ( ihc ) on tissue samples have been suggested as gold standard [ 8 , 9 ] .
antemortem , culture is used as gold standard for many bacteria but is presently not applicable in the routine diagnosis of the obligate intracellular l. intracellularis and presently , pcr or immunperoxidase staining of faecal smears is the only option available [ 6 , 10 ] .
today , most laboratories utilize pcr that is fast , sensitive , and specific in the demonstration of bacterial dna in tissue or faeces . however , when applied on complex biological samples such as faeces , false negative results constitute a problem [ 5 , 6 , 1113 ] .
in addition , the technique does not distinguish between dead or live bacteria and the quality and yield of dna may depend on the target , the sample composition , and the method used for purification of dna [ 1 , 5 ] .
recently , a new method to separate particles and bacteria based on their buoyant density has been described .
the method was successfully applied to separate yersinia enterocolitica and campylobacter species from food particles and inhibitors [ 14 , 15 ] and was stated to enable the separation between live and dead bacteria as well as free bacterial dna .
the purpose of this study was to develop and evaluate a new method in the diagnosis of l. intracellularis , based on the separation of l. intracellularis cells from faecal samples by flotation prior to pcr .
0.1 g of all samples was also lysed by boiling and analysed for l. intracellularis by nested pcr combined with agaros gel electrophoresis and ethidium - bromide staining , as previously described . media of various densities were prepared from two colloidal density gradient media stock solutions ; bactxtractor - low density ( bx - l ) with a density of 1.057 g / ml and bactxtractor - high density ( bx - h ) with a density of 1.309 g / ml ( fertipro n.v .
, industriepark noord 32 , 8730 beernem , belgium ) . to obtain other calculated densities required
, bx - h was diluted with physiologic saline ( 0.86 0.90 % nacl , ph 7.5 ) .
all experiments were performed at room temperature . to estimate the buoyant density of l. intracellularis the methodology described by pertoft was used .
briefly , 0.2 gram of a known pcr - positive faecal sample was mixed with 1.5 ml saline and incubated at room temperature for 10 minutes .
one ml of the supernatant was mixed with 5.675 ml saline and 2.225 ml bx - h in a 15 ml tube . in a second tube , 6.625 ml saline and 2.225 ml bx - h were mixed with 50 l density marker beads ( amersham biosciences ab , uppsala , sweden ) .
the tubes were centrifuged in an ultracentrifuge with fixed angle rotor ( beckman optima l80-xp , beckman coulter inc . ,
fullerton , calif , usa ) at 15 000 g for 30 minutes to create a self - generated continuous density gradient .
following centrifugation , the distance from the bottom of the second tube to the coloured bands created by the separated marker beads was measured and plotted against their densities . the volume per mm
aliquots of the sample were drawn from the bottom , and the volumes were measured and plotted in the density curve .
the procedure was repeated with two additional faecal samples known to be pcr positive for l. intracellularis . in a fourth tube , a sample previously shown to be strongly pcr inhibiting , as judged by the repeated failure to amplify the internal standard , was included to determine the buoyant density of major inhibitory components .
the amount of l. intracellularis - specific dna was estimated semiquantitatively by comparisons to an internal standard ( mimic ) .
pcr analysis for l. intracellularis was performed in accordance with jacobson et al . and 10 internal controls ( mimics )
were included in each pcr reaction . a positive control , originating from a tissue sample diagnosed as proliferative enteropathy at necropsy that was extracted by phenol - chloroform before pcr and containing ~10l .
the forward primer sequence was 5-tat ggc tgt caa aca ctc cg-3 and the reverse primer sequence was 5-tga agg tat tgg tat tct cc-3 .
the reaction conditions consisted of 10 mm tris - hcl ( ph 8.3 ) , 50 mm kcl , 2 mm mgcl2 , 0.2 mm of each dntp , and 1 u amplitaq gold dna polymerase ( applied biosystems , foster city , calif , usa ) .
one l of the template was added and pcr was performed in a dna thermal cycler ( ptc-200 , mj research inc , watertown , mass , usa ) starting with 10 minutes at 95c followed by 35 cycles consisting of 95c for 30 s , 55c for 30 s , and 72c for 30 s. when undiluted density media was included in the template , a prolonged annealing time ( 45 s ) was used .
all analyses were performed in duplicate or triplicate . the discontinuous density gradient setup consisted of a high - density bottom phase , a middle phase of intermediate density , and a low - density top phase . based on the estimated buoyant density of l. intracellularis , the densities were designed to float the bacterium from the bottom phase and concentrate the microbe to the interphase between the top and middle phases .
components of lower densities would be concentrated in the top phase while components of higher densities would be located in the middle and bottom phases .
small and water - soluble components such as free dna and various pcr - inhibitors that were not affected by the applied g - force were expected to be located in the bottom phase . to maximize the separation of l. intracellularis from inhibitory factors , different densities of the middle and top phases
approximately 0.5 g faecal sample was homogenized in 1 ml saline incubated for 10 minutes , and 1 ml supernatant was used as a sample applied in the bottom phase . in five tubes ,
a strongly positive sample was used and in one tube , a strongly inhibited sample was included .
the two upper phases were carefully layered one by one to avoid disturbing the gradients .
the samples were centrifuged in a swinging bucket rotor ( # 3047 , biofuge stratos , heraus instruments gmbh , hanau , germany ) at 5000 rpm ( ~4863 g ) or 30 minutes . from the upper interphase , samples ( 0.51 ml ) were drawn with a sterile needle through the tube wall to avoid mixing the phases . except for tube 1 , the top , middle , and bottom phases
the samples were pelleted by centrifugation at 10000 rpm for 5 minutes and washed twice in saline , and the final pellet was resuspended in 0.20.3 ml saline and lysed at 95c for 20 minutes prior to pcr .
in addition , six tubes were prepared and centrifuged in accordance with tube 4 , table 1 , to further analyse the overall distribution and recovery of l. intracellularis dna in the gradient ( figure 1 ) . to compare the amount of dna recovered by flotation to the amount recovered by boiling [ 5 , 18 ] , tenfold dilutions of the sample prepared by either method were subjected to nested pcr together with 10 mimics ( figure 1(1 ) ) . to determine if whole l. intracellularis - containing enterocytes were present , the top phase from tube 2 was analysed by pcr ( undiluted and diluted 1:100 ) and likewise , the bottom phase was analysed to determine if free dna was present . to confirm that free dna would be allocated to the bottom phase
only , a lysed sample was included in tube 3 , the phases were collected separately , and undiluted samples and samples diluted 1:100 were subjected to pcr . to determine if l. intracellularis dna was lost during the process , the interphase from tube 4 was collected and subjected to a second flotation .
subsequently , corresponding amounts of the interphases from the first and the second centrifugations were serially diluted and subjected to pcr . to determine if faecal particles would influence the recovery , 100 l sample from the bottom and top fractions , respectively ( tube 4 ) , was mixed with 1 ml supernatant from a negative faecal sample prior to centrifugation ( tube 5 and 6 ) .
further , to determine if the colloidal media would interfere with pcr , 1 l of l. intracellularis dna precipitated by phenol / chloroform was subjected to pcr without being diluted , diluted 1:1 with saline , 1:1 with bx - l , or 1:1 with bx - h . to adopt the procedure to standard laboratory equipment , a small - scale setup ( bottom phase : 100 l sample mixed with 300 l bx - h , density 1.20 g / ml ; middle phase : 304.1 l saline plus 95.9 l bx - h , density 1.074 g / ml ; top phase : 315.8 l saline mixed with 84.2 l bx - h , density 1.065 g / ml ) was tested on a panel of selected samples ( table 2 ) .
six samples had previously been strongly positive for l. intracellularis when prepared by boiling and analysed by nested pcr , six samples had a weak positive reaction caused by a small amount of dna in the sample as judged by comparison to the mimic , six had a weak positive reaction caused by partial inhibition of the pcr , and six samples had previously been completely inhibited .
the samples were run in duplicate in a standard centrifuge with a fixed angle rotor ( hermle z 160 m , hermle labortechnik gmbh , wehingen , germany ) at 5 000 g for 30 minutes . following removal of 300 l of the top phase ,
the next 200 l sample was recovered from the upper interphase , lysed and centrifuged at 15 000 rpm for 30 s. before analysis by pcr .
further , 116 clinical samples originating from seven different farms were prepared by the optimized small - scale setup .
the bottom phase consisted of 150 l supernatant from the sample mixed with 450 l bx - h , the middle phase consisted of 450 l saline and 150 l bx - h with a density of 1.0772 g / ml , and the top phase was increased to 600 l bx - l .
following centrifugation at ~5000 g for 30 minutes , the top phase was removed and the underlying 200400 l was transferred to a new tube and lysed , and samples ( undiluted and diluted 1:100 ) were analysed by pcr .
aliquots of the samples had previously also been prepared by a commercial kit ( qiaamp dna stool mini kit , qiagen inc . , valencia , calif , usa ) and by boiling and these results were included for comparison ( table 3 ) .
following centrifugation , the self - generated continuous density gradient in the first tube yielded 25 fractions with volumes ranging from 155 to 510 l .
following pcr , all fractions in the density range of 1.138 to 1.051 g / ml were positive for l. intracellularis .
fractions with densities higher than 1.074 g / ml and lower than 1.065 g / ml contained less bacterial dna ( < 10target templates / ml ) as determined semiquantitatively . in the repeated run on two additional positive samples , all fractions with densities
> 1.102 g / ml and < 1.064 g / ml were negative and all fractions between these values were positive , each fraction containing ~1010 dna target templates . in the inhibited sample , weak inhibition was seen in four out of six fractions with densities between 1.102 and 1.074 g / ml , one fraction in this interval was completely inhibited , and one fraction was not inhibited .
fractions with densities around and slightly below 1.074 g / ml were not inhibited , but weak or complete inhibition was demonstrated in fractions with densities approaching 1.064 g / ml . following centrifugation ,
in the first setup , positive pcr reactions were demonstrated in all phases in tubes 14 ( table 1 ) . as determined semiquantitatively ,
the bottom phases from tubes 2 and 3 contained less amount of dna ( ~10 target templates ) . in tube 5 ,
the upper interphase was positive , the top phase was weakly positive , and the middle and bottom phases were negative .
hence , the buoyant density of l. intracellularis seems to be between 1.064 and 1.077 g / ml . in tube 6 ,
the upper interphase and the top phase were inhibited whereas the middle and bottom phases were negative .
based on the results obtained , the selected setup consisted of a bottom phase with a density of 1.23 g / ml , a middle phase with a density of 1.077 g / ml , and a top phase with a density of 1.057 g / ml ( tube 4 , table 1 ) . in the comparison between the recoveries from samples prepared by buoyant density gradient centrifugation and from samples prepared by boiled lysate ( figure 1 ) , both methods yielded positive pcr results in the undiluted sample and in samples diluted 1:10 and 1:100 .
however , a slightly stronger signal was achieved in samples prepared by boiling in the 1:100 dilution . in tube 2 ,
a strong signal from the l. intracellularis amplicon was seen in the undiluted sample from the top phase .
a positive result was also obtained from the undiluted bottom fraction , with a slightly stronger signal obtained in the lysed sample .
all samples diluted 1:100 were negative . in tube 3 , a positive result was achieved in all phases in both dilutions .
the strongest amplicon signal was achieved in the top phase . in the undiluted samples , an unspecific band of ~1500 base pair
following the first centrifugation of the fourth tube , a weakly pcr - positive signal was achieved from the upper interphase diluted 1:100 .
it was not possible to retrieve lawsonia dna following mixing with the negative sample and centrifugation ( tube 5 and 6 ) .
pcr on the undiluted positive control or the control diluted with saline resulted in a single band of expected size ( 319 b p ) .
when the control was mixed with bx - l , a weak unspecific band ( ~1500 b p ) was seen and when the control was mixed with bx - h , a strong band of the same size was seen in addition to the positive control .
the results from the analyses of the panel of selected samples are shown in table 2 . the selected setup consisted of 150 l sample mixed with 450
l bx - h as bottom phase , 450 l saline mixed with 150 l bx - h as middle phase , and 300 l bx - l as top phase . of 24 samples previously lysed by boiling and judged as positive by nested pcr ,
15 were judged as positive when prepared by the small - scale setup followed by conventional pcr .
of 17 samples previously judged as inhibited , two remained inhibited whereas 15 were judged as negative when prepared by the new method . in the previous experiment using a commercial kit for dna extraction followed by pcr , 13 out of 24 samples
were judged as positive and of the 17 previously inhibited samples , 16 were judged as negative and one as inhibited .
all samples judged as negative by pcr on boiled lysate remained negative ( table 3 ) . utilising the preparation by boiling as gold standard and assuming that all inhibited samples are positive , the preparation by boiling would have a sensitivity of 70.7% and a specificity of 100% , the preparation by the commercial kit would have a sensitivity of 59.4% and a specificity of 98.7% , and the new method would have a sensitivity of 61.2% and a specificity of 97.4% . utilising the preparation by boiling as
gold standard and assuming that all inhibited samples are negative , the preparation by boiling would have a sensitivity of 100% and a specificity of 84.4% , the preparation by the commercial kit would have a sensitivity of 68.6% and a specificity of 98.9% , and the new method would have a sensitivity of 72.7 % and a specificity of 97.9% .
one of the major challenges in the diagnosis of l. intracellularis in faecal specimens is to recover a small number of pathogens in the bulk of faeces without the possibility to multiply the microbe by cultivation .
further , amplification of microbial dna by pcr may be prohibited by inhibitors present in faeces [ 1113 , 2023 ] .
flotation can be used to concentrate all microbes within a defined density range at an easily identifiable position in the tube and separate them from soluble inhibitors and particles of other densities .
the technique is comparably fast and easy to perform . in the small scale design ,
standard laboratory equipment is used and the different phases seem to remain stable during collection of the interphase .
the total time used for preparation is approximately the same as for the commercial kit ( i.e. 1.5 hours/10 samples ) ; however , excluding the time for centrifugation and incubation , the time used for manual preparation is about halved ( i.e. 0.5 versus 1 hour/10 samples
. data not shown . ) the method enables the demonstration of l. intracellularis dna with a high specificity . in our study
, however , the sensitivity seemed to be lower when compared to nested pcr performed on samples lysed by boiling .
this may be explained by the fact that only the fraction containing a concentrate of whole l. intracellularis cells is analysed .
the results are however still comparable to , or slightly better than , those obtained following dna preparation by the commercial kit .
although both flotation and the kit combined with conventional pcr performed poorer as compared to boiling combined with nested pcr , the latter method is not suited for routine diagnosis [ 5 , 24 ] .
it is not clear why pcr - positive reactions for l. intracellularis were obtained from all phases in some samples .
theoretically , the time or the speed of centrifugation could be too short to allow the bacteria to pass through the higher density layers and concentrate in the upper interphase .
however , positive pcr results for l. intracellularis were also achieved from the top phase .
further , incautious handling of the tubes might cause stirring of the phases . however , the different phases were clearly visible both before and after centrifugation .
in addition , collection of the sample through the tube wall did not improve the results .
this phenomenon has not been described in similar setups with pure cultures of bacteria of similar density .
a third explanation could be that l. intracellularis may be shed both within sloughed epithelial cells , as free organisms and as bacterial dna from disintegrated bacteria , thereby obtaining a range of densities .
further , it is not known if l. intracellularisis extruded from the intestines at a particular growth phase .
longitudinal studies on c. jejuni have shown that the buoyant densities changed over a range of 1.076 to 1.130 g / ml , indicating that the density may alter depending on the growth phase .
hence , free dna originating from lysed bacteria would be located in the bottom phase , whereas bacteria in exfoliated enterocytes shed in faeces would be retained in the top phase .
in fact , this has proven to be a possible way to separate dead and live bacteria , which would be an advantage , for instance , in the validation of treatment regimens . in the continuous gradient ,
all fractions from one sample were pcr positive for l. intracellularis although less dna was demonstrated at higher and lower densities .
however , the density marker beads simultaneously ran in a separate tube and under similar conditions created distinct , coloured bands at predicted locations .
hence , it seems that in clinical specimens , l. intracellularis is not of homogenous density .
it would have been preferable to test this hypothesis by performing the studies on pure culture of bacteria ; however , due to the difficulties to cultivate l. intracellularis , this was not possible .
it has been speculated that faeces contain several inhibitors of different composition [ 11 , 12 , 2629 ] .
this was also indicated in the present study , where pcr detection of l. intracellularis was slightly or completely inhibited in three fractions with densities between 1.102 and 1.074 and in two fractions with densities just below 1.064 g / ml .
further , we have previously demonstrated the presence of inhibitory factors in the protein , lipid , fibre and mineral fraction of faeces ( data not shown ) .
this may explain the difficulties described in several studies to overcome the pcr inhibition in faecal samples , since most methods are designed to circumvent one single inhibitory mechanism [ 5 , 27 ] .
the results in the present study may also indicate that some inhibitory substances have a density close to that of l. intracellularis .
the nature of the unspecific band of approximately 1500 bp seen following electrophoresis and ethidium bromide staining of the colloidal media has not been elucidated .
ethidium bromide specifically stains dna , but attempts to purify and identify the product have been unsuccessful ( data not shown ) .
however , it does not seem to interfere with pcr or the detection of l. intracellularis .
theoretically it may therefore have a potential for purification and retention of l. intracellularis , for instance , prior to cultivation .
this may be a desirable property , since purification of l. intracellularis traditionally is based on filtering through sequentially smaller membranes , which is time consuming and requires special equipment . in
opposite to percoll , the present technique is based on silanised silicon particles resulting in a clear solution . hence , the media do not interfere with fluorescence - based methods such as real - time pcr . as compared to percoll ,
, when used without further washing , convection in the hot solution is slower and therefore the annealing time in the pcr reaction might need to be adjusted .
in addition , caution should be paid to samples with high lipid content that may disrupt the gradients . in conclusion ,
the performance of the buoyant density gradient centrifugation combined with conventional pcr is comparable to or slightly better than the method presently used for routine diagnosis of l. intracellularis in faecal samples , that is , preparation by a commercial kit combined with conventional pcr . in our hands , the new method is also faster and easier to perform and can be used to separate the microbe from soluble inhibitors and particles of other densities . in addition
based on the results obtained in this study , the buoyant density of l. intracellularis seems to be between 1.064 and 1.077 g / ml . | the obligate intracellular bacterium lawsonia intracellularis causes enteritis and poor growth in weaned pigs .
cultivation is difficult and diagnosis ante mortem is mainly based on techniques such as polymerase chain reaction .
however , false negative results caused by the presence of pcr - inhibitory factors constitute a problem . this study aimed to develop and evaluate a new technique , flotation , to separate l. intracellularis from inhibitors in faeces prior to pcr .
the technique was evaluated by comparison to two previously evaluated and commonly used methods , preparation by boiling lysate combined with nested pcr and preparation by a commercial kit combined with conventional pcr .
continuous density centrifugation of faecal samples containing l. intracellularis suggested the buoyant density of the microbe to be between 1.064 and 1.077 g / ml .
several flotation setups were tested to achieve optimal separation of the microbe from inhibitors and faecal particles .
the finally selected setup floated whole l. intracellularis from the application site at the bottom to the upper part of the gradient while inhibitory components mainly remained in the bottom .
pcr was performed directly on material recovered from the upper interphase .
the method was evaluated on 116 clinical samples .
as compared to sample preparation by boiling combined with nested pcr , fewer samples were inhibited but also fewer positives were identified . in comparison to preparation by a commercial kit combined with conventional pcr , presently used for routine diagnosis , similar results were obtained .
however , the new method was comparably faster to perform .
the new method , based on flotation of lawsonia intracellularis combined with conventional pcr , was well suited for routine diagnosis . | 1. Introduction
2. Material and Methods
3. Results
4. Discussion | however , clinical signs such as diarrhoea and poor growth may be caused by a wide range of microorganisms such as parasites , bacteria , and viruses . hence , to implement adequate treatment and prophylactic strategies it is important to identify the causative organism and the necessity of a reliable diagnosis is obvious . the bacterium lawsonia intracellularis is a major cause of enteritis in weaned pigs and is demonstrated in up to 94% of the herds . necropsy is used to establish a relationship between clinical signs and the presence of representative lesions but to specifically identify the causative agent ; other techniques must be added [ 3 , 4 ] . however , a causal relationship will not be established and false reactions may be difficult to interpret [ 57 ] . identification of specific dna by polymerase chain reaction ( pcr ) and immunohistochemistry ( ihc ) on tissue samples have been suggested as gold standard [ 8 , 9 ] . antemortem , culture is used as gold standard for many bacteria but is presently not applicable in the routine diagnosis of the obligate intracellular l. intracellularis and presently , pcr or immunperoxidase staining of faecal smears is the only option available [ 6 , 10 ] . however , when applied on complex biological samples such as faeces , false negative results constitute a problem [ 5 , 6 , 1113 ] . in addition , the technique does not distinguish between dead or live bacteria and the quality and yield of dna may depend on the target , the sample composition , and the method used for purification of dna [ 1 , 5 ] . recently , a new method to separate particles and bacteria based on their buoyant density has been described . the method was successfully applied to separate yersinia enterocolitica and campylobacter species from food particles and inhibitors [ 14 , 15 ] and was stated to enable the separation between live and dead bacteria as well as free bacterial dna . the purpose of this study was to develop and evaluate a new method in the diagnosis of l. intracellularis , based on the separation of l. intracellularis cells from faecal samples by flotation prior to pcr . 0.1 g of all samples was also lysed by boiling and analysed for l. intracellularis by nested pcr combined with agaros gel electrophoresis and ethidium - bromide staining , as previously described . media of various densities were prepared from two colloidal density gradient media stock solutions ; bactxtractor - low density ( bx - l ) with a density of 1.057 g / ml and bactxtractor - high density ( bx - h ) with a density of 1.309 g / ml ( fertipro n.v . to estimate the buoyant density of l. intracellularis the methodology described by pertoft was used . following centrifugation , the distance from the bottom of the second tube to the coloured bands created by the separated marker beads was measured and plotted against their densities . the volume per mm
aliquots of the sample were drawn from the bottom , and the volumes were measured and plotted in the density curve . the procedure was repeated with two additional faecal samples known to be pcr positive for l. intracellularis . in a fourth tube , a sample previously shown to be strongly pcr inhibiting , as judged by the repeated failure to amplify the internal standard , was included to determine the buoyant density of major inhibitory components . pcr analysis for l. intracellularis was performed in accordance with jacobson et al . one l of the template was added and pcr was performed in a dna thermal cycler ( ptc-200 , mj research inc , watertown , mass , usa ) starting with 10 minutes at 95c followed by 35 cycles consisting of 95c for 30 s , 55c for 30 s , and 72c for 30 s. when undiluted density media was included in the template , a prolonged annealing time ( 45 s ) was used . based on the estimated buoyant density of l. intracellularis , the densities were designed to float the bacterium from the bottom phase and concentrate the microbe to the interphase between the top and middle phases . small and water - soluble components such as free dna and various pcr - inhibitors that were not affected by the applied g - force were expected to be located in the bottom phase . to maximize the separation of l. intracellularis from inhibitory factors , different densities of the middle and top phases
approximately 0.5 g faecal sample was homogenized in 1 ml saline incubated for 10 minutes , and 1 ml supernatant was used as a sample applied in the bottom phase . from the upper interphase , samples ( 0.51 ml ) were drawn with a sterile needle through the tube wall to avoid mixing the phases . except for tube 1 , the top , middle , and bottom phases
the samples were pelleted by centrifugation at 10000 rpm for 5 minutes and washed twice in saline , and the final pellet was resuspended in 0.20.3 ml saline and lysed at 95c for 20 minutes prior to pcr . in addition , six tubes were prepared and centrifuged in accordance with tube 4 , table 1 , to further analyse the overall distribution and recovery of l. intracellularis dna in the gradient ( figure 1 ) . to compare the amount of dna recovered by flotation to the amount recovered by boiling [ 5 , 18 ] , tenfold dilutions of the sample prepared by either method were subjected to nested pcr together with 10 mimics ( figure 1(1 ) ) . to determine if whole l. intracellularis - containing enterocytes were present , the top phase from tube 2 was analysed by pcr ( undiluted and diluted 1:100 ) and likewise , the bottom phase was analysed to determine if free dna was present . to confirm that free dna would be allocated to the bottom phase
only , a lysed sample was included in tube 3 , the phases were collected separately , and undiluted samples and samples diluted 1:100 were subjected to pcr . to determine if l. intracellularis dna was lost during the process , the interphase from tube 4 was collected and subjected to a second flotation . subsequently , corresponding amounts of the interphases from the first and the second centrifugations were serially diluted and subjected to pcr . to determine if faecal particles would influence the recovery , 100 l sample from the bottom and top fractions , respectively ( tube 4 ) , was mixed with 1 ml supernatant from a negative faecal sample prior to centrifugation ( tube 5 and 6 ) . further , to determine if the colloidal media would interfere with pcr , 1 l of l. intracellularis dna precipitated by phenol / chloroform was subjected to pcr without being diluted , diluted 1:1 with saline , 1:1 with bx - l , or 1:1 with bx - h . to adopt the procedure to standard laboratory equipment , a small - scale setup ( bottom phase : 100 l sample mixed with 300 l bx - h , density 1.20 g / ml ; middle phase : 304.1 l saline plus 95.9 l bx - h , density 1.074 g / ml ; top phase : 315.8 l saline mixed with 84.2 l bx - h , density 1.065 g / ml ) was tested on a panel of selected samples ( table 2 ) . six samples had previously been strongly positive for l. intracellularis when prepared by boiling and analysed by nested pcr , six samples had a weak positive reaction caused by a small amount of dna in the sample as judged by comparison to the mimic , six had a weak positive reaction caused by partial inhibition of the pcr , and six samples had previously been completely inhibited . following removal of 300 l of the top phase ,
the next 200 l sample was recovered from the upper interphase , lysed and centrifuged at 15 000 rpm for 30 s. before analysis by pcr . further , 116 clinical samples originating from seven different farms were prepared by the optimized small - scale setup . the bottom phase consisted of 150 l supernatant from the sample mixed with 450 l bx - h , the middle phase consisted of 450 l saline and 150 l bx - h with a density of 1.0772 g / ml , and the top phase was increased to 600 l bx - l . following centrifugation at ~5000 g for 30 minutes , the top phase was removed and the underlying 200400 l was transferred to a new tube and lysed , and samples ( undiluted and diluted 1:100 ) were analysed by pcr . aliquots of the samples had previously also been prepared by a commercial kit ( qiaamp dna stool mini kit , qiagen inc . , valencia , calif , usa ) and by boiling and these results were included for comparison ( table 3 ) . following centrifugation , the self - generated continuous density gradient in the first tube yielded 25 fractions with volumes ranging from 155 to 510 l . following pcr , all fractions in the density range of 1.138 to 1.051 g / ml were positive for l. intracellularis . fractions with densities higher than 1.074 g / ml and lower than 1.065 g / ml contained less bacterial dna ( < 10target templates / ml ) as determined semiquantitatively . in the repeated run on two additional positive samples , all fractions with densities
> 1.102 g / ml and < 1.064 g / ml were negative and all fractions between these values were positive , each fraction containing ~1010 dna target templates . in the inhibited sample , weak inhibition was seen in four out of six fractions with densities between 1.102 and 1.074 g / ml , one fraction in this interval was completely inhibited , and one fraction was not inhibited . fractions with densities around and slightly below 1.074 g / ml were not inhibited , but weak or complete inhibition was demonstrated in fractions with densities approaching 1.064 g / ml . following centrifugation ,
in the first setup , positive pcr reactions were demonstrated in all phases in tubes 14 ( table 1 ) . as determined semiquantitatively ,
the bottom phases from tubes 2 and 3 contained less amount of dna ( ~10 target templates ) . in tube 5 ,
the upper interphase was positive , the top phase was weakly positive , and the middle and bottom phases were negative . hence , the buoyant density of l. intracellularis seems to be between 1.064 and 1.077 g / ml . in tube 6 ,
the upper interphase and the top phase were inhibited whereas the middle and bottom phases were negative . based on the results obtained , the selected setup consisted of a bottom phase with a density of 1.23 g / ml , a middle phase with a density of 1.077 g / ml , and a top phase with a density of 1.057 g / ml ( tube 4 , table 1 ) . in the comparison between the recoveries from samples prepared by buoyant density gradient centrifugation and from samples prepared by boiled lysate ( figure 1 ) , both methods yielded positive pcr results in the undiluted sample and in samples diluted 1:10 and 1:100 . however , a slightly stronger signal was achieved in samples prepared by boiling in the 1:100 dilution . in tube 2 ,
a strong signal from the l. intracellularis amplicon was seen in the undiluted sample from the top phase . a positive result was also obtained from the undiluted bottom fraction , with a slightly stronger signal obtained in the lysed sample . the strongest amplicon signal was achieved in the top phase . in the undiluted samples , an unspecific band of ~1500 base pair
following the first centrifugation of the fourth tube , a weakly pcr - positive signal was achieved from the upper interphase diluted 1:100 . when the control was mixed with bx - l , a weak unspecific band ( ~1500 b p ) was seen and when the control was mixed with bx - h , a strong band of the same size was seen in addition to the positive control . the results from the analyses of the panel of selected samples are shown in table 2 . of 24 samples previously lysed by boiling and judged as positive by nested pcr ,
15 were judged as positive when prepared by the small - scale setup followed by conventional pcr . of 17 samples previously judged as inhibited , two remained inhibited whereas 15 were judged as negative when prepared by the new method . in the previous experiment using a commercial kit for dna extraction followed by pcr , 13 out of 24 samples
were judged as positive and of the 17 previously inhibited samples , 16 were judged as negative and one as inhibited . utilising the preparation by boiling as gold standard and assuming that all inhibited samples are positive , the preparation by boiling would have a sensitivity of 70.7% and a specificity of 100% , the preparation by the commercial kit would have a sensitivity of 59.4% and a specificity of 98.7% , and the new method would have a sensitivity of 61.2% and a specificity of 97.4% . utilising the preparation by boiling as
gold standard and assuming that all inhibited samples are negative , the preparation by boiling would have a sensitivity of 100% and a specificity of 84.4% , the preparation by the commercial kit would have a sensitivity of 68.6% and a specificity of 98.9% , and the new method would have a sensitivity of 72.7 % and a specificity of 97.9% . one of the major challenges in the diagnosis of l. intracellularis in faecal specimens is to recover a small number of pathogens in the bulk of faeces without the possibility to multiply the microbe by cultivation . flotation can be used to concentrate all microbes within a defined density range at an easily identifiable position in the tube and separate them from soluble inhibitors and particles of other densities . the technique is comparably fast and easy to perform . in the small scale design ,
standard laboratory equipment is used and the different phases seem to remain stable during collection of the interphase . the total time used for preparation is approximately the same as for the commercial kit ( i.e. 1.5 hours/10 samples ) ; however , excluding the time for centrifugation and incubation , the time used for manual preparation is about halved ( i.e. the method enables the demonstration of l. intracellularis dna with a high specificity . in our study
, however , the sensitivity seemed to be lower when compared to nested pcr performed on samples lysed by boiling . this may be explained by the fact that only the fraction containing a concentrate of whole l. intracellularis cells is analysed . the results are however still comparable to , or slightly better than , those obtained following dna preparation by the commercial kit . although both flotation and the kit combined with conventional pcr performed poorer as compared to boiling combined with nested pcr , the latter method is not suited for routine diagnosis [ 5 , 24 ] . it is not clear why pcr - positive reactions for l. intracellularis were obtained from all phases in some samples . theoretically , the time or the speed of centrifugation could be too short to allow the bacteria to pass through the higher density layers and concentrate in the upper interphase . however , positive pcr results for l. intracellularis were also achieved from the top phase . however , the different phases were clearly visible both before and after centrifugation . a third explanation could be that l. intracellularis may be shed both within sloughed epithelial cells , as free organisms and as bacterial dna from disintegrated bacteria , thereby obtaining a range of densities . longitudinal studies on c. jejuni have shown that the buoyant densities changed over a range of 1.076 to 1.130 g / ml , indicating that the density may alter depending on the growth phase . hence , free dna originating from lysed bacteria would be located in the bottom phase , whereas bacteria in exfoliated enterocytes shed in faeces would be retained in the top phase . in fact , this has proven to be a possible way to separate dead and live bacteria , which would be an advantage , for instance , in the validation of treatment regimens . in the continuous gradient ,
all fractions from one sample were pcr positive for l. intracellularis although less dna was demonstrated at higher and lower densities . however , the density marker beads simultaneously ran in a separate tube and under similar conditions created distinct , coloured bands at predicted locations . it would have been preferable to test this hypothesis by performing the studies on pure culture of bacteria ; however , due to the difficulties to cultivate l. intracellularis , this was not possible . this was also indicated in the present study , where pcr detection of l. intracellularis was slightly or completely inhibited in three fractions with densities between 1.102 and 1.074 and in two fractions with densities just below 1.064 g / ml . further , we have previously demonstrated the presence of inhibitory factors in the protein , lipid , fibre and mineral fraction of faeces ( data not shown ) . the results in the present study may also indicate that some inhibitory substances have a density close to that of l. intracellularis . the nature of the unspecific band of approximately 1500 bp seen following electrophoresis and ethidium bromide staining of the colloidal media has not been elucidated . however , it does not seem to interfere with pcr or the detection of l. intracellularis . theoretically it may therefore have a potential for purification and retention of l. intracellularis , for instance , prior to cultivation . this may be a desirable property , since purification of l. intracellularis traditionally is based on filtering through sequentially smaller membranes , which is time consuming and requires special equipment . in
opposite to percoll , the present technique is based on silanised silicon particles resulting in a clear solution . hence , the media do not interfere with fluorescence - based methods such as real - time pcr . as compared to percoll ,
, when used without further washing , convection in the hot solution is slower and therefore the annealing time in the pcr reaction might need to be adjusted . in conclusion ,
the performance of the buoyant density gradient centrifugation combined with conventional pcr is comparable to or slightly better than the method presently used for routine diagnosis of l. intracellularis in faecal samples , that is , preparation by a commercial kit combined with conventional pcr . in our hands , the new method is also faster and easier to perform and can be used to separate the microbe from soluble inhibitors and particles of other densities . in addition
based on the results obtained in this study , the buoyant density of l. intracellularis seems to be between 1.064 and 1.077 g / ml . | [
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the field of nanotechnology has been growing rapidly within various industries over the last decade .
various types of nanoparticles , including titanium oxide , zinc oxide , and silica dioxide ( sio2 ) , are used in cosmetics , sports equipment , and building materials.1 due to their widespread applications in various industries , use of nanoparticles continues to increase .
however , nanoparticles may be hazardous to human health because of their unusual physicochemical properties , eg , small size , high surface to volume ratio , chemical composition , crystallinity , electronic properties , surface structure reactivity and functional groups , inorganic or organic coatings , solubility , shape , and aggregation behavior.24 the risk to human health arising from exposure to nanoparticles through ingestion , inhalation , and dermal absorption may increase as the applications of nanoparticles continue to increase.57 the organization for economic cooperation and development ( oecd ) has also paid attention to this problem and organized a special session in a joint meeting on the potential implications of manufactured nanomaterials for human health and environmental safety in 2005 .
the oecd established the working party on manufactured nanomaterials in 2006 for dealing with human and environmental safety issues associated with manufactured nanomaterials.8 sio2 nanoparticles have been widely used in a variety of industrial fields , including the plastics , rubber , ceramics , coatings , adhesives , and medical industries.9,10 numerous studies have reported the cytotoxicity , genotoxicity , and alterations in protein expression associated with sio2 nanoparticles in both in vitro and in vivo systems.11,12 further , it has been suggested that the genotoxicity caused by sio2 nanoparticles may be due to proinflammatory effects , modification of chromatin structure , and liberation of dnase or a potent inducer of cytogenetic damage.13,14 however , these studies were controversial due to the slightly different physicochemical features involved in the synthesis , dispersion , and stability of nanoparticles in biological media.1518 nanoparticle - related studies reported that these differences were generated by the size and surface charge of the specific nanoparticles.1922 the size of nanoparticles could influence their absorption , distribution , metabolism , and excretion.2325 the hydrodynamic size distributions of nanoparticles could be also changed by surface charge and properties , affecting agglomeration .
moreover , the surface charge of nanoparticles could affect uptake and translocation within organisms.26,27 several nanotoxicology reports have proposed that more detailed characterization of nanoparticles would yield a better understanding of their properties and interactions in vivo.2830 therefore , the physicochemical features of nanoparticles would be the critical factor in studying nanotoxicology . in the present study ,
four genotoxicity tests were conducted with well characterized sio2 nanoparticles 20 nm ( sio2 ) and 100 nm ( sio2 ) in size , each having a negative surface charge . in order to accurately evaluate toxicity , physicochemical characterization of nanoparticles and their behavior under physiological conditions
were determined by following experimental protocols similar to those used in our previous study.31 for accurate evaluation of genotoxicity , four types of in vitro and in vivo genotoxicity tests , ie , the bacterial mutation assay , in vitro chromosomal aberration test , in vivo comet assay , and in vivo micronucleus test , were performed according to oecd guidelines3234 or international validation study guideline35 with the good laboratory practice ( glp ) .
all animals used in this study were cared for in accordance with the guide for the care and use of laboratory animals issued by the animal care and use committee of the national veterinary research and quarantine service .
sio2 colloids 20 nm ( sio2 ) and 100 nm ( sio2 ) in size were obtained from e&b nanotech co ltd ( gyeonggi - do , republic of korea ) . the preparation process and physicochemical characterizations were conducted as described in a previous report.31 briefly , a jsm-6700f field emission scanning electron microscope ( joel ltd , tokyo , japan ) and zetasizer nanozs ( malvern instruments ltd , malvern , uk ) were used to determine the particle size , morphology , and surface charge of sio2 .
the bacterial reverse mutation test was conducted in compliance with the korea food and drug administration notification no .
2009 - 116 testing guideline and oecd testing guideline 471.32 the tester strains used in this study were ames salmonella typhimurium ta98 , ta100 , ta1535 , and ta1537 , and escherichia coli wp2uvra in the absence and presence of metabolic activation system .
the metabolic activation system was prepared by mixing s9 metabolic activation ( molecular toxicology inc . ) with cofactor 1 from wako pure chemical industries ltd ( osaka , japan ) , giving a final concentration of 10% ( volume / volume ) s9 .
the tester strains were cultured in 2.5% nutrient broth no 2 ( oxoid ltd , basingstoke , uk ) in a 37c shaking incubator ( 120 rpm ) for approximately 11 hours .
the mutagenicity test was performed by mixing test substance and tester strains , which was cultured overnight in the presence and absence of the s9 mix .
next , the mixture was incubated in a water bath for 20 minutes at 37c , mixed with top agar and a minimal amount of histidine - biotin ( for s. typhimurium strains ) or tryptophan ( for e. coli strain ) , and then poured onto the surface of a gamma - ray sterile falcon petri dish ( thermo fisher scientific , waltham , ma , usa ) containing about 15 ml of solidified bottom agar .
all plates were prepared in triplicate , and the results were tabulated as the mean standard deviation for each condition . the in vitro chromosomal aberration test was performed according to the korea food and drug administration notification no .
2009 - 116 testing guideline and oecd testing guideline 473.33 the clastogenicity of sio2 nanoparticles was evaluated for its ability to induce chromosomal aberrations in chinese hamster lung ( chl ) fibroblast cells .
a clonal subline of chl cells was obtained from the american type culture collection ( rockville , md , usa ) .
the chl cells were grown in minimum essential eagle s medium , supplemented with 10% fetal bovine serum , 50 u / ml penicillin , and 50 g / ml streptomycin ( all from gibco brl life technologies inc . ,
gaithersburg , md , usa ) at 37c in a humidified atmosphere containing 5% co2 .
mitomycin c ( cas no . 50 - 07 - 7 ; sigma - aldrich co. , st louis , mo , usa ) was used as a positive control , both with and without the s9 mixture .
after 22 hours of incubation , colcemid was added to the cultures at a final concentration of 0.2 g / ml , and meta - phase cells were harvested by trypsinization and centrifugation .
the cells were swelled by adding hypotonic ( 0.075 m ) kcl solution for 20 minutes at 37c , and then washed three times in ice - cold fixative ( methanol to glacial acetic acid , 3:1 ) .
a few drops of cell pellet suspension were dropped onto precleaned glass microscope slides and air - dried .
the slides were stained with 5% giemsa buffer solution ( thermo fisher scientific , waltham , ma , usa ) .
the classification of aberration types referred to jems - mms ( japanese environmental mutagen society - mammalian mutagenesis study group ) .
aberration frequencies , defined as aberrations observed , were divided by the number of cells counted , and were analyzed using fisher s exact test with dunnett s adjustment , and compared with results from the solvent controls .
the alkaline ( ph > 13 ) comet assay was conducted according to international validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens ( version 14.2).35 male crl : cd ( sprague dawley [ sd ] ) rats ( aged 6 weeks ) were used for the in vivo alkaline comet assay .
after a 7-day acclimation period , the test substance was administered three times by gavage at 0 , 24 , and 45 hours .
the test doses ( 500 , 1,000 , and 2,000 mg / kg ) were selected by a range - finding experiment . following the guideline ,
ethyl methanesulfonate was used for the positive control at a dose of 200 mg / kg .
the liver and stomach were collected from each animal and maintained in cold mincing buffer ( mg - free and ca - free hanks balanced salt solution ( gibco , carlsbad , ca , usa ) with 20 mm ethylenediaminetetraacetic acid ( edta)-na2 and 10% volume / volume dimethylsulfoxide ( sigma - aldridge , st louis , mo , usa ) .
the forestomach was discarded , and the glandular stomach was placed into cold mincing buffer and incubated on ice for 1530 minutes .
this layer was discarded subsequently , and the stomach epithelium was carefully scoured with a scraper to release the cells .
the cell suspension was stored on ice for 1530 seconds to allow large clumps to settle , and the supernatant was used to prepare the comet slides .
a 10 l aliquot of single cell suspension was mixed with 0.5% low melting agarose ( invitrogen , carlsbad , ca , usa ) and spread on comet assay slides ( travigen , gaithersburg , md , usa ) .
the slides were immersed in cold lysis solution ( 2.5 m nacl , 100 mm edta - na2 , 10 mm tris - base , 10% dimethylsulfoxide , 1% triton - x [ ph10 ] ) overnight . after this incubation , the slides were placed in electrophoresis solution ( 0.3 m naoh , 1 mm edta [ ph > 13 ] ) for 20 minutes to allow for unwinding of dna .
the slides were then immersed in neutralization solution ( 0.4 m tris - base [ ph 7.5 ] ) for at least 5 minutes and then dehydrated with absolute ethanol to fix .
the cells were stained with sybr gold ( invitrogen ) according to the manufacturer s specifications .
the comet was observed via fluorescence microscopy ( nikon , tokyo , japan ) at a magnification of 200 and analyzed by comet assay iv software ( perceptive instruments ltd , bury st edmunds , uk ) . for each sample ( animal / tissue ) ,
a positive response is defined as a statistically significant change in the percent tail dna in at least one dose group in comparison with the vehicle control value using dunnett s test ( two - sided , p<0.05 ) as well as a statistically significant linear trend test ( two - sided , p<0.05 ) . the positive control should produce a statistically significant increase as determined by the student s t - test ( one - sided , p<0.025 ) . the in vivo micronucleus test was performed in compliance with the korea food and drug administration notification no .
2009 - 116 testing guideline and oecd testing guideline 474.34 out - bred 67-week - old mice of strain icr were used in this study .
the test substance was administered orally in three doses in volumes of 10 ml / kg . it was given twice with a 24-hour interval in between , and test subjects were sacrificed by cervical dislocation .
preparation and staining of bone marrow was carried out according to the method described by schmid.36 in scoring the preparations , micronuclei were counted in polychromatic erythrocytes and separately in monochromatic erythrocytes .
the rate of micronucleated cells , expressed as a percentage , was based on the total of polychromatic erythrocytes present in the scored optic fields .
this method of scoring , which must always be followed where the test substance markedly influences the proliferation rate in bone marrow , prevented distortion of the results by the influx of peripheral blood into the damaged marrow .
the scoring of micronucleated normocytes was used to recognize the presence of artifacts ( which is rare in mouse preparations ) , which provided additional interesting information on the mode of action of the test substances .
generally , an incidence of more than one micronucleated normocyte per 1,000 polychromatic erythrocytes indicates an effect on cell stages , especially post - s - phase .
the result of the statistical evaluation was deemed to be statistically significant when the p - value was less than 0.05 .
wallis h test and dunnett s test for differences in numbers of micronucleated polychromatic erythrocytes between the treated and negative control groups ; the mann
whitney u test for differences in numbers of micronucleated polychromatic erythrocytes between the positive and negative control groups ; analysis of variance and dunnett s test for differences in the polychromatic erythrocyte ( pce)/(pce + normochromatic erythrocyte [ nce ] ) ratio between the treated and negative control groups ; the student s t - test for differences in the pce/(pce + nce ) ratio between the positive and negative control groups ; and analysis of variance and dunnett s test for comparison of animal body weight at the time of euthanasia .
all animals used in this study were cared for in accordance with the guide for the care and use of laboratory animals issued by the animal care and use committee of the national veterinary research and quarantine service .
sio2 colloids 20 nm ( sio2 ) and 100 nm ( sio2 ) in size were obtained from e&b nanotech co ltd ( gyeonggi - do , republic of korea ) . the preparation process and physicochemical characterizations were conducted as described in a previous report.31 briefly , a jsm-6700f field emission scanning electron microscope ( joel ltd , tokyo , japan ) and zetasizer nanozs ( malvern instruments ltd , malvern , uk ) were used to determine the particle size , morphology , and surface charge of sio2 .
the bacterial reverse mutation test was conducted in compliance with the korea food and drug administration notification no .
2009 - 116 testing guideline and oecd testing guideline 471.32 the tester strains used in this study were ames salmonella typhimurium ta98 , ta100 , ta1535 , and ta1537 , and escherichia coli wp2uvra in the absence and presence of metabolic activation system .
the metabolic activation system was prepared by mixing s9 metabolic activation ( molecular toxicology inc . ) with cofactor 1 from wako pure chemical industries ltd ( osaka , japan ) , giving a final concentration of 10% ( volume / volume ) s9 .
the tester strains were cultured in 2.5% nutrient broth no 2 ( oxoid ltd , basingstoke , uk ) in a 37c shaking incubator ( 120 rpm ) for approximately 11 hours .
the mutagenicity test was performed by mixing test substance and tester strains , which was cultured overnight in the presence and absence of the s9 mix .
next , the mixture was incubated in a water bath for 20 minutes at 37c , mixed with top agar and a minimal amount of histidine - biotin ( for s. typhimurium strains ) or tryptophan ( for e. coli strain ) , and then poured onto the surface of a gamma - ray sterile falcon petri dish ( thermo fisher scientific , waltham , ma , usa ) containing about 15 ml of solidified bottom agar .
all plates were prepared in triplicate , and the results were tabulated as the mean standard deviation for each condition .
the in vitro chromosomal aberration test was performed according to the korea food and drug administration notification no .
2009 - 116 testing guideline and oecd testing guideline 473.33 the clastogenicity of sio2 nanoparticles was evaluated for its ability to induce chromosomal aberrations in chinese hamster lung ( chl ) fibroblast cells .
a clonal subline of chl cells was obtained from the american type culture collection ( rockville , md , usa ) .
the chl cells were grown in minimum essential eagle s medium , supplemented with 10% fetal bovine serum , 50 u / ml penicillin , and 50 g / ml streptomycin ( all from gibco brl life technologies inc . , gaithersburg , md , usa ) at 37c in a humidified atmosphere containing 5% co2 .
mitomycin c ( cas no . 50 - 07 - 7 ; sigma - aldrich co. , st louis , mo , usa ) was used as a positive control , both with and without the s9 mixture .
after 22 hours of incubation , colcemid was added to the cultures at a final concentration of 0.2 g / ml , and meta - phase cells were harvested by trypsinization and centrifugation .
the cells were swelled by adding hypotonic ( 0.075 m ) kcl solution for 20 minutes at 37c , and then washed three times in ice - cold fixative ( methanol to glacial acetic acid , 3:1 ) .
a few drops of cell pellet suspension were dropped onto precleaned glass microscope slides and air - dried .
the slides were stained with 5% giemsa buffer solution ( thermo fisher scientific , waltham , ma , usa ) .
the classification of aberration types referred to jems - mms ( japanese environmental mutagen society - mammalian mutagenesis study group ) .
aberration frequencies , defined as aberrations observed , were divided by the number of cells counted , and were analyzed using fisher s exact test with dunnett s adjustment , and compared with results from the solvent controls .
the alkaline ( ph > 13 ) comet assay was conducted according to international validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens ( version 14.2).35 male crl : cd ( sprague dawley [ sd ] ) rats ( aged 6 weeks ) were used for the in vivo alkaline comet assay .
the sd rats were randomized into groups containing five rats each . after a 7-day acclimation period ,
the test substance was administered three times by gavage at 0 , 24 , and 45 hours .
the test doses ( 500 , 1,000 , and 2,000 mg / kg ) were selected by a range - finding experiment . following the guideline ,
ethyl methanesulfonate was used for the positive control at a dose of 200 mg / kg .
the liver and stomach were collected from each animal and maintained in cold mincing buffer ( mg - free and ca - free hanks balanced salt solution ( gibco , carlsbad , ca , usa ) with 20 mm ethylenediaminetetraacetic acid ( edta)-na2 and 10% volume / volume dimethylsulfoxide ( sigma - aldridge , st louis , mo , usa ) .
the forestomach was discarded , and the glandular stomach was placed into cold mincing buffer and incubated on ice for 1530 minutes .
this layer was discarded subsequently , and the stomach epithelium was carefully scoured with a scraper to release the cells .
the cell suspension was stored on ice for 1530 seconds to allow large clumps to settle , and the supernatant was used to prepare the comet slides . a 10 l aliquot of single cell suspension was mixed with 0.5% low melting agarose ( invitrogen , carlsbad , ca , usa ) and spread on comet assay slides ( travigen , gaithersburg , md , usa ) . the slides were immersed in cold lysis solution ( 2.5 m nacl , 100 mm edta - na2 , 10 mm tris - base , 10% dimethylsulfoxide , 1% triton - x [ ph10 ] ) overnight . after this incubation , the slides were placed in electrophoresis solution ( 0.3 m naoh , 1 mm edta [ ph > 13 ] ) for 20 minutes to allow for unwinding of dna .
the slides were then immersed in neutralization solution ( 0.4 m tris - base [ ph 7.5 ] ) for at least 5 minutes and then dehydrated with absolute ethanol to fix .
the cells were stained with sybr gold ( invitrogen ) according to the manufacturer s specifications .
the comet was observed via fluorescence microscopy ( nikon , tokyo , japan ) at a magnification of 200 and analyzed by comet assay iv software ( perceptive instruments ltd , bury st edmunds , uk ) . for each sample ( animal / tissue ) ,
a positive response is defined as a statistically significant change in the percent tail dna in at least one dose group in comparison with the vehicle control value using dunnett s test ( two - sided , p<0.05 ) as well as a statistically significant linear trend test ( two - sided , p<0.05 ) . the positive control should produce a statistically significant increase as determined by the student s t - test ( one - sided , p<0.025 ) .
the in vivo micronucleus test was performed in compliance with the korea food and drug administration notification no .
2009 - 116 testing guideline and oecd testing guideline 474.34 out - bred 67-week - old mice of strain icr were used in this study .
the test substance was administered orally in three doses in volumes of 10 ml / kg . it was given twice with a 24-hour interval in between , and test subjects were sacrificed by cervical dislocation . preparation and staining of bone marrow was carried out according to the method described by schmid.36 in scoring the preparations , micronuclei were counted in polychromatic erythrocytes and separately in monochromatic erythrocytes .
the rate of micronucleated cells , expressed as a percentage , was based on the total of polychromatic erythrocytes present in the scored optic fields .
this method of scoring , which must always be followed where the test substance markedly influences the proliferation rate in bone marrow , prevented distortion of the results by the influx of peripheral blood into the damaged marrow .
the scoring of micronucleated normocytes was used to recognize the presence of artifacts ( which is rare in mouse preparations ) , which provided additional interesting information on the mode of action of the test substances .
generally , an incidence of more than one micronucleated normocyte per 1,000 polychromatic erythrocytes indicates an effect on cell stages , especially post - s - phase .
the result of the statistical evaluation was deemed to be statistically significant when the p - value was less than 0.05 .
wallis h test and dunnett s test for differences in numbers of micronucleated polychromatic erythrocytes between the treated and negative control groups ; the mann
whitney u test for differences in numbers of micronucleated polychromatic erythrocytes between the positive and negative control groups ; analysis of variance and dunnett s test for differences in the polychromatic erythrocyte ( pce)/(pce + normochromatic erythrocyte [ nce ] ) ratio between the treated and negative control groups ; the student s t - test for differences in the pce/(pce + nce ) ratio between the positive and negative control groups ; and analysis of variance and dunnett s test for comparison of animal body weight at the time of euthanasia .
particle size and surface charge are significant factors when determining the biological behavior of nanoparticles.31 the physicochemical properties of sio2 nanoparticles were investigated in a previous study , as described briefly below.31 the average size and morphology of the sio2 nanoparticles was investigated by field emission scanning electron microscopy .
the results showed a monodispersed size distribution and spherical morphology with a mean size of ~33 nm and ~90 nm for sio2 and sio2 , respectively .
dynamic light scattering analysis was determined to be 230.1 nm and 91.60.5 nm for sio2 and sio2 , respectively .
the surface charge on the sio2 nanoparticles in distilled water suspension was determined to be negative ( around 40 mv ) .
in the preliminary dose - ranging tests ( data not shown ) , two kinds of sio2 nanoparticles , ie , sio2 and sio2 , were found to have a nontoxic effect in all tester strains of s. typhimurium ( ta98 , ta100 , ta1535 , and ta1537 ) and in e. coli wp2uvra at a dose of 5,000 g per plate in the presence and absence of s9 .
based on the data from the preliminary dose - ranging tests , we selected 5,000 g per plate as the highest dose .
as shown in table 1 , none of the tester strains showed any increase in the number of revertant colonies in comparison with the solvent control ( 0 g per plate ) when the bacteria were treated with the two types of sio2 nanoparticles at 313 , 625 , 1,250 , 2,500 , and 5,000 g per plate , regardless of metabolic activation . on the other hand ,
the positive control group resulted in more revertant colonies than the solvent control or other sio2 treatment groups .
initially , a dose - ranging test was performed to determine the test doses for use in the in vitro chromosome aberration test .
the highest concentration was determined to be 1,400 g / ml , as it showed less than 50% cytotoxicity in chl cells ( data not shown ) .
the in vitro chromosome aberration test was conducted with sio2 and sio2 at concentrations of 175 , 350 , 700 , and 1,400 g / ml , both with and without metabolic activation ( s9 ) . except for the positive control group ,
the results indicated no significant increase in sio2 nanoparticles in the treatment groups in comparison with the solvent control at the four concentrations , regardless of metabolic activation ( table 2 ) . on the other hand , the positive control group showed significantly increased structural aberrations in comparison with the solvent control and with the other sio2 treatment groups .
a dose - ranging study was conducted in sd rats ( data not shown ) , and the test doses were determined as 500 , 1,000 , and 2,000 mg / kg body weight .
dna damage was measured by the in vivo alkaline comet assay , following the international validation study protocol ( version 14.2).35 our data showed no significant differences in percent tail dna in single cells from the liver and stomach treated with sio2 ( figure 1a ) and sio2 ( figure 1b ) .
the positive control showed a significant increase in tail dna intensity ( percent tail dna ) in comparison with the negative control .
therefore , no genotoxic effect of sio2 nanoparticles ( sio2 and sio2 ) was observed using the in vivo comet assay . in the preliminary dose - ranging test , acute oral toxicity , death , or apparent abnormality in appearance
could not be determined in mice at doses ranging from 500 mg / kg to 2,000 mg / kg body weight .
the pce/(nce + pce ) ratios were used as an index of bone marrow cytotoxicity .
the ratios did not show any significant difference in the sio2 treatment groups in comparison with the solvent control group , for either size of sio2 nanoparticle .
the micronucleated pce frequencies were not statistically significant and did not yield any dose - dependent pattern among the three treatment groups in comparison with the solvent control group for either size of sio2 nanoparticle .
on the other hand , the positive control group showed a significantly increased micronucleated pce frequency in comparison with the solvent control and sio2 treatment groups ( table 3 ) .
therefore , both types of sio2 nanoparticles were regarded to have no genotoxic or cytotoxic potential using the current in vivo system .
particle size and surface charge are significant factors when determining the biological behavior of nanoparticles.31 the physicochemical properties of sio2 nanoparticles were investigated in a previous study , as described briefly below.31 the average size and morphology of the sio2 nanoparticles was investigated by field emission scanning electron microscopy .
the results showed a monodispersed size distribution and spherical morphology with a mean size of ~33 nm and ~90 nm for sio2 and sio2 , respectively .
dynamic light scattering analysis was determined to be 230.1 nm and 91.60.5 nm for sio2 and sio2 , respectively .
the surface charge on the sio2 nanoparticles in distilled water suspension was determined to be negative ( around 40 mv ) .
in the preliminary dose - ranging tests ( data not shown ) , two kinds of sio2 nanoparticles , ie , sio2 and sio2 , were found to have a nontoxic effect in all tester strains of s. typhimurium ( ta98 , ta100 , ta1535 , and ta1537 ) and in e. coli wp2uvra at a dose of 5,000 g per plate in the presence and absence of s9 . based on the data from the preliminary dose - ranging tests , we selected 5,000 g per plate as the highest dose .
as shown in table 1 , none of the tester strains showed any increase in the number of revertant colonies in comparison with the solvent control ( 0 g per plate ) when the bacteria were treated with the two types of sio2 nanoparticles at 313 , 625 , 1,250 , 2,500 , and 5,000 g per plate , regardless of metabolic activation . on the other hand
, the positive control group resulted in more revertant colonies than the solvent control or other sio2 treatment groups .
initially , a dose - ranging test was performed to determine the test doses for use in the in vitro chromosome aberration test .
the highest concentration was determined to be 1,400 g / ml , as it showed less than 50% cytotoxicity in chl cells ( data not shown ) .
the in vitro chromosome aberration test was conducted with sio2 and sio2 at concentrations of 175 , 350 , 700 , and 1,400 g / ml , both with and without metabolic activation ( s9 ) . except for the positive control group ,
the results indicated no significant increase in sio2 nanoparticles in the treatment groups in comparison with the solvent control at the four concentrations , regardless of metabolic activation ( table 2 ) . on the other hand , the positive control group showed significantly increased structural aberrations in comparison with the solvent control and with the other sio2 treatment groups .
a dose - ranging study was conducted in sd rats ( data not shown ) , and the test doses were determined as 500 , 1,000 , and 2,000 mg / kg body weight .
dna damage was measured by the in vivo alkaline comet assay , following the international validation study protocol ( version 14.2).35 our data showed no significant differences in percent tail dna in single cells from the liver and stomach treated with sio2 ( figure 1a ) and sio2 ( figure 1b ) .
the positive control showed a significant increase in tail dna intensity ( percent tail dna ) in comparison with the negative control .
therefore , no genotoxic effect of sio2 nanoparticles ( sio2 and sio2 ) was observed using the in vivo comet assay .
in the preliminary dose - ranging test , acute oral toxicity , death , or apparent abnormality in appearance could not be determined in mice at doses ranging from 500 mg / kg to 2,000 mg / kg body weight .
the pce/(nce + pce ) ratios were used as an index of bone marrow cytotoxicity .
the ratios did not show any significant difference in the sio2 treatment groups in comparison with the solvent control group , for either size of sio2 nanoparticle .
the micronucleated pce frequencies were not statistically significant and did not yield any dose - dependent pattern among the three treatment groups in comparison with the solvent control group for either size of sio2 nanoparticle .
on the other hand , the positive control group showed a significantly increased micronucleated pce frequency in comparison with the solvent control and sio2 treatment groups ( table 3 ) .
therefore , both types of sio2 nanoparticles were regarded to have no genotoxic or cytotoxic potential using the current in vivo system .
nanotoxicity studies have been necessary , although their results are still controversial due to variations in the physicochemical properties of the materials under investigation.1921,3739 further , their results could differ according to the protocol used for evaluation .
hence , well characterized nanoparticles and approved methods are necessary when conducting nanotoxicity studies . in this study
, nanogenotoxicity was investigated using well characterized sio2 nanoparticles , ie , sio2 and sio2 .
characterization of these nanoparticles and their behavior was done under physiological conditions , as described in our previous study.31 next , four different end point genotoxic tests , ie , the bacterial mutation assay , in vitro chromosomal aberration test , in vivo alkaline comet assay , and in vivo micronucleus test , were conducted in accordance with oecd guidelines and an international validation study protocol using the glp system .
the bacterial mutation assay is used to evaluate the mutagenicity of a chemical compound.1 it is also reported to be an essential test within the current battery of assays required for evaluation of genotoxicity.40 to date , there have been a few reports on the mutagenicity of nanoparticles such as aluminum oxide , cobalt oxide , titanium oxide , and zinc oxide based on the bacterial mutation assay .
hence , we performed this assay using strains of s. typhimurium ta98 , ta100 , ta1535 , and ta1537 , and e. coli wp2uvra , with and without metabolic activation by s9 mixture.4143 it has recently been reported that no mutagenic potential is observed in the ames test with and without metabolic activation.4446 in addition , li et al investigated the genotoxic potential of exfoliated silicate nanoclay.47 our data also showed no significantly increased mutagenicity in any of the strains exposed to sio2 and sio2 with and without s9 ( table 1 ) .
the in vitro chromosomal aberration test is another standard genotoxicity test , and is commonly used to observe variations in chromosomal structure induced by toxic chemicals.33 our data show that sio2 and sio2 did not induce any structural chromosomal aberrations in chl cells , with or without s9 mixture ( table 2 ) .
another previous report showed no induction of chromosomal aberrations in mammalian cells.4446 although different types of sio2 were used in the previous studies , similar results were obtained .
hence , sio2 nanoparticles might not induce clastogenesis in mammalian cells . in vitro genotoxicity tests
the in vivo alkaline comet assay and in vivo micronucleus test were included in the current study in order to conduct and evaluate genotoxicity tests accurately .
the comet assay , a single cell gel electrophoresis assay , has been widely used for detection of dna damage due to its simplicity , low cost , and high sensitivity.48 the in vivo comet assay has been regarded as a potential replacement for the in vivo rodent hepatocyte unscheduled dna synthesis assay , and the protocol for the in vivo comet assay has been standardized and published by an international expert group . in the present study , the in vivo comet assay was conducted using sio2 and sio2 and followed the standard protocol , revealing no genotoxic effect of sio2 and sio2 in rat liver and stomach cells ( figure 1a and 1b ) .
even though the majority of the studies have reported negative results for the genotoxicity of sio2,11,17,47 a few investigations have indicated otherwise.49 downs et al investigated the genotoxic effects of exposure to three consecutive intravenous injections of sio2 nanoparticles by in vivo comet assay in rat liver and lung tissues , and observed the induction of dna damage by sio2.50 hence , the different administration route can lead to different results for genotoxicity evaluation .
the micronucleus test was used to evaluate genotoxicity by scoring the number of micronuclei under chemical exposure . regarding the genotoxicity induced by sio2 nanoparticles , several publications have reported on in vitro micronucleus tests.11,15,47,51,52 in contrast ,
downs et al conducted in vivo micronucleus testing of sio2 nanoparticles , where they analyzed micronuclei in rat blood.50 in the present study , in vivo micronucleus testing was performed with rat bone marrow cells for the first time to determine whether sio2 and sio2 could induce chromosome abnormalities or mitotic apparatus in mouse model . based on the frequencies of micronucleated pce , no genotoxic effect of sio2 was found ( table 3 ) .
nanotoxicology study results can be influenced by physicochemical properties , and the contrast between our results and the previous data could be due to the different administration methods used or different organs investigated , as mentioned above .
in the present study , the genotoxic effects of sio2 and sio2 were elucidated using four genotoxicity tests , ie , the bacterial mutation assay , in vitro chromosomal aberration test , in vivo comet assay , and in vivo micronucleus test , under standardized protocols with glp system .
although the different exposure routes from our study can induce sio2 genotoxicity in different organs in in vivo systems , our data suggest that sio2 and sio2 are not genotoxic substances based on the oecd test guidelines . | backgroundsilica dioxide ( sio2 ) has been used in various industrial products , including paints and coatings , plastics , synthetic rubbers , and adhesives .
several studies have investigated the genotoxic effects of sio2 ; however , the results remain controversial due to variations in the evaluation methods applied in determining its physicochemical properties .
thus , well characterized chemicals and standardized methods are needed for better assessment of the genotoxicity of nanoparticles.methodsthe genotoxicity of sio2 was evaluated using two types of well characterized sio2 , ie , 20 nm ( ) charge ( sio en20()2 ) and 100 nm ( ) charge ( sio en100()2 ) .
four end point genotoxicity tests , ie , the bacterial mutation assay , in vitro chromosomal aberration test , in vivo comet assay , and in vivo micronucleus test , were conducted following the test guidelines of the organization for economic cooperation and development ( oecd ) with application of good laboratory practice.resultsno statistically significant differences were found in the bacterial mutation assay , in vitro chromosomal aberration test , in vivo comet assay , and in vivo micronucleus test when tested for induction of genotoxicity in both two types of sio2 nanoparticles.conclusionthese results suggest that sio2 nanoparticles , in particular sio2en20( ) and sio2en100( ) , are not genotoxic in both in vitro and in vivo systems under oecd guidelines .
further , the results were generated in accordance with oecd test guidelines , and good laboratory practice application ; it can be accepted as reliable information regarding sio2-induced genotoxicity . | Introduction
Materials and methods
Animal care
Preparation and characterization of nanoparticles
Bacterial reverse mutation test
In vitro chromosomal aberration test
In vivo alkaline comet assay
In vivo micronucleus test
Results
Characterization of nanoparticles
Bacterial reverse mutation test
In vitro chromosome aberration test
In vivo alkaline comet assay
In vivo micronucleus test
Discussion
Conclusion | various types of nanoparticles , including titanium oxide , zinc oxide , and silica dioxide ( sio2 ) , are used in cosmetics , sports equipment , and building materials.1 due to their widespread applications in various industries , use of nanoparticles continues to increase . however , nanoparticles may be hazardous to human health because of their unusual physicochemical properties , eg , small size , high surface to volume ratio , chemical composition , crystallinity , electronic properties , surface structure reactivity and functional groups , inorganic or organic coatings , solubility , shape , and aggregation behavior.24 the risk to human health arising from exposure to nanoparticles through ingestion , inhalation , and dermal absorption may increase as the applications of nanoparticles continue to increase.57 the organization for economic cooperation and development ( oecd ) has also paid attention to this problem and organized a special session in a joint meeting on the potential implications of manufactured nanomaterials for human health and environmental safety in 2005 . the oecd established the working party on manufactured nanomaterials in 2006 for dealing with human and environmental safety issues associated with manufactured nanomaterials.8 sio2 nanoparticles have been widely used in a variety of industrial fields , including the plastics , rubber , ceramics , coatings , adhesives , and medical industries.9,10 numerous studies have reported the cytotoxicity , genotoxicity , and alterations in protein expression associated with sio2 nanoparticles in both in vitro and in vivo systems.11,12 further , it has been suggested that the genotoxicity caused by sio2 nanoparticles may be due to proinflammatory effects , modification of chromatin structure , and liberation of dnase or a potent inducer of cytogenetic damage.13,14 however , these studies were controversial due to the slightly different physicochemical features involved in the synthesis , dispersion , and stability of nanoparticles in biological media.1518 nanoparticle - related studies reported that these differences were generated by the size and surface charge of the specific nanoparticles.1922 the size of nanoparticles could influence their absorption , distribution , metabolism , and excretion.2325 the hydrodynamic size distributions of nanoparticles could be also changed by surface charge and properties , affecting agglomeration . in the present study ,
four genotoxicity tests were conducted with well characterized sio2 nanoparticles 20 nm ( sio2 ) and 100 nm ( sio2 ) in size , each having a negative surface charge . in order to accurately evaluate toxicity , physicochemical characterization of nanoparticles and their behavior under physiological conditions
were determined by following experimental protocols similar to those used in our previous study.31 for accurate evaluation of genotoxicity , four types of in vitro and in vivo genotoxicity tests , ie , the bacterial mutation assay , in vitro chromosomal aberration test , in vivo comet assay , and in vivo micronucleus test , were performed according to oecd guidelines3234 or international validation study guideline35 with the good laboratory practice ( glp ) . all animals used in this study were cared for in accordance with the guide for the care and use of laboratory animals issued by the animal care and use committee of the national veterinary research and quarantine service . sio2 colloids 20 nm ( sio2 ) and 100 nm ( sio2 ) in size were obtained from e&b nanotech co ltd ( gyeonggi - do , republic of korea ) . the preparation process and physicochemical characterizations were conducted as described in a previous report.31 briefly , a jsm-6700f field emission scanning electron microscope ( joel ltd , tokyo , japan ) and zetasizer nanozs ( malvern instruments ltd , malvern , uk ) were used to determine the particle size , morphology , and surface charge of sio2 . 2009 - 116 testing guideline and oecd testing guideline 471.32 the tester strains used in this study were ames salmonella typhimurium ta98 , ta100 , ta1535 , and ta1537 , and escherichia coli wp2uvra in the absence and presence of metabolic activation system . next , the mixture was incubated in a water bath for 20 minutes at 37c , mixed with top agar and a minimal amount of histidine - biotin ( for s. typhimurium strains ) or tryptophan ( for e. coli strain ) , and then poured onto the surface of a gamma - ray sterile falcon petri dish ( thermo fisher scientific , waltham , ma , usa ) containing about 15 ml of solidified bottom agar . all plates were prepared in triplicate , and the results were tabulated as the mean standard deviation for each condition . the in vitro chromosomal aberration test was performed according to the korea food and drug administration notification no . 2009 - 116 testing guideline and oecd testing guideline 473.33 the clastogenicity of sio2 nanoparticles was evaluated for its ability to induce chromosomal aberrations in chinese hamster lung ( chl ) fibroblast cells . the alkaline ( ph > 13 ) comet assay was conducted according to international validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens ( version 14.2).35 male crl : cd ( sprague dawley [ sd ] ) rats ( aged 6 weeks ) were used for the in vivo alkaline comet assay . after a 7-day acclimation period , the test substance was administered three times by gavage at 0 , 24 , and 45 hours . for each sample ( animal / tissue ) ,
a positive response is defined as a statistically significant change in the percent tail dna in at least one dose group in comparison with the vehicle control value using dunnett s test ( two - sided , p<0.05 ) as well as a statistically significant linear trend test ( two - sided , p<0.05 ) . the in vivo micronucleus test was performed in compliance with the korea food and drug administration notification no . this method of scoring , which must always be followed where the test substance markedly influences the proliferation rate in bone marrow , prevented distortion of the results by the influx of peripheral blood into the damaged marrow . the scoring of micronucleated normocytes was used to recognize the presence of artifacts ( which is rare in mouse preparations ) , which provided additional interesting information on the mode of action of the test substances . all animals used in this study were cared for in accordance with the guide for the care and use of laboratory animals issued by the animal care and use committee of the national veterinary research and quarantine service . sio2 colloids 20 nm ( sio2 ) and 100 nm ( sio2 ) in size were obtained from e&b nanotech co ltd ( gyeonggi - do , republic of korea ) . the preparation process and physicochemical characterizations were conducted as described in a previous report.31 briefly , a jsm-6700f field emission scanning electron microscope ( joel ltd , tokyo , japan ) and zetasizer nanozs ( malvern instruments ltd , malvern , uk ) were used to determine the particle size , morphology , and surface charge of sio2 . 2009 - 116 testing guideline and oecd testing guideline 471.32 the tester strains used in this study were ames salmonella typhimurium ta98 , ta100 , ta1535 , and ta1537 , and escherichia coli wp2uvra in the absence and presence of metabolic activation system . next , the mixture was incubated in a water bath for 20 minutes at 37c , mixed with top agar and a minimal amount of histidine - biotin ( for s. typhimurium strains ) or tryptophan ( for e. coli strain ) , and then poured onto the surface of a gamma - ray sterile falcon petri dish ( thermo fisher scientific , waltham , ma , usa ) containing about 15 ml of solidified bottom agar . all plates were prepared in triplicate , and the results were tabulated as the mean standard deviation for each condition . the in vitro chromosomal aberration test was performed according to the korea food and drug administration notification no . 2009 - 116 testing guideline and oecd testing guideline 473.33 the clastogenicity of sio2 nanoparticles was evaluated for its ability to induce chromosomal aberrations in chinese hamster lung ( chl ) fibroblast cells . the alkaline ( ph > 13 ) comet assay was conducted according to international validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens ( version 14.2).35 male crl : cd ( sprague dawley [ sd ] ) rats ( aged 6 weeks ) were used for the in vivo alkaline comet assay . after a 7-day acclimation period ,
the test substance was administered three times by gavage at 0 , 24 , and 45 hours . for each sample ( animal / tissue ) ,
a positive response is defined as a statistically significant change in the percent tail dna in at least one dose group in comparison with the vehicle control value using dunnett s test ( two - sided , p<0.05 ) as well as a statistically significant linear trend test ( two - sided , p<0.05 ) . the in vivo micronucleus test was performed in compliance with the korea food and drug administration notification no . this method of scoring , which must always be followed where the test substance markedly influences the proliferation rate in bone marrow , prevented distortion of the results by the influx of peripheral blood into the damaged marrow . the scoring of micronucleated normocytes was used to recognize the presence of artifacts ( which is rare in mouse preparations ) , which provided additional interesting information on the mode of action of the test substances . particle size and surface charge are significant factors when determining the biological behavior of nanoparticles.31 the physicochemical properties of sio2 nanoparticles were investigated in a previous study , as described briefly below.31 the average size and morphology of the sio2 nanoparticles was investigated by field emission scanning electron microscopy . in the preliminary dose - ranging tests ( data not shown ) , two kinds of sio2 nanoparticles , ie , sio2 and sio2 , were found to have a nontoxic effect in all tester strains of s. typhimurium ( ta98 , ta100 , ta1535 , and ta1537 ) and in e. coli wp2uvra at a dose of 5,000 g per plate in the presence and absence of s9 . as shown in table 1 , none of the tester strains showed any increase in the number of revertant colonies in comparison with the solvent control ( 0 g per plate ) when the bacteria were treated with the two types of sio2 nanoparticles at 313 , 625 , 1,250 , 2,500 , and 5,000 g per plate , regardless of metabolic activation . initially , a dose - ranging test was performed to determine the test doses for use in the in vitro chromosome aberration test . the in vitro chromosome aberration test was conducted with sio2 and sio2 at concentrations of 175 , 350 , 700 , and 1,400 g / ml , both with and without metabolic activation ( s9 ) . except for the positive control group ,
the results indicated no significant increase in sio2 nanoparticles in the treatment groups in comparison with the solvent control at the four concentrations , regardless of metabolic activation ( table 2 ) . a dose - ranging study was conducted in sd rats ( data not shown ) , and the test doses were determined as 500 , 1,000 , and 2,000 mg / kg body weight . dna damage was measured by the in vivo alkaline comet assay , following the international validation study protocol ( version 14.2).35 our data showed no significant differences in percent tail dna in single cells from the liver and stomach treated with sio2 ( figure 1a ) and sio2 ( figure 1b ) . therefore , no genotoxic effect of sio2 nanoparticles ( sio2 and sio2 ) was observed using the in vivo comet assay . therefore , both types of sio2 nanoparticles were regarded to have no genotoxic or cytotoxic potential using the current in vivo system . particle size and surface charge are significant factors when determining the biological behavior of nanoparticles.31 the physicochemical properties of sio2 nanoparticles were investigated in a previous study , as described briefly below.31 the average size and morphology of the sio2 nanoparticles was investigated by field emission scanning electron microscopy . in the preliminary dose - ranging tests ( data not shown ) , two kinds of sio2 nanoparticles , ie , sio2 and sio2 , were found to have a nontoxic effect in all tester strains of s. typhimurium ( ta98 , ta100 , ta1535 , and ta1537 ) and in e. coli wp2uvra at a dose of 5,000 g per plate in the presence and absence of s9 . as shown in table 1 , none of the tester strains showed any increase in the number of revertant colonies in comparison with the solvent control ( 0 g per plate ) when the bacteria were treated with the two types of sio2 nanoparticles at 313 , 625 , 1,250 , 2,500 , and 5,000 g per plate , regardless of metabolic activation . initially , a dose - ranging test was performed to determine the test doses for use in the in vitro chromosome aberration test . the in vitro chromosome aberration test was conducted with sio2 and sio2 at concentrations of 175 , 350 , 700 , and 1,400 g / ml , both with and without metabolic activation ( s9 ) . except for the positive control group ,
the results indicated no significant increase in sio2 nanoparticles in the treatment groups in comparison with the solvent control at the four concentrations , regardless of metabolic activation ( table 2 ) . a dose - ranging study was conducted in sd rats ( data not shown ) , and the test doses were determined as 500 , 1,000 , and 2,000 mg / kg body weight . dna damage was measured by the in vivo alkaline comet assay , following the international validation study protocol ( version 14.2).35 our data showed no significant differences in percent tail dna in single cells from the liver and stomach treated with sio2 ( figure 1a ) and sio2 ( figure 1b ) . therefore , no genotoxic effect of sio2 nanoparticles ( sio2 and sio2 ) was observed using the in vivo comet assay . therefore , both types of sio2 nanoparticles were regarded to have no genotoxic or cytotoxic potential using the current in vivo system . nanotoxicity studies have been necessary , although their results are still controversial due to variations in the physicochemical properties of the materials under investigation.1921,3739 further , their results could differ according to the protocol used for evaluation . hence , well characterized nanoparticles and approved methods are necessary when conducting nanotoxicity studies . in this study
, nanogenotoxicity was investigated using well characterized sio2 nanoparticles , ie , sio2 and sio2 . characterization of these nanoparticles and their behavior was done under physiological conditions , as described in our previous study.31 next , four different end point genotoxic tests , ie , the bacterial mutation assay , in vitro chromosomal aberration test , in vivo alkaline comet assay , and in vivo micronucleus test , were conducted in accordance with oecd guidelines and an international validation study protocol using the glp system . the bacterial mutation assay is used to evaluate the mutagenicity of a chemical compound.1 it is also reported to be an essential test within the current battery of assays required for evaluation of genotoxicity.40 to date , there have been a few reports on the mutagenicity of nanoparticles such as aluminum oxide , cobalt oxide , titanium oxide , and zinc oxide based on the bacterial mutation assay . hence , we performed this assay using strains of s. typhimurium ta98 , ta100 , ta1535 , and ta1537 , and e. coli wp2uvra , with and without metabolic activation by s9 mixture.4143 it has recently been reported that no mutagenic potential is observed in the ames test with and without metabolic activation.4446 in addition , li et al investigated the genotoxic potential of exfoliated silicate nanoclay.47 our data also showed no significantly increased mutagenicity in any of the strains exposed to sio2 and sio2 with and without s9 ( table 1 ) . the in vitro chromosomal aberration test is another standard genotoxicity test , and is commonly used to observe variations in chromosomal structure induced by toxic chemicals.33 our data show that sio2 and sio2 did not induce any structural chromosomal aberrations in chl cells , with or without s9 mixture ( table 2 ) . another previous report showed no induction of chromosomal aberrations in mammalian cells.4446 although different types of sio2 were used in the previous studies , similar results were obtained . in vitro genotoxicity tests
the in vivo alkaline comet assay and in vivo micronucleus test were included in the current study in order to conduct and evaluate genotoxicity tests accurately . the comet assay , a single cell gel electrophoresis assay , has been widely used for detection of dna damage due to its simplicity , low cost , and high sensitivity.48 the in vivo comet assay has been regarded as a potential replacement for the in vivo rodent hepatocyte unscheduled dna synthesis assay , and the protocol for the in vivo comet assay has been standardized and published by an international expert group . in the present study , the in vivo comet assay was conducted using sio2 and sio2 and followed the standard protocol , revealing no genotoxic effect of sio2 and sio2 in rat liver and stomach cells ( figure 1a and 1b ) . even though the majority of the studies have reported negative results for the genotoxicity of sio2,11,17,47 a few investigations have indicated otherwise.49 downs et al investigated the genotoxic effects of exposure to three consecutive intravenous injections of sio2 nanoparticles by in vivo comet assay in rat liver and lung tissues , and observed the induction of dna damage by sio2.50 hence , the different administration route can lead to different results for genotoxicity evaluation . regarding the genotoxicity induced by sio2 nanoparticles , several publications have reported on in vitro micronucleus tests.11,15,47,51,52 in contrast ,
downs et al conducted in vivo micronucleus testing of sio2 nanoparticles , where they analyzed micronuclei in rat blood.50 in the present study , in vivo micronucleus testing was performed with rat bone marrow cells for the first time to determine whether sio2 and sio2 could induce chromosome abnormalities or mitotic apparatus in mouse model . nanotoxicology study results can be influenced by physicochemical properties , and the contrast between our results and the previous data could be due to the different administration methods used or different organs investigated , as mentioned above . in the present study , the genotoxic effects of sio2 and sio2 were elucidated using four genotoxicity tests , ie , the bacterial mutation assay , in vitro chromosomal aberration test , in vivo comet assay , and in vivo micronucleus test , under standardized protocols with glp system . although the different exposure routes from our study can induce sio2 genotoxicity in different organs in in vivo systems , our data suggest that sio2 and sio2 are not genotoxic substances based on the oecd test guidelines . | [
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] |
nuclear magnetic resonance spectra
were recorded on a 400 mhz nmr instrument at rt .
all h
nmr spectra were measured in parts per million ( ppm ) relative to the
signals for residual dmso in dmso - d6 ( 2.50
ppm ) .
data for h nmr were
reported as follows : chemical shift , multiplicity ( s = singlet , d
= doublet , t = triplet , q = quartet , qu = quintet , sex = sextet , m
= multiplet , ovrlp = overlap , br = broad ) , coupling constants , and
integration .
all c nmr spectra were reported in ppm relative
to dmso - d6 ( 39.52 ppm ) .
tbubrettphos ( l5 ) , pd precatalyst 1 , and 3-chloro-1-trityl - indazole ( s1 ) were prepared according to the literature procedures . in a nitrogen - filled glovebox ,
an oven - dried 25
ml round - bottom flask ( a ) capped with a rubber septum was charged
with csohh2o ( 16.7 g , 99.5 mmol ) .
the flask
was then evacuated and backfilled with argon ( this process was repeated
a total of three times ) , and degassed deionized water ( 4.17 ml , 232
mmol ) was then added into the flask .
the resulting reaction mixture
was agitated until all csohh2o dissolved
to form a homogeneous solution .
the mole ratio of csoh and h2o in the
standard csoh solution is 99.5 mmol : [ 232 mmol ( from water added )
+ 99.5 mmol ( from csohh2o ) ] = 99.5 mmol :
331.5 mmol = 3:10 ( or 1:3.33 ) .
thus , the mole ratio of csoh to water
in the standard csoh solution is always kept at 3:10 for simultaneous
transfers of both csoh and water in a desired mole ratio . following the procedure of ( i ) , the standard csoh
solution ( mole ratio of csoh : h2o = 3:20 ) was prepared using
csohh2o ( 520 mg , 3.1 mmol ) and deionized
water ( 0.32 ml , 17.8 mmol ) .
following
the procedure of ( i ) , the standard csoh
solution ( mole ratio of csoh : h2o = 3:30 ) was prepared using
csohh2o ( 520 mg , 3.1 mmol ) and deionized
water ( 0.50 ml , 27.8 mmol ) . an oven - dried
10 ml resealable screw - cap
test tube ( a ) equipped with a teflon - coated magnetic
stir bar
was charged with l5 ( 2.4 mg , 0.005 mmol , 2 mol
% ) and koh ( 24 equiv , 28.156.1 mg ) .
tube a was evacuated and backfilled with argon ( this process was repeated
a total of three times ) , and 4-chloroanisole ( 36 mg , 31 l ,
0.25 mmol , 1 equiv ) and degassed deionized water ( 20111 equiv ,
90 l to 0.50 ml ) were then added into tube a via
syringe .
a second oven - dried 10 ml resealable screw - cap test tube
( b ) equipped with a teflon - coated magnetic stir bar was
charged with 1 ( 4.3 mg , 0.005 mmol , 2 mol % ) .
tube b was then evacuated and backfilled with argon ( this process
was repeated a total of three times ) , and 1,4-dioxane ( 0.50 ml ) was
added into tube b via syringe . the reaction mixture in
tube b
was stirred at rt for 1 min to form a
homogeneous solution . the precatalyst solution from tube b
was transferred into tube a via syringe . the resulting
reaction mixture in tube a was stirred at 50 or 80 c
in an oil bath for 20 h. after cooling to rt
, the
crude product was diluted with etoac ( 2 ml ) , 1,4-dimethoxybenzene
( 34.6 mg , 0.25 mmol , 1.0 equiv ) was added , and the mixture was then
acidified with aqueous hcl solution ( 1 m , 2 ml ) .
the resulting reaction
mixture in the capped test tube was agitated until all of the solid
was dissolved into the reaction mixture .
the reaction mixture was
then neutralized with saturated nahco3 solution ( 2 ml ) .
a small fraction of the upper organic layer was filtered through a
plug of silica gel and then subjected to gc analysis to determine
the reaction conversion and the gc yields of product and 4-methoxyphenol ,
using 1,4-dimethoxybenzene as an internal standard . an oven - dried 10 ml resealable screw - cap
test tube ( a ) equipped with a teflon - coated magnetic
stir bar
was charged with l5 ( 2.4 mg , 0.005 mmol , 2 mol
% ) [ except for table 2 , entries 1 and 5 , in
which the tube was then transferred into the n2-filled
glovebox , and csohh2o ( 126 mg , 0.75 mmol , 3 equiv )
was added ] .
tube a was evacuated and backfilled with
argon ( this process was repeated a total of three times ) , and 1-bromo-2,4-dimethylbenzene
( 46.3 mg , 34 l , 0.25 mmol ) and standard aqueous csoh solution
prepared in the preceding procedures were then added into tube a via syringe ( except for table 2 ,
entries 1 and 5 ) [ for table 2 , entries 2 and
6 : csoh : h2o = 3 equiv:10 equiv ; csoh ( 112.4 mg , 0.75 mmol , 3 equiv )
dissolved in deionized water ( 45 mg , 2.5 mmol , 10 equiv ) . for table 2 , entry 3 : csoh : h2o = 3 equiv:20 equiv ;
csoh ( 112.4 mg , 0.75 mmol , 3 equiv ) dissolved in deionized water ( 90
mg , 5.0 mmol , 20 equiv ) . for table 2 , entry
4 : csoh : h2o = 3 equiv:30 equiv ; csoh ( 112.4 mg , 0.75 mmol ,
3 equiv ) dissolved in deionized water ( 135 mg , 7.5 mmol , 30 equiv ) ] .
a second oven - dried 10 ml resealable screw - cap test tube ( b ) equipped with a teflon - coated magnetic stir bar was charged with 1 ( 4.3 mg , 0.005 mmol , 2 mol % ) .
tube b was then
evacuated and backfilled with argon ( this process was repeated a total
of three times ) , and the solvent ( 1,4-dioxane or thf , 0.50 ml ) was
added into tube b via syringe . the reaction mixture in
tube b
was stirred at rt for 1 min to form a
homogeneous solution . the precatalyst solution from tube b
was transferred into tube a via syringe . the resulting
reaction mixture in tube a was stirred at rt for 20 h.
the crude
product
was diluted with etoac ( 2 ml ) , 1,3,5-trimethoxybenzene ( 42.1
mg , 0.25 mmol , 1.0 equiv ) was added , and the mixture was then acidified
with aqueous hcl solution ( 1 m , 2 ml ) .
the resulting reaction mixture
in the capped test tube was agitated until all of the solid was dissolved .
a small fraction of the upper organic layer
was filtered through a plug of silica gel and then subjected to gc
analysis to determine the reaction conversion and the gc yields of
product and 2,4-dimethylphenol , using 1,3,5-trimethoxybenzene as an
internal standard . an oven - dried 20 ml resealable
screw - cap test tube ( a ) equipped with a teflon - coated
magnetic stir bar
was charged with l5 ( 9.7 mg , 0.020
mmol , 2 mol % ) , koh ( 168.3 mg , 3.0 mmol , 3 equiv ) , and ( hetero)aryl
halide ( if solid ) ( 1.0 mmol , 1 equiv ) .
tube a was evacuated
and backfilled with argon ( this process was repeated a total of three
times ) , and degassed deionized water ( 0.36 ml , 20.0 mmol , 20 equiv )
and ( hetero)aryl halide ( if liquid ) ( 1.0 mmol , 1 equiv ) were
then added into tube a via syringe .
a second oven - dried
10 ml resealable screw - cap test tube ( b ) equipped with
a teflon - coated magnetic stir bar was charged with 1 ( 17.1
mg , 0.020 mmol , 2 mol % ) .
tube b was then evacuated and
backfilled with argon ( this process was repeated a total of three
times ) , and 1,4-dioxane ( 2.0 ml ) was added into tube b via syringe .
the reaction mixture in tube b was stirred
at rt for 1 min to form a homogeneous solution .
the precatalyst
solution from tube b was transferred into tube a via syringe . the resulting reaction mixture in tube a was stirred at 80 c in an oil bath for 18 h. after
cooling to rt
, the crude product was diluted with etoac ( 5 ml ) and
then acidified with aqueous hcl solution ( 1 m , 5 ml ) .
the resulting
reaction mixture in the capped test tube was agitated until all of
the solid was dissolved .
the reaction mixture was transferred into
a separatory funnel and then neutralized with saturated nahco3 solution ( 5 ml ) .
the organic fraction was isolated , and the
aqueous fraction was rinsed with etoac ( 2 10 ml ) .
the
crude product residue was purified by flash chromatography using a
solvent mixture ( etoac / hexanes / methanol ) as an eluent to afford the
isolated product .
an oven - dried
20 ml resealable
screw - cap test tube ( a ) equipped with a teflon - coated
magnetic stir bar was charged with l5 ( 9.7 mg , 0.020
mmol , 2 mol % ) and ( hetero)aryl halide ( if solid ) ( 1.0 mmol ,
1 equiv ) . tube a was evacuated and backfilled with argon
( this process was repeated a total of three times ) , and standard aqueous
csoh solution [ mole ratio of csoh to h2o = 3:10 ; csoh ( 450
mg , 3.0 mmol , 3 equiv ) dissolved in deionized water ( 180 mg , 10.0
mmol , 10 equiv ) as prepared in the preceding procedures ] was then
added into tube a via syringe , followed by the addition
of ( hetero)aryl halide ( if liquid ) ( 1.0 mmol , 1 equiv ) . a second
oven - dried 10 ml resealable screw - cap test tube ( b ) equipped
with a teflon - coated magnetic stir bar was charged with 1 ( 17.1 mg , 0.020 mmol , 2 mol % ) .
tube b was then evacuated
and backfilled with argon ( this process was repeated a total of three
times ) , and 1,4-dioxane ( 2.0 ml ) was added into tube b via syringe . the reaction mixture in tube b
was stirred
at rt for 1 min to form a homogeneous solution . the precatalyst
solution from tube b
was transferred into tube a via syringe . the resulting reaction mixture in tube a was stirred at rt for 18 h.
the crude product was diluted
with etoac ( 5 ml ) and then acidified with aqueous hcl solution ( 1
m , 5 ml ) .
the resulting reaction mixture in the capped test tube was
agitated until all of the solid was dissolved .
the reaction mixture
was transferred into a separatory funnel and then neutralized with
saturated nahco3 solution ( 5 ml ) .
the organic fraction
was isolated , and the aqueous fraction was rinsed with etoac ( 2
10 ml ) .
the crude product residue was purified by flash chromatography
using a solvent mixture ( etoac / hexanes ) as an eluent to afford the
isolated product .
the reported yields are of the isolated product
and averages of two runs . following general procedure
a , the title compound was prepared using 10-n - butyl-2-chloroacridin-9(10h)-one ( 286 mg , 1.0 mmol ) ; etoac / hexanes ( 1:3 ) , then etoac ;
263 mg , 98% ; sunset - yellow solid . m.p . :
239240 c . h nmr ( 400 mhz , dmso - d6 ) :
9.70 ( br s , 1 h ) , 8.32 ( dd , j = 8.6 hz , j = 1.2 hz , 1 h ) , 7.787.71 ( ovrlp , 3 h ) , 7.70 ( d , j = 4.0 hz , 1 h ) , 7.35 ( dd , j = 9.2 hz , j = 2.8 hz , 1 h ) , 7.25 ( ddd , j = 8.0 hz , j = 6.0 hz , j = 1.6 hz , 1 h ) , 4.41 ( t , j = 7.6 hz , 2 h ) , 1.74 ( qu , j = 7.6 hz ,
2 h ) , 1.49 ( sex , j = 7.6 hz , 2 h ) , 0.96 ( t , j = 7.6 hz , 3 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 175.9 , 152.0 , 141.0 , 135.2 , 133.7 ,
126.7 , 124.1 , 122.8 , 120.6 , 120.4 , 117.5 , 115.5 , 109.2 , 45.0 , 29.1 ,
19.4 , 13.7
ir ( neat cm ) 3278 , 1591 , 1568 , 1499 , 1460 , 1357 , 1270 , 1229 ,
1171 , 861 , 810 , 752 , 684 , 638 . following general
procedure
a , the title compound was prepared using 2-chloro-9h - thioxanthen-9-one ( 247 mg , 1.0 mmol ) ; etoac / hexanes ( 1:4 ) ,
then etoac ; 224 mg , 98% ; sunset - yellow solid . m.p .
h nmr ( 400 mhz , dmso - d6 ) : 10.16 ( br s , 1 h ) , 8.45 ( dd , j = 8.4
hz , j = 1.2 hz , 1 h ) , 7.86 ( d , j = 2.8 hz , 1 h ) , 7.79 ( dd , j = 8.0 hz , j = 0.8 hz , 1 h ) , 7.73 ( ddd , j = 8.4 hz , j = 6.8 hz , j = 1.6 hz , 1 h ) , 7.67 ( d , j = 8.8 hz , 1 h ) , 7.54 ( ddd , j = 8.4 hz , j = 7.2 hz , j = 1.2 hz , 1 h ) , 7.27 ( dd , j = 8.8 hz , j = 2.8 hz , 1 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 178.6 ,
156.5 , 137.0 , 132.6 , 129.6 , 129.0 , 128.0 , 127.7 , 126.5 , 126.3 , 126.1 ,
122.7 , 113.2 . anal .
calcd for c13h8o2s : c , 68.40 ; h , 3.53 . found : c , 68.14 ; h , 3.55 .
ir ( neat cm ) 3303 , 1620 , 1568 , 1484 , 1436 , 1341 , 1216 , 1150 ,
1121 , 1075 , 814 , 736 , 633 . following general
procedure a
, the
title compound was prepared using 2-(4-chlorophenyl)benzothiazole
( 246 mg , 1.0 mmol ) ; etoac / hexanes ( 1:6 ) , then ( 1:4 ) ; 223 mg , 98% ;
pale - yellow solid . m.p . :
227229 c ( lit : 228229
c).h nmr ( 400 mhz ,
dmso - d6 ) : 10.23 ( br s , 1 h ) , 8.05
( dd , j = 7.6 hz , 1 h ) , 7.98 ( dd , j = 8.0 hz , 1 h ) , 7.93 ( d , j = 8.4 hz , 2 h ) , 7.49
( td , j = 8.0 hz , j = 1.2 hz , 1 h ) ,
7.39 ( td , j = 8.0 hz , j = 1.2 hz ,
1 h ) , 6.94 ( d , j = 8.8 hz , 2 h ) .
c nmr
( 100 mhz , dmso - d6 ) : 167.5 , 160.6 ,
153.7 , 134.1 , 129.1 , 126.4 , 124.9 , 124.1 , 122.3 , 122.1 , 116.1 .
calcd for c13h9nos : c , 68.70 ; h , 3.99 .
found : c , 68.83 h , 4.13 .
ir ( neat cm ) 1604 , 1480 ,
1429 , 1283 , 1224 , 1167 , 977 , 826 , 756 , 723 . following
general procedure
a , the
title compound was prepared using 1-bromo-4-methoxy-2,5-dimethylbenzene
( 215.1 mg , 1.0 mmol ) ; etoac / hexanes ( 1:6 ) ; 127 mg , 83% ; white solid .
m.p . : 8990 c ( lit : 8688 c).h nmr ( 400 mhz , dmso - d6 ) : 8.58 ( br s , 1 h ) , 6.62 ( s , 1 h ) , 6.55 ( s ,
1 h ) , 3.66 ( s , 3 h ) , 2.08 ( s , 3 h ) , 2.03 ( s , 3 h ) . c
nmr ( 100 mhz , dmso - d6 ) : 150.0 ,
148.5 , 123.1 , 121.1 , 117.1 , 113.1 , 55.6 , 16.0 , 15.7 .
hrms ( esi ) calcd
for c9h13o2 [ m + h ] : 153.0910 . found :
153.0915 .
ir ( neat cm ) 3238 , 1519 , 1452 , 1412 ,
1198 , 1103 , 1018 , 864 , 834 , 671 . following general
procedure
a , the title compound was prepared
using 2-bromo-1,3-dimethoxybenzene ( 217 mg , 1.0 mmol ) ; etoac / hexanes
( 1:6 ) , then ( 1:4 ) ; 134 mg , 86% ; pale - brown solid . m.p .
: 5657
c ( lit : 5456 c).h nmr ( 400 mhz , dmso - d6 ) :
8.25 ( br s , 1 h ) , 6.69 ( t , j = 8.4 hz , 1 h ) , 6.59
( d , j = 8.4 hz , 2 h ) , 3.74 ( s , 6 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 148.2 ,
135.7 , 118.1 , 105.7 , 55.9 .
hrms ( esi ) calcd for c8h11o3 [ m + h ] : 155.0703 . found : 155.0706 .
ir ( neat
cm ) 3452 , 1612 , 1508 , 1478 , 1438 , 1360 , 1279 ,
1210 , 1097 , 1027 , 825 , 767 , 726 , 706 , 609 .
following general
procedure a , the title compound was prepared
using 1-bromonaphthalene ( 207 mg , 1.0 mmol ) ; etoac / hexanes ( 1:6 ) ;
133 mg , 92% ; pale - brown solid . m.p
: 95 c ( lit : 9193
c).h nmr ( 400 mhz ,
dmso - d6 ) : 10.10 ( br s , 1 h ) , 8.14
( dd , j = 7.2 hz , j = 1.2 hz , 1 h ) ,
7.80 ( dd , j = 7.2 hz , j = 1.6 hz ,
1 h ) , 7.487.41 ( ovrlp , 2 h ) , 7.347.28 ( ovrlp , 2 h ) ,
6.88 ( dd , j = 7.2 hz , j = 1.6 hz ,
1 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 153.2 , 134.4 , 127.4 , 126.4 , 126.1 , 124.6 , 124.5 , 122.0 ,
118.3 , 108.0 .
hrms ( esi ) calcd for c10h9o [ m
+ h ] : 145.0648 . found : 145.0646 .
ir ( neat cm )
3277 , 1598 , 1457 , 1385 , 1268 , 1148 , 1083 , 1043 , 1015 , 876 , 860 , 789 ,
764 , 710 . following
general procedure
a , the title compound was prepared using 1-bromo-2,3-dimethoxynaphthalene
( 267 mg , 1.0 mmol ) ; etoac / hexanes ( 1:6 ) ; 202 mg , 99% ; yellow oil .
h nmr ( 400 mhz , dmso - d6 ) :
9.53 ( br s , 1 h ) , 8.01 ( d , j = 8.4 hz , 1 h ) , 7.69
( d , j = 8.0 hz , 1 h ) , 7.35 ( ddd , j = 8.0 hz , j = 6.8 hz , j = 1.2
hz , 1 h ) , 7.28 ( ddd , j = 8.4 hz , j = 6.8 hz , j = 1.2 hz , 1 h ) , 6.87 ( s , 1 h ) , 3.89
( s , 3 h ) , 3.77 ( s , 3 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 152.7 , 144.6 , 133.8 , 130.7 , 126.3 ,
125.5 , 122.5 , 121.6 , 121.1 , 98.2 , 60.5 , 55.5 .
hrms ( esi ) calcd for
c12h13o3 [ m + h ] : 205.0859 . found :
205.0852 .
ir ( neat cm ) 3500 , 1739 , 1635 , 1600 ,
1509 , 1476 , 1295 , 1265 , 1231 , 1195 , 1120 , 1095 , 1032 , 1015 , 975 , 859 ,
812 , 735 , 700 . following general procedure a ,
the
title compound was prepared
using 6-bromoquinoxaline ( 209 mg , 1.0 mmol ) ; etoac / meoh ( 20:1 ) ; 130.5
mg , 89% ; brown solid . m.p .
: 251253 c ( lit : 252254
c).h nmr ( 400 mhz ,
dmso - d6 ) : 10.53 ( br s , 1 h ) , 8.77
( d , j = 2.0 hz , 1 h ) , 8.68 ( d , j = 1.6 hz , 1 h ) , 7.93 ( d , j = 9.2 hz , 1 h ) , 7.41
( dd , j = 9.2 hz , j = 2.8 hz , 1 h ) ,
7.27 ( d , j = 2.8 hz , 1 h ) .
c nmr ( 100
mhz , dmso - d6 ) : 158.8 , 145.4 , 144.0 ,
142.2 , 137.6 , 130.4 , 122.9 , 109.5 . anal .
calcd for c8h6n2o : c , 65.75 ; h , 4.14 . found : c , 65.63 ;
h , 4.12 . hrms ( esi ) calcd for c8h7n2o [ m + h ] : 147.0553 .
ir ( neat cm ) 1610 , 1478 , 1409 , 1304 , 1231 , 1197 , 1117 , 1036 , 941 , 831 , 750 . following general procedure a , the title compound was prepared
using 1-benzyl-6-chloro-1h - indole ( 241 mg , 1.0 mmol ) ;
etoac / hexanes ( 1:4 ) ; 182.1 mg , 82% ; pale - brown solid .
dmso - d6 ) : 8.99
( br s , 1 h ) , 7.35 ( d , j = 8.4 hz , 1 h ) , 7.30 ( t , j = 7.2 hz , 2 h ) , 7.26 ( d , j = 3.2 hz ,
1 h ) , 7.24 ( t , j = 7.2 hz , 1 h ) , 7.15 ( d , j = hz , 2 h ) , 6.73 ( d , j = 2.0 hz , 1 h ) ,
6.60 ( dd , j = 8.4 hz , j = 2.0 hz ,
1 h ) , 6.36 ( dd , j = 3.2 hz , j =
0.4 hz , 1 h ) , 5.29 ( s , 2 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 153.2 , 138.4 , 136.9 , 128.5 , 127.3 ,
127.2 , 126.8 , 121.6 , 120.9 , 109.7 , 100.8 , 95.4 , 49.1 .
hrms ( esi ) calcd for c15h12no
[ m h ] : 222.0924 . found : 222.0941 .
ir ( neat cm ) 3308 , 1625 , 1580 , 1507 , 1468 , 1354 , 1321 , 1267 , 1168 , 1074 , 943 ,
827 , 799 , 745 , 705 . following general procedure
a , the
title compound was prepared
using 5-chlorobenzothiophene ( 169 mg , 1.0 mmol ) at 100 c ; etoac / hexanes
( 1:6 ) ; 143.6 mg , 96% ; off - white solid .
h nmr ( 400 mhz ,
dmso - d6 ) : 9.42 ( br s , 1 h ) , 7.74
( d , j = 8.8 hz , 1 h ) , 7.65 ( d , j = 5.6 hz , 1 h ) , 7.27 ( dd , j = 5.6 hz , j = 0.4 hz , 1 h ) , 7.21 ( d , j = 2.0 hz , 1 h ) , 6.88
( ddd , j = 8.4 hz , j = 2.0 hz , j = 0.4 hz , 1 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 154.9 , 140.9 , 129.4 , 128.0 , 123.5 ,
123.1 , 114.8 , 108.2 .
hrms ( esi ) calcd for c8h6os [ m ] : 150.0134 . found : 150.0133 .
ir ( neat
cm ) 3235 , 1599 , 1565 , 1502 , 1422 , 1231 , 1149 ,
945 , 889 , 849 , 748 , 692 . following general procedure a , the
title compound was prepared
using 7-chlorobenzofuran ( 153 mg , 1.0 mmol ) ; etoac / hexanes ( 1:4 ) ;
125 mg , 93% ; pale - yellow oil .
h nmr ( 400 mhz , dmso - d6 ) : 9.96 ( br s , 1 h ) , 7.91 ( d , j = 2.0 hz , 1 h ) , 7.06 ( d , j = 7.6 hz ,
1 h ) , 7.02 ( t , j = 7.6 hz , 1 h ) , 6.87 ( d , j = 2.0 hz , 1 h ) , 6.74 ( d , j = 7.6 hz ,
1 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 145.4 , 143.4 , 142.8 , 129.1 , 123.5 , 111.6 , 110.3 , 107.0 .
hrms ( esi ) calcd for c8h7o2 [ m +
h ] : 135.0441 . found : 135.0440 .
ir ( neat cm ) 3323 ,
1593 , 1443 , 1297 , 1217 , 1122 , 841 . following
general procedure
a , the title compound was prepared using 5-chlorobenzo-2 - 1 - 3-thiadiazole
( 171 mg , 1.0 mmol ) ; etoac / hexanes ( 1:6 ) ; 145 mg , 95% ; pale - yellow
solid .
h nmr ( 400 mhz , dmso - d6 ) : 10.55 ( br s , 1 h ) , 7.90 ( d , j = 9.2 hz ,
1 h ) , 7.33 ( dd , j = 9.2 hz , j =
2.4 hz , 1 h ) , 7.15 ( dd , j = 2.4 hz , j = 0.4 hz , 1 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 159.3 , 156.0 , 150.1 , 125.2 , 121.5 , 100.4 .
hrms ( esi ) calcd for c6h5n2os [ m + h ] : 153.0117 . found : 153.0114 .
ir ( neat cm ) 3133 , 1613 , 1495 , 1403 , 1226 , 1187 ,
1127 , 874 , 833 , 814 , 753 , 664 , 626 . following general
procedure a , the title compound was prepared
using 2-bromodibenzothiophene ( 263 mg , 1.0 mmol ) .
after work up , the
crude product was purified by flash chromatography using etoac / hexanes
( 1:6 ) as an eluent to afford dibenzothiophen-2-ol ( 3 m ) ( 185 mg , 92% ) as a pale - brown solid .
h nmr ( 400 mhz ,
dmso - d6 ) : 9.68 ( br s , 1 h ) , 8.238.18
( m , 1 h ) , 7.977.91 ( m , 1 h ) , 7.78 ( d , j =
8.8 hz , 1 h ) , 7.68 ( d , j = 2.4 hz , 1 h ) , 7.497.42
( ovrlp , 2 h ) , 7.03 ( dd , j = 8.4 hz , j = 2.4 hz , 1 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 155.4 , 139.7 , 136.3 , 135.0 , 128.5 , 126.8 ,
124.4 , 123.6 , 123.0 , 121.9 , 116.7 , 107.4 .
ir ( neat cm ) 3385 , 1602 , 1470 , 1427 ,
1336 , 1187 , 849 , 810 , 755 , 733 . following general procedure
a , the title compound was prepared
using 3-chloro-1-trityl-1h - indazole ( s1 ) ( 395 mg , 1.0 mmol ) ; etoac / hexanes ( 1:6 ) ; 297 mg , 0.79 mmol , 79% ;
pale - brown solid . m.p . :
nmr
( 400 mhz , dmso - d6 ) : 10.98 ( s ,
1 h ) , 7.58 ( d , j = 8.0 hz , 1 h ) , 7.36 ( d , j = 7.2 hz , 6 h ) , 7.28 ( t , j = 7.2 hz ,
6 h ) , 7.22 ( t , j = 7.2 hz , 3 h ) , 7.00 ( t , j = 7.2 hz , 1 h ) , 6.93 ( t , j = 7.2 hz ,
1 h ) , 6.40 ( d , j = 8.4 hz , 1 h ) . c nmr
( 100 mhz , dmso - d6 ) : 144.9 , 133.2 ,
132.7 , 119.2 , 117.6 , 116.9 , 116.3 , 110.1 , 109.5 , 105.8 , 103.6 , 67.0 .
hrms ( esi ) calcd for c26h19n2o [ m h ] : 375.1503 . found : 375.1507 .
ir ( neat cm ) 1671 , 1489 , 1450 , 1217 , 1001 , 906 , 746 , 705 , 632 . following
general procedure b ,
the title compound was prepared
using 1-bromo-2,4-dimethylbenzene ( 185 mg , 1.0 mmol ) ; etoac / hexanes
( 1:6 ) ; 87 mg , 71% ) ; volatile , pale - brown oil .
the h nmr
yield of the title product was determined to be 96% based on 0.25
mmol arbr .
h nmr ( 400 mhz , dmso - d6 ) : 8.93 ( br s , 1 h ) , 6.84 ( s , 1 h ) , 6.76 ( dd , j = 8.0 hz , j = 1.6 hz , 1 h ) , 6.64 ( d , j = 8.0 hz , 1 h ) , 2.15 ( s , 3 h ) , 2.07 ( s , 3 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 153.0 ,
131.1 , 127.0 , 126.8 , 123.4 , 114.4 , 20.1 , 15.9 . hrms ( esi )
ir ( neat
cm ) 3377 , 2921 , 1506 , 1325 , 1262 , 1204 , 1117 ,
929 , 808 , 766 . following
general
procedure b ,
the title compound was prepared using 3-bromo-4-methylacetophenone
( 213 mg , 1.0 mmol ) .
etoac / hexanes ( 1:2 ) ; 150 mg , 100% ; off - white solid .
m.p . : 121122 c ( lit : 119120 c).h nmr ( 400 mhz , dmso - d6 ) : 9.68 ( br s , 1 h ) , 7.347.32 ( ovrlp ,
2 h ) , 7.18 ( d , j = 8.0 hz , 1 h ) , 2.48 ( s , 3 h ) , 2.17
( s , 3 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 197.4 , 155.5 , 135.9 , 130.7 , 130.3 , 119.6 , 113.2 ,
26.5 , 16.2 .
hrms ( esi ) calcd for c9h11o2 [ m + h ] : 151.0754 . found : 151.0752 .
ir ( neat cm ) 3404 , 1659 , 1580 , 1418 , 1349 , 1285 , 1238 , 1129 , 899 , 884 , 813 ,
712 , 688 , 608 . following general procedure b ,
the
title compound
was prepared using 5-bromo-1,2,3-trimethoxybenzene ( 247 mg , 1.0 mmol ) , 1 ( 25.7 mg , 0.03 mmol , 3 mol % ) , and l5 ( 14.6
mg , 0.03 mmol , 3 mol % ) ; etoac / hexanes ( 1:1 ) ; 168 mg , 91% ; pale - brown
solid . m.p . :
147148 c ( lit : 146147 c).h nmr ( 400 mhz , dmso - d6 ) : 9.19 ( br s , 1 h ) , 6.05 ( s , 2 h ) , 3.69 ( s ,
6 h ) , 3.55 ( s , 3 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 153.8 , 153.3 , 130.3 , 92.8 , 60.1 , 55.5 .
hrms
( esi ) calcd for c9h11o4 [ m
h ] : 183.0663 . found : 183.0671 .
ir ( neat cm ) 3254 ,
1599 , 1428 , 1218 , 1177 , 1121 , 990 , 811 , 777 . following
general procedure b ,
the title compound was prepared using 4-bromo - n , n - diethylbenzamide ( 256 mg , 1.0 mmol ) ;
etoac / hexanes ( 3:1 ) ; 173 mg , 89% ; pale - brown solid . m.p . :
121123
c ( lit : 120 c).h nmr ( 400 mhz , dmso - d6 ) : 9.74
( br s , 1 h ) , 7.18 ( d , j = 8.4 hz , 2 h ) , 6.78 ( d , j = 8.8 hz , 2 h ) , 3.30 ( br s , 4 h ) , 1.08 ( t , j = 7.2 hz , 6 h ) .
c nmr ( 100 mhz , dmso - d6 ) : 170.2 , 158.2 , 128.1 , 127.7 , 114.8 , 13.5 .
hrms
( esi ) calcd for c11h14no2 [ m
h ] : 192.1030 . found : 192.1039 .
ir ( neat cm ) 3076 , 1570 , 1434 , 1361 , 1316 , 1277 , 1244 , 1170 , 1101 , 837 , 764 ,
597 . following general procedure b ,
the title compound was prepared
using 4-bromobenzophenone ( 261 mg , 1.0 mmol ) ; etoac / hexanes ( 1:4 ) ;
196 mg , 99% ; pale - brown solid . m.p . :
133134 c ( lit :
133.5134.5 c).h nmr ( 400 mhz , dmso - d6 ) : 10.45
( br s , 1 h ) , 7.687.64 ( ovrlp , 4 h ) , 7.62 ( t , j = 7.2 hz , 1 h ) , 7.52 ( t , j = 7.2 hz , 2 h ) , 6.90
( d , j = 8.4 hz , 2 h ) .
c nmr ( 100 mhz ,
dmso - d6 ) : 194.3 , 162.0 , 138.1 ,
132.5 , 131.8 , 129.1 , 128.4 , 127.9 , 115.3 . hrms
( esi ) calcd for c13h9o2 [ m h ] : 197.0608 . found :
197.0609 .
ir ( neat cm ) 3137 , 1632 , 1599 , 1557 ,
1512 , 1444 , 1312 , 1286 , 1232 , 1172 , 1148 , 939 , 924 , 848 , 740 , 694 ,
608 . following general procedure b ,
the title compound was prepared
using 4-bromobenzonitrile ( 364 mg , 2.0 mmol , 2 equiv ) , 1 ( 68.4 mg , 0.08 mmol , 4 mol % ) , l5 ( 38.8 mg , 0.08 mmol ,
4 mol % ) , aqueous csoh solution [ csoh ( 900 mg , 6.0 mmol , 6 equiv )
dissolved in deionized water ( 360 mg , 20.0 mmol , 20 equiv ) as prepared
in the preceding procedures ] , and 1,4-dioxane ( 4.0 ml ) ; etoac / hexanes
( 1:2 ) ; 230 mg , 97% ; pale - brown solid . m.p . : 109110 c
( lit . :
111112 c).h nmr ( 400 mhz , dmso - d6 ) :
10.61 ( br s , 1 h ) , 7.62 ( d , j = 9.2 hz , 2 h ) , 6.90
( d , j = 8.8 hz , 2 h ) .
c nmr ( 100 mhz ,
dmso - d6 ) : 161.7 , 134.2 , 119.6 ,
116.4 , 101.0 .
hrms ( esi ) calcd for c7h9n2o [ m + nh4 ] : 137.0709 . found : 137.0709 .
ir ( neat
cm ) 3263 , 2230 , 1585 , 1507 , 1438 , 1365 , 1281 ,
1219 , 1165 , 835 , 750 , 701 . | a method
for the hydroxylation of aryl and heteroaryl halides ,
promoted by a catalyst based on a biarylphosphine ligand tbubrettphos ( l5 ) and its corresponding palladium
precatalyst ( 1 ) , is described .
the reactions allow the
cross - coupling of both potassium and cesium hydroxides with ( hetero)aryl
halides to afford a variety of phenols and hydroxylated heteroarenes
in high to excellent yield . | Experimental
Section | nuclear magnetic resonance spectra
were recorded on a 400 mhz nmr instrument at rt . data for h nmr were
reported as follows : chemical shift , multiplicity ( s = singlet , d
= doublet , t = triplet , q = quartet , qu = quintet , sex = sextet , m
= multiplet , ovrlp = overlap , br = broad ) , coupling constants , and
integration . tbubrettphos ( l5 ) , pd precatalyst 1 , and 3-chloro-1-trityl - indazole ( s1 ) were prepared according to the literature procedures . the flask
was then evacuated and backfilled with argon ( this process was repeated
a total of three times ) , and degassed deionized water ( 4.17 ml , 232
mmol ) was then added into the flask . thus , the mole ratio of csoh to water
in the standard csoh solution is always kept at 3:10 for simultaneous
transfers of both csoh and water in a desired mole ratio . following the procedure of ( i ) , the standard csoh
solution ( mole ratio of csoh : h2o = 3:20 ) was prepared using
csohh2o ( 520 mg , 3.1 mmol ) and deionized
water ( 0.32 ml , 17.8 mmol ) . following
the procedure of ( i ) , the standard csoh
solution ( mole ratio of csoh : h2o = 3:30 ) was prepared using
csohh2o ( 520 mg , 3.1 mmol ) and deionized
water ( 0.50 ml , 27.8 mmol ) . an oven - dried
10 ml resealable screw - cap
test tube ( a ) equipped with a teflon - coated magnetic
stir bar
was charged with l5 ( 2.4 mg , 0.005 mmol , 2 mol
% ) and koh ( 24 equiv , 28.156.1 mg ) . tube a was evacuated and backfilled with argon ( this process was repeated
a total of three times ) , and 4-chloroanisole ( 36 mg , 31 l ,
0.25 mmol , 1 equiv ) and degassed deionized water ( 20111 equiv ,
90 l to 0.50 ml ) were then added into tube a via
syringe . tube b was then evacuated and backfilled with argon ( this process
was repeated a total of three times ) , and 1,4-dioxane ( 0.50 ml ) was
added into tube b via syringe . the resulting
reaction mixture in tube a was stirred at 50 or 80 c
in an oil bath for 20 h. after cooling to rt
, the
crude product was diluted with etoac ( 2 ml ) , 1,4-dimethoxybenzene
( 34.6 mg , 0.25 mmol , 1.0 equiv ) was added , and the mixture was then
acidified with aqueous hcl solution ( 1 m , 2 ml ) . tube a was evacuated and backfilled with
argon ( this process was repeated a total of three times ) , and 1-bromo-2,4-dimethylbenzene
( 46.3 mg , 34 l , 0.25 mmol ) and standard aqueous csoh solution
prepared in the preceding procedures were then added into tube a via syringe ( except for table 2 ,
entries 1 and 5 ) [ for table 2 , entries 2 and
6 : csoh : h2o = 3 equiv:10 equiv ; csoh ( 112.4 mg , 0.75 mmol , 3 equiv )
dissolved in deionized water ( 45 mg , 2.5 mmol , 10 equiv ) . tube b was then
evacuated and backfilled with argon ( this process was repeated a total
of three times ) , and the solvent ( 1,4-dioxane or thf , 0.50 ml ) was
added into tube b via syringe . the resulting
reaction mixture in tube a was stirred at rt for 20 h.
the crude
product
was diluted with etoac ( 2 ml ) , 1,3,5-trimethoxybenzene ( 42.1
mg , 0.25 mmol , 1.0 equiv ) was added , and the mixture was then acidified
with aqueous hcl solution ( 1 m , 2 ml ) . an oven - dried 20 ml resealable
screw - cap test tube ( a ) equipped with a teflon - coated
magnetic stir bar
was charged with l5 ( 9.7 mg , 0.020
mmol , 2 mol % ) , koh ( 168.3 mg , 3.0 mmol , 3 equiv ) , and ( hetero)aryl
halide ( if solid ) ( 1.0 mmol , 1 equiv ) . tube a was evacuated
and backfilled with argon ( this process was repeated a total of three
times ) , and degassed deionized water ( 0.36 ml , 20.0 mmol , 20 equiv )
and ( hetero)aryl halide ( if liquid ) ( 1.0 mmol , 1 equiv ) were
then added into tube a via syringe . tube b was then evacuated and
backfilled with argon ( this process was repeated a total of three
times ) , and 1,4-dioxane ( 2.0 ml ) was added into tube b via syringe . the resulting reaction mixture in tube a was stirred at 80 c in an oil bath for 18 h. after
cooling to rt
, the crude product was diluted with etoac ( 5 ml ) and
then acidified with aqueous hcl solution ( 1 m , 5 ml ) . an oven - dried
20 ml resealable
screw - cap test tube ( a ) equipped with a teflon - coated
magnetic stir bar was charged with l5 ( 9.7 mg , 0.020
mmol , 2 mol % ) and ( hetero)aryl halide ( if solid ) ( 1.0 mmol ,
1 equiv ) . tube a was evacuated and backfilled with argon
( this process was repeated a total of three times ) , and standard aqueous
csoh solution [ mole ratio of csoh to h2o = 3:10 ; csoh ( 450
mg , 3.0 mmol , 3 equiv ) dissolved in deionized water ( 180 mg , 10.0
mmol , 10 equiv ) as prepared in the preceding procedures ] was then
added into tube a via syringe , followed by the addition
of ( hetero)aryl halide ( if liquid ) ( 1.0 mmol , 1 equiv ) . the resulting reaction mixture in tube a was stirred at rt for 18 h.
the crude product was diluted
with etoac ( 5 ml ) and then acidified with aqueous hcl solution ( 1
m , 5 ml ) . h nmr ( 400 mhz , dmso - d6 ) :
9.70 ( br s , 1 h ) , 8.32 ( dd , j = 8.6 hz , j = 1.2 hz , 1 h ) , 7.787.71 ( ovrlp , 3 h ) , 7.70 ( d , j = 4.0 hz , 1 h ) , 7.35 ( dd , j = 9.2 hz , j = 2.8 hz , 1 h ) , 7.25 ( ddd , j = 8.0 hz , j = 6.0 hz , j = 1.6 hz , 1 h ) , 4.41 ( t , j = 7.6 hz , 2 h ) , 1.74 ( qu , j = 7.6 hz ,
2 h ) , 1.49 ( sex , j = 7.6 hz , 2 h ) , 0.96 ( t , j = 7.6 hz , 3 h ) . following general
procedure
a , the title compound was prepared using 2-chloro-9h - thioxanthen-9-one ( 247 mg , 1.0 mmol ) ; etoac / hexanes ( 1:4 ) ,
then etoac ; 224 mg , 98% ; sunset - yellow solid . h nmr ( 400 mhz , dmso - d6 ) : 10.16 ( br s , 1 h ) , 8.45 ( dd , j = 8.4
hz , j = 1.2 hz , 1 h ) , 7.86 ( d , j = 2.8 hz , 1 h ) , 7.79 ( dd , j = 8.0 hz , j = 0.8 hz , 1 h ) , 7.73 ( ddd , j = 8.4 hz , j = 6.8 hz , j = 1.6 hz , 1 h ) , 7.67 ( d , j = 8.8 hz , 1 h ) , 7.54 ( ddd , j = 8.4 hz , j = 7.2 hz , j = 1.2 hz , 1 h ) , 7.27 ( dd , j = 8.8 hz , j = 2.8 hz , 1 h ) . following general
procedure a
, the
title compound was prepared using 2-(4-chlorophenyl)benzothiazole
( 246 mg , 1.0 mmol ) ; etoac / hexanes ( 1:6 ) , then ( 1:4 ) ; 223 mg , 98% ;
pale - yellow solid . :
227229 c ( lit : 228229
c).h nmr ( 400 mhz ,
dmso - d6 ) : 10.23 ( br s , 1 h ) , 8.05
( dd , j = 7.6 hz , 1 h ) , 7.98 ( dd , j = 8.0 hz , 1 h ) , 7.93 ( d , j = 8.4 hz , 2 h ) , 7.49
( td , j = 8.0 hz , j = 1.2 hz , 1 h ) ,
7.39 ( td , j = 8.0 hz , j = 1.2 hz ,
1 h ) , 6.94 ( d , j = 8.8 hz , 2 h ) . : 8990 c ( lit : 8688 c).h nmr ( 400 mhz , dmso - d6 ) : 8.58 ( br s , 1 h ) , 6.62 ( s , 1 h ) , 6.55 ( s ,
1 h ) , 3.66 ( s , 3 h ) , 2.08 ( s , 3 h ) , 2.03 ( s , 3 h ) . following general
procedure
a , the title compound was prepared
using 2-bromo-1,3-dimethoxybenzene ( 217 mg , 1.0 mmol ) ; etoac / hexanes
( 1:6 ) , then ( 1:4 ) ; 134 mg , 86% ; pale - brown solid . : 5657
c ( lit : 5456 c).h nmr ( 400 mhz , dmso - d6 ) :
8.25 ( br s , 1 h ) , 6.69 ( t , j = 8.4 hz , 1 h ) , 6.59
( d , j = 8.4 hz , 2 h ) , 3.74 ( s , 6 h ) . m.p
: 95 c ( lit : 9193
c).h nmr ( 400 mhz ,
dmso - d6 ) : 10.10 ( br s , 1 h ) , 8.14
( dd , j = 7.2 hz , j = 1.2 hz , 1 h ) ,
7.80 ( dd , j = 7.2 hz , j = 1.6 hz ,
1 h ) , 7.487.41 ( ovrlp , 2 h ) , 7.347.28 ( ovrlp , 2 h ) ,
6.88 ( dd , j = 7.2 hz , j = 1.6 hz ,
1 h ) . : 251253 c ( lit : 252254
c).h nmr ( 400 mhz ,
dmso - d6 ) : 10.53 ( br s , 1 h ) , 8.77
( d , j = 2.0 hz , 1 h ) , 8.68 ( d , j = 1.6 hz , 1 h ) , 7.93 ( d , j = 9.2 hz , 1 h ) , 7.41
( dd , j = 9.2 hz , j = 2.8 hz , 1 h ) ,
7.27 ( d , j = 2.8 hz , 1 h ) . dmso - d6 ) : 8.99
( br s , 1 h ) , 7.35 ( d , j = 8.4 hz , 1 h ) , 7.30 ( t , j = 7.2 hz , 2 h ) , 7.26 ( d , j = 3.2 hz ,
1 h ) , 7.24 ( t , j = 7.2 hz , 1 h ) , 7.15 ( d , j = hz , 2 h ) , 6.73 ( d , j = 2.0 hz , 1 h ) ,
6.60 ( dd , j = 8.4 hz , j = 2.0 hz ,
1 h ) , 6.36 ( dd , j = 3.2 hz , j =
0.4 hz , 1 h ) , 5.29 ( s , 2 h ) . h nmr ( 400 mhz ,
dmso - d6 ) : 9.42 ( br s , 1 h ) , 7.74
( d , j = 8.8 hz , 1 h ) , 7.65 ( d , j = 5.6 hz , 1 h ) , 7.27 ( dd , j = 5.6 hz , j = 0.4 hz , 1 h ) , 7.21 ( d , j = 2.0 hz , 1 h ) , 6.88
( ddd , j = 8.4 hz , j = 2.0 hz , j = 0.4 hz , 1 h ) . h nmr ( 400 mhz , dmso - d6 ) : 10.55 ( br s , 1 h ) , 7.90 ( d , j = 9.2 hz ,
1 h ) , 7.33 ( dd , j = 9.2 hz , j =
2.4 hz , 1 h ) , 7.15 ( dd , j = 2.4 hz , j = 0.4 hz , 1 h ) . after work up , the
crude product was purified by flash chromatography using etoac / hexanes
( 1:6 ) as an eluent to afford dibenzothiophen-2-ol ( 3 m ) ( 185 mg , 92% ) as a pale - brown solid . h nmr ( 400 mhz ,
dmso - d6 ) : 9.68 ( br s , 1 h ) , 8.238.18
( m , 1 h ) , 7.977.91 ( m , 1 h ) , 7.78 ( d , j =
8.8 hz , 1 h ) , 7.68 ( d , j = 2.4 hz , 1 h ) , 7.497.42
( ovrlp , 2 h ) , 7.03 ( dd , j = 8.4 hz , j = 2.4 hz , 1 h ) . :
nmr
( 400 mhz , dmso - d6 ) : 10.98 ( s ,
1 h ) , 7.58 ( d , j = 8.0 hz , 1 h ) , 7.36 ( d , j = 7.2 hz , 6 h ) , 7.28 ( t , j = 7.2 hz ,
6 h ) , 7.22 ( t , j = 7.2 hz , 3 h ) , 7.00 ( t , j = 7.2 hz , 1 h ) , 6.93 ( t , j = 7.2 hz ,
1 h ) , 6.40 ( d , j = 8.4 hz , 1 h ) . the h nmr
yield of the title product was determined to be 96% based on 0.25
mmol arbr . h nmr ( 400 mhz , dmso - d6 ) : 8.93 ( br s , 1 h ) , 6.84 ( s , 1 h ) , 6.76 ( dd , j = 8.0 hz , j = 1.6 hz , 1 h ) , 6.64 ( d , j = 8.0 hz , 1 h ) , 2.15 ( s , 3 h ) , 2.07 ( s , 3 h ) . : 121122 c ( lit : 119120 c).h nmr ( 400 mhz , dmso - d6 ) : 9.68 ( br s , 1 h ) , 7.347.32 ( ovrlp ,
2 h ) , 7.18 ( d , j = 8.0 hz , 1 h ) , 2.48 ( s , 3 h ) , 2.17
( s , 3 h ) . :
147148 c ( lit : 146147 c).h nmr ( 400 mhz , dmso - d6 ) : 9.19 ( br s , 1 h ) , 6.05 ( s , 2 h ) , 3.69 ( s ,
6 h ) , 3.55 ( s , 3 h ) . :
121123
c ( lit : 120 c).h nmr ( 400 mhz , dmso - d6 ) : 9.74
( br s , 1 h ) , 7.18 ( d , j = 8.4 hz , 2 h ) , 6.78 ( d , j = 8.8 hz , 2 h ) , 3.30 ( br s , 4 h ) , 1.08 ( t , j = 7.2 hz , 6 h ) . :
133134 c ( lit :
133.5134.5 c).h nmr ( 400 mhz , dmso - d6 ) : 10.45
( br s , 1 h ) , 7.687.64 ( ovrlp , 4 h ) , 7.62 ( t , j = 7.2 hz , 1 h ) , 7.52 ( t , j = 7.2 hz , 2 h ) , 6.90
( d , j = 8.4 hz , 2 h ) . | [
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] |
non - alcoholic fatty liver disease ( nafld ) , the most common form of liver disease , is now recognized as a major public health problem across the world ( 1 - 4 ) . as a hepatic manifestation of the metabolic syndrome
as such , nafld patients are at high risk of death due to cardiovascular disease ( cvd ) ( 8) .
tumor necrosis factor - alpha ( tnf- ) is a proinflammatory cytokine that is produced and secreted mainly by inflammatory cells .
the tnf- promotes inflammatory responses to injury and regulates insulin resistance ( ir ) through insulin signal transduction pathways in the liver ( 10 ) .
therefore , tnf- may play an important role in the development and progression of metabolic disorders in humans ( 11 - 13 ) .
expression of tnf- is upregulated by inflammatory mediators , as well as genetic variations in the tnf- promoter region , which alter the transcriptional activity and protein expression levels of tnf- ( 14 - 18 ) .
previous studies have shown a strong association between the presence of tnf- polymorphisms and the risk of developing nafld , especially in patients of chinese origin ( 19 - 21 ) . a recent meta - analysis further confirmed these data ( 22 ) .
however , other studies examining the association between tnf- polymorphisms and risk of coronary artery disease ( cad ) have yielded discrepant results ( 23 - 31 ) .
while in caucasians , tnf- variants were indeed associated with cad development , progression , and complications ( 26 ) , such an association did not appear to exist in the chinese population ( 31 ) .
considering the high incidence of cad - related morality in nafld patients , it is of particular importance to further identify the potential association between tnf- polymorphisms and relative risk of developing cad in nafld patients .
such studies may ultimately provide novel means to combat the development of cad and to decrease the morality rate of nafld patients .
in the present study , we selected and genotyped two of the most widely studied tnf- polymorphisms , namely the substitution of guanine ( g ) by adenine ( a ) at residues 238 and 308 of the promoter region , in nafld patients , with or without cad , versus healthy controls .
we aimed to assess the effect of these two tnf- polymorphisms for their association with the risk of chinese han nafld patients for developing cad .
this study was approved by the ethical committee of qingdao municipal hospital ( qingdao , china ) and a written informed consent form was obtained from each subject before participation in the study .
this study was performed in accordance with the principles of the declaration of helsinki ( 32 ) . to test our hypothesis , we selected a total of 493 unrelated adult chinese nafld patients of both genders and 304 healthy controls matched for sex and age with the patients ( 152 males , 152 females , mean age 61.31 9.40 years ) between april 2010 and may 2014 .
these cases included 493 nafld patients diagnosed by b - type ultrasonography [ 246 patients with cad ( 127 males , 118 females , mean age 61.54 10.28 years ) and 247 patients without cad ( 126 males , 120 females , mean age 62.13 9.74 years ) ] .
these healthy controls were volunteers recruited from the departments of gastroenterology and cardiology of qingdao municipal hospital .
the diagnosis of nafld was based on the standard clinical evaluation according to the chinese association of medicine in 2010 ( 33 ) .
the cad was diagnosed by a percutaneous coronary angiogram , analyzed by two experienced interventional cardiologists , and defined by presence of at least 50% stenosis in at least one of the coronary arteries .
the control group consisted of individuals who were confirmed as being healthy by echocardiography , medical history , and general and laboratory examinations at the same hospital .
subjects were excluded if they had other liver diseases , cardiac disorders , diabetes mellitus , infectious disease , concurrent major renal and malignant disease , or a history of medication . basic clinicopathological information ( name , age , etc . ) was obtained using a standard study questionnaire .
blood samples were taken from each subject after a 12-hour overnight fast and collected into ethylene diamine tetraacetic acid - containing tubes . for biochemical analyses ,
blood samples were obtained and assessed for serum levels of total cholesterol ( tc ) , triglyceride ( tg ) , low - density lipoprotein ( ldl ) and high - density lipoprotein ( hdl ) using routine enzymatic methods .
serum alanine aminotransferase ( alt ) and aspartate aminotransferase ( ast ) concentrations were also measured , as described previously ( 34 ) .
genomic dna was extracted from peripheral blood using the genomic dna purification kit ( beijing bioteke biotechnology , beijing , china ) following the manufacturer s instructions and stored at -20 c until use . to genotype tnf- single nucleotide polymorphisms ( snps )
, we performed polymerase chain reaction ( pcr ) analysis using the following primers for tnf--238 polymorphism : 5'-agaagacccccctcggaacc-3 ' and 5'-atctggaggaagcggtagtg-3 ' .
the pcr amplification profile was achieved by 35 cycles of denaturation , annealing and extension at 94 c , 59 c and 70 c for 1 min , respectively .
the resulting pcr products were then separated in 2% gel with a 152-base pair product in size .
the pcr conditions were 35 cycles of denaturation , annealing and extension for 1 min each at 94 c , 60 c and 72 c to obtain a 134-base pair pcr product .
the tnf- genotypes were then detected by direct dna sequencing using the abi prism sequence detection system abi3730 ( foster city , ca , usa ) .
statistical analyses were performed using spss statistical software , version 20.0 for mac ( spss inc . ,
genotype and allele frequencies were estimated by counting the dna sequencing data of each subject .
the distributions between patients and controls were analyzed by pearson s test or fisher s exact test , where appropriate .
the hardy - weinberg equilibrium between expected and observed genotype distributions was assessed using the test .
differences in characteristics between different groups were examined using the student s t test , paired samples t test or the test .
the strength of the association between the polymorphism and presence / absence of cad was evaluated by logistic regression analysis and estimated by the odds ratio ( or ) with 95% confidence interval ( ci ) . a p < 0.05 was considered as statistically significant .
this study was approved by the ethical committee of qingdao municipal hospital ( qingdao , china ) and a written informed consent form was obtained from each subject before participation in the study .
this study was performed in accordance with the principles of the declaration of helsinki ( 32 ) . to test our hypothesis , we selected a total of 493 unrelated adult chinese nafld patients of both genders and 304 healthy controls matched for sex and age with the patients ( 152 males , 152 females , mean age 61.31 9.40 years ) between april 2010 and may 2014 .
these cases included 493 nafld patients diagnosed by b - type ultrasonography [ 246 patients with cad ( 127 males , 118 females , mean age 61.54 10.28 years ) and 247 patients without cad ( 126 males , 120 females , mean age 62.13 9.74 years ) ] .
these healthy controls were volunteers recruited from the departments of gastroenterology and cardiology of qingdao municipal hospital .
the diagnosis of nafld was based on the standard clinical evaluation according to the chinese association of medicine in 2010 ( 33 ) .
the cad was diagnosed by a percutaneous coronary angiogram , analyzed by two experienced interventional cardiologists , and defined by presence of at least 50% stenosis in at least one of the coronary arteries .
the control group consisted of individuals who were confirmed as being healthy by echocardiography , medical history , and general and laboratory examinations at the same hospital .
subjects were excluded if they had other liver diseases , cardiac disorders , diabetes mellitus , infectious disease , concurrent major renal and malignant disease , or a history of medication . basic clinicopathological information ( name , age , etc . ) was obtained using a standard study questionnaire .
blood samples were taken from each subject after a 12-hour overnight fast and collected into ethylene diamine tetraacetic acid - containing tubes . for biochemical analyses ,
blood samples were obtained and assessed for serum levels of total cholesterol ( tc ) , triglyceride ( tg ) , low - density lipoprotein ( ldl ) and high - density lipoprotein ( hdl ) using routine enzymatic methods .
serum alanine aminotransferase ( alt ) and aspartate aminotransferase ( ast ) concentrations were also measured , as described previously ( 34 ) .
genomic dna was extracted from peripheral blood using the genomic dna purification kit ( beijing bioteke biotechnology , beijing , china ) following the manufacturer s instructions and stored at -20 c until use . to genotype tnf- single nucleotide polymorphisms ( snps ) , we performed polymerase chain reaction ( pcr ) analysis using the following primers for tnf--238 polymorphism : 5'-agaagacccccctcggaacc-3 ' and 5'-atctggaggaagcggtagtg-3 ' .
the pcr amplification profile was achieved by 35 cycles of denaturation , annealing and extension at 94 c , 59 c and 70 c for 1 min , respectively .
the resulting pcr products were then separated in 2% gel with a 152-base pair product in size .
the pcr conditions were 35 cycles of denaturation , annealing and extension for 1 min each at 94 c , 60 c and 72 c to obtain a 134-base pair pcr product .
the tnf- genotypes were then detected by direct dna sequencing using the abi prism sequence detection system abi3730 ( foster city , ca , usa ) .
statistical analyses were performed using spss statistical software , version 20.0 for mac ( spss inc . ,
genotype and allele frequencies were estimated by counting the dna sequencing data of each subject .
the distributions between patients and controls were analyzed by pearson s test or fisher s exact test , where appropriate .
the hardy - weinberg equilibrium between expected and observed genotype distributions was assessed using the test .
differences in characteristics between different groups were examined using the student s t test , paired samples t test or the test .
the strength of the association between the polymorphism and presence / absence of cad was evaluated by logistic regression analysis and estimated by the odds ratio ( or ) with 95% confidence interval ( ci ) . a p < 0.05 was considered as statistically significant .
healthy controls were matched for sex and age with the nafld patient group , with or without cad ( p > 0.05 ) . as expected , nafld patients had a high prevalence of traditional risk factors for fatty liver , including hypertension , increased body - mass index ( bmi ) , serum tg , tc , and ldl levels and decreased hdl levels , compared to controls ( all p < 0.05 ) .
moreover , serum alt and ast levels were also higher in patients compared to controls ( p < 0.05 ) .
importantly , nafld patients with cad had a higher prevalence of tobacco use and hypertension , increased bmi , alt , ast , tg , tc , and ldl , and decreased hdl levels compared to healthy controls ( all p < 0.05 ) . in nafld patients without cad , we also found an increased prevalence of tobacco use , hypertension , tg , and ldl , and a lower hdl level compared to those with cad ( all p < 0.05 ) .
abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; p1 , cad + nafld vs. cad nafld ; p2 , cad + nafld vs. control ; p3 , cad nafld vs. control ; tc , total cholesterol ; tg , triglyceride .
the genotype distributions of the two tnf- variants were in accordance with the hardy - weinberg equilibrium in both patients and controls ( all p > 0.05 ) .
we found no evidence of the aa homozygote for these two tnf- variants in our study population .
to ensure the accuracy of our genotyping , we randomly repeated the dna sequencing in 100 subjects for reverse sequencing .
as shown in table 2 , at position tnf--238 , there was significant difference in genotypic and allelic distributions between the nafld patients and their control counterparts ( or : 3.007 , 95% ci : 1.8194.971 , p = 0.000 ; or : 2.760 , 95% ci : 1.7014.478 , p = 0.000 ; or : 1.824 , 95% ci : 1.0683.116 , p = 0.026 ; or : 1.759 , 95% ci : 1.0472.957 , p = 0.031 , respectively ) .
moreover , a statistically significant difference was observed between nafld patients with and without cad ( or : 1.648 , 95% ci : 1.0362.624 , p = 0.034 ; or : 1.568 , 95% ci : 1.0092.439 , p = 0.044 , respectively ) .
in contrast , there was no statistical difference observed for tnf--308 snps between cases and controls ( all p > 0.05 ) .
abbreviations : cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; p1 , cad + nafld vs. cad nafld ; p2 , cad + nafld vs. control ; p3 , cad
( % ) . to assess whether these two tnf- polymorphisms associate with clinical parameters , we compared tnf- genotype with the clinical characteristics between nafld patients with or without cad and the control group ( table 3 ) . in nafld patients with cad ,
serum tg and ldl levels were significantly higher in the ga genotype carriers than those with tnf- gg genotype at position -238 ( p = 0.025 and 0.001 , respectively ) .
the tnf--238 ga genotype of nafld patients with cad showed higher tc levels ( ga , 5.34 1.15 vs. gg , 5.18 0.90 ) and lower hdl levels ( ga , 1.18 0.41 vs. gg , 1.22 0.37 ) than those with the gg genotype .
furthermore , cad - free nafld patients with the tnf--238 ga genotype exhibited higher tg levels than those with the gg genotype ( p = 0.017 ) .
the tnf--308 ga genotype of nafld patients with cad was higher than in those of the gg genotype ( p = 0.030 ) .
no statistically significant differences were observed between the controls and nafld patients without cad , with regards to presence of tnf--308 ( p > 0.05 ) .
abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; tg , triglyceride ; tc , total cholesterol .
healthy controls were matched for sex and age with the nafld patient group , with or without cad ( p > 0.05 ) . as expected , nafld patients had a high prevalence of traditional risk factors for fatty liver , including hypertension , increased body - mass index ( bmi ) , serum tg , tc , and ldl levels and decreased hdl levels , compared to controls ( all p < 0.05 ) .
moreover , serum alt and ast levels were also higher in patients compared to controls ( p < 0.05 ) .
importantly , nafld patients with cad had a higher prevalence of tobacco use and hypertension , increased bmi , alt , ast , tg , tc , and ldl , and decreased hdl levels compared to healthy controls ( all p < 0.05 ) . in nafld patients without cad , we also found an increased prevalence of tobacco use , hypertension , tg , and ldl , and a lower hdl level compared to those with cad ( all p < 0.05 ) .
abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; p1 , cad + nafld vs. cad nafld ; p2 , cad + nafld vs. control ; p3 , cad nafld vs. control ; tc , total cholesterol ; tg , triglyceride .
the genotype distributions of the two tnf- variants were in accordance with the hardy - weinberg equilibrium in both patients and controls ( all p > 0.05 ) .
we found no evidence of the aa homozygote for these two tnf- variants in our study population .
to ensure the accuracy of our genotyping , we randomly repeated the dna sequencing in 100 subjects for reverse sequencing .
as shown in table 2 , at position tnf--238 , there was significant difference in genotypic and allelic distributions between the nafld patients and their control counterparts ( or : 3.007 , 95% ci : 1.8194.971 , p = 0.000 ; or : 2.760 , 95% ci : 1.7014.478 , p = 0.000 ; or : 1.824 , 95% ci : 1.0683.116 , p = 0.026 ; or : 1.759 , 95% ci : 1.0472.957 , p = 0.031 , respectively ) .
moreover , a statistically significant difference was observed between nafld patients with and without cad ( or : 1.648 , 95% ci : 1.0362.624 , p = 0.034 ; or : 1.568 , 95% ci : 1.0092.439 , p = 0.044 , respectively ) .
in contrast , there was no statistical difference observed for tnf--308 snps between cases and controls ( all p > 0.05 ) .
abbreviations : cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; p1 , cad + nafld vs. cad nafld ; p2 , cad + nafld vs. control ; p3 , cad
to assess whether these two tnf- polymorphisms associate with clinical parameters , we compared tnf- genotype with the clinical characteristics between nafld patients with or without cad and the control group ( table 3 ) . in nafld patients with cad ,
serum tg and ldl levels were significantly higher in the ga genotype carriers than those with tnf- gg genotype at position -238 ( p = 0.025 and 0.001 , respectively ) .
the tnf--238 ga genotype of nafld patients with cad showed higher tc levels ( ga , 5.34 1.15 vs. gg , 5.18 0.90 ) and lower hdl levels ( ga , 1.18 0.41 vs. gg , 1.22 0.37 ) than those with the gg genotype .
furthermore , cad - free nafld patients with the tnf--238 ga genotype exhibited higher tg levels than those with the gg genotype ( p = 0.017 ) .
the tnf--308 ga genotype of nafld patients with cad was higher than in those of the gg genotype ( p = 0.030 ) .
no statistically significant differences were observed between the controls and nafld patients without cad , with regards to presence of tnf--308 ( p > 0.05 ) .
abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; tg , triglyceride ; tc , total cholesterol .
the current study is the first report to associate tnf- polymorphisms with risk of developing cad in nafld patients .
we found that in these patients , presence of the tnf--238 ga genotype and the a allele increased the risk of cad compared with the gg homozygotes .
future studies are required to address whether and how this polymorphism modulates tnf- expression , as well as the role of tnf- in the pathogenesis of cad in the setting of nafld .
the human tnf- gene is localized at chromosome 6p21.3 and codes a protein that acts as a proinflammatory cytokine .
it is mainly produced by monocytes and macrophages after gram - negative bacteria infection or lipopolysaccharide ( lps ) ( 35 , 36 ) . as a biomarker of systemic inflammation , tnf-
is centrally involved in the pathophysiology of cardiovascular and metabolic syndromes , including cad and nafld ( 37 ) .
overexpression of tnf- may contribute to the development of nafld , atherosclerosis and cad by affecting major lipid metabolism , reducing insulin receptor signaling , and blocking insulin actions ( 38 - 40 ) .
valenti et al . ( 41 ) showed that tnf--238 , and not -308 , had higher prevalence in italian patients with nafld compared to controls . and carriers of tnf--238 a allele had higher insulin resistance indices .
we also found that compared to the control subjects , the frequencies of tnf--238 a allele , instead of -308 a allele were higher in subjects with nafld .
moreover , tnf- promoter polymorphism at position -238 a allele increased tg and ldl levels in patients with nafld , indicating that a allele of tnf--238 may contribute to the development of nafld .
these results are in accordance with other chinese data ( 19 - 21 ) .
studies examining the association between tnf- polymorphisms and the risk cad or other cardiovascular events have generated controversial results .
a previous study in non - diabetic japanese males showed that presence of a tnf--863 variant was significantly related to cad .
specifically , these investigators found that the tnf--863 a allele may protect against the progression of cad ( 23 ) . on the other hand , vendrell et al .
( 26 ) reported that tnf--308 polymorphism may increase the risk of developing cad in european women with type 2 diabetes mellitus . in agreement with previous studies
, korean researchers showed that tnf- g to a polymorphism at position -238 was significantly associated with cad and that carriers of the -238 a allele exhibited an increased risk of developing cad .
it was therefore argued that this allele could be used as a predictive marker for cad in koreans ( 25 ) .
however , because of differences in ethnicity , phenotype , and environment factors , discrepant results have been reported in other studies ( 27 - 30 ) . in the present report ,
the frequency of tnf--238 ga heterozygote genotype and a allele in nafld patients with cad was substantially higher than in those without cad . also , nafld patients with cad who carried the tnf--238 a allele had an increase in serum tg and ldl levels than those who did not carry the a allele .
however , we observed detrimental effects of tnf--308 a allele on lipid levels in nafld patients with cad , suggesting that tnf- a allele could contribute to insulin resistance in cad patients , which in turn , may exacerbate the risk of cardiovascular complications . in a previous study , it was also shown that tnf- induced phosphorylation of p38 mitogen - activated protein kinase ( mapk ) and extracellular signal - regulated kinase ( erk ) in human aortic smooth muscle cells , and that these post - translational modifications were inhibited by phaseolus vulgaris extract treatment ( 42 ) . in chronic heart failure patients , high tnf- levels
interestingly , anti - tnf- therapy significantly controlled the aortic stiffness , carotid atherosclerosis , and calprotectin in patients with inflammatory arthropathy , indicating that long - term anti - tnf- therapy reduced aortic stiffness and carotid intima media thickness progression in patients with inflammatory arthropathy ( 44 ) .
all of these studies demonstrated that tnf- contributed to cad and thus , further study is needed to associate tnf--238 snp with expression of tnf- protein .
our current study is subject to several methodological limitations that are worth nothing ( 45 ) .
first , due to difficulty in obtaining liver biopsy in this epidemiological survey , we resorted to the use of ultrasonography for diagnosing nafld .
secondly , we did not associate tnf- polymorphisms and the level of expression with insulin resistance or disease severity in nafld patients .
thirdly , future studies with larger sample sizes and multiple ethnic groups are required to confirm our current data . in conclusion , this study provided preliminary evidence in favor of an association between presence of a tnf--238 polymorphism and the development of cad in nafld patients of chinese han origin .
in addition , the tnf--308 ga heterozygote genotype was positively associated with increased levels of tg in nafld patients with cad , suggesting the potential role of tnf--308 ga genotype in the development of cad in these patients .
however , mechanisms underlying the association between tnf- gene polymorphisms and the risk of cad in nafld patients will require further investigation . | background : cardiovascular events account for the main cause of death in patients with non - alcoholic fatty liver disease ( nafld ) , and are largely influenced by genetic factors .
although multiple studies showed that tumor necrosis factor - alpha ( tnf- ) polymorphisms are risk factors in the progression of nafld , few papers on the association of the polymorphisms and the developing coronary artery disease ( cad ) in nafld patients have been reported.objectives:the present study was designed to evaluate the association of tnf- polymorphisms at residues -238 and -308 , with the risk of developing cad in chinese patients with nafld.patients and methods : the tnf- polymorphisms at residues 238 and 308 were genotyped in b - type ultrasonography proven nafld patients with ( n = 246 ) , without ( n = 247 ) cad and healthy controls ( n = 304 ) , using polymerase chain reaction ( pcr ) .
serum lipid profiles were determined using biochemical methods .
statistical analyses were performed using spss statistical software , version 20.0 for mac.results:we found a significant association between tnf--238 guanine to alanine ( ga ) polymorphism and carriers of variant allele a between nafld patients with and without cad ( p < 0.05 ) .
carriers of the a allele of tnf--238 had higher serum triglycerides ( tg ) and low density lipoprotein ( ldl ) levels in nafld patients with cad ( p = 0.025 and 0.001 , respectively ) and a higher tg level in nafld patients without cad ( p = 0.017 ) , than their non - carrier counterparts.conclusions:in the chinese han population that we studied , nafld patients who carry the tnf--238 ga polymorphism have an increased risk of developing cad .
mechanisms underlying this potentially important association require further investigation . | 1. Background
2. Objectives
3. Patients and Methods
3.1. Study Subjects
3.2. Biochemical Analyses
3.3. Genomic DNA Extraction and Genotyping
3.4. Statistical Analysis
4. Results
4.1. Characteristics of the Study Population
4.2. TNF--238 and -308 Genotypes and Allele Distribution
4.3. TNF- Polymorphism Association With Clinical Parameters in Non-alcoholic Fatty Liver Disease Patients
5. Discussion | non - alcoholic fatty liver disease ( nafld ) , the most common form of liver disease , is now recognized as a major public health problem across the world ( 1 - 4 ) . as a hepatic manifestation of the metabolic syndrome
as such , nafld patients are at high risk of death due to cardiovascular disease ( cvd ) ( 8) . tumor necrosis factor - alpha ( tnf- ) is a proinflammatory cytokine that is produced and secreted mainly by inflammatory cells . previous studies have shown a strong association between the presence of tnf- polymorphisms and the risk of developing nafld , especially in patients of chinese origin ( 19 - 21 ) . however , other studies examining the association between tnf- polymorphisms and risk of coronary artery disease ( cad ) have yielded discrepant results ( 23 - 31 ) . while in caucasians , tnf- variants were indeed associated with cad development , progression , and complications ( 26 ) , such an association did not appear to exist in the chinese population ( 31 ) . considering the high incidence of cad - related morality in nafld patients , it is of particular importance to further identify the potential association between tnf- polymorphisms and relative risk of developing cad in nafld patients . in the present study , we selected and genotyped two of the most widely studied tnf- polymorphisms , namely the substitution of guanine ( g ) by adenine ( a ) at residues 238 and 308 of the promoter region , in nafld patients , with or without cad , versus healthy controls . we aimed to assess the effect of these two tnf- polymorphisms for their association with the risk of chinese han nafld patients for developing cad . this study was approved by the ethical committee of qingdao municipal hospital ( qingdao , china ) and a written informed consent form was obtained from each subject before participation in the study . to test our hypothesis , we selected a total of 493 unrelated adult chinese nafld patients of both genders and 304 healthy controls matched for sex and age with the patients ( 152 males , 152 females , mean age 61.31 9.40 years ) between april 2010 and may 2014 . these cases included 493 nafld patients diagnosed by b - type ultrasonography [ 246 patients with cad ( 127 males , 118 females , mean age 61.54 10.28 years ) and 247 patients without cad ( 126 males , 120 females , mean age 62.13 9.74 years ) ] . the diagnosis of nafld was based on the standard clinical evaluation according to the chinese association of medicine in 2010 ( 33 ) . for biochemical analyses ,
blood samples were obtained and assessed for serum levels of total cholesterol ( tc ) , triglyceride ( tg ) , low - density lipoprotein ( ldl ) and high - density lipoprotein ( hdl ) using routine enzymatic methods . to genotype tnf- single nucleotide polymorphisms ( snps )
, we performed polymerase chain reaction ( pcr ) analysis using the following primers for tnf--238 polymorphism : 5'-agaagacccccctcggaacc-3 ' and 5'-atctggaggaagcggtagtg-3 ' . statistical analyses were performed using spss statistical software , version 20.0 for mac ( spss inc . the strength of the association between the polymorphism and presence / absence of cad was evaluated by logistic regression analysis and estimated by the odds ratio ( or ) with 95% confidence interval ( ci ) . this study was approved by the ethical committee of qingdao municipal hospital ( qingdao , china ) and a written informed consent form was obtained from each subject before participation in the study . to test our hypothesis , we selected a total of 493 unrelated adult chinese nafld patients of both genders and 304 healthy controls matched for sex and age with the patients ( 152 males , 152 females , mean age 61.31 9.40 years ) between april 2010 and may 2014 . these cases included 493 nafld patients diagnosed by b - type ultrasonography [ 246 patients with cad ( 127 males , 118 females , mean age 61.54 10.28 years ) and 247 patients without cad ( 126 males , 120 females , mean age 62.13 9.74 years ) ] . the diagnosis of nafld was based on the standard clinical evaluation according to the chinese association of medicine in 2010 ( 33 ) . for biochemical analyses ,
blood samples were obtained and assessed for serum levels of total cholesterol ( tc ) , triglyceride ( tg ) , low - density lipoprotein ( ldl ) and high - density lipoprotein ( hdl ) using routine enzymatic methods . to genotype tnf- single nucleotide polymorphisms ( snps ) , we performed polymerase chain reaction ( pcr ) analysis using the following primers for tnf--238 polymorphism : 5'-agaagacccccctcggaacc-3 ' and 5'-atctggaggaagcggtagtg-3 ' . statistical analyses were performed using spss statistical software , version 20.0 for mac ( spss inc . the strength of the association between the polymorphism and presence / absence of cad was evaluated by logistic regression analysis and estimated by the odds ratio ( or ) with 95% confidence interval ( ci ) . a p < 0.05 was considered as statistically significant . healthy controls were matched for sex and age with the nafld patient group , with or without cad ( p > 0.05 ) . as expected , nafld patients had a high prevalence of traditional risk factors for fatty liver , including hypertension , increased body - mass index ( bmi ) , serum tg , tc , and ldl levels and decreased hdl levels , compared to controls ( all p < 0.05 ) . moreover , serum alt and ast levels were also higher in patients compared to controls ( p < 0.05 ) . importantly , nafld patients with cad had a higher prevalence of tobacco use and hypertension , increased bmi , alt , ast , tg , tc , and ldl , and decreased hdl levels compared to healthy controls ( all p < 0.05 ) . in nafld patients without cad , we also found an increased prevalence of tobacco use , hypertension , tg , and ldl , and a lower hdl level compared to those with cad ( all p < 0.05 ) . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; p1 , cad + nafld vs. cad nafld ; p2 , cad + nafld vs. control ; p3 , cad nafld vs. control ; tc , total cholesterol ; tg , triglyceride . the genotype distributions of the two tnf- variants were in accordance with the hardy - weinberg equilibrium in both patients and controls ( all p > 0.05 ) . as shown in table 2 , at position tnf--238 , there was significant difference in genotypic and allelic distributions between the nafld patients and their control counterparts ( or : 3.007 , 95% ci : 1.8194.971 , p = 0.000 ; or : 2.760 , 95% ci : 1.7014.478 , p = 0.000 ; or : 1.824 , 95% ci : 1.0683.116 , p = 0.026 ; or : 1.759 , 95% ci : 1.0472.957 , p = 0.031 , respectively ) . moreover , a statistically significant difference was observed between nafld patients with and without cad ( or : 1.648 , 95% ci : 1.0362.624 , p = 0.034 ; or : 1.568 , 95% ci : 1.0092.439 , p = 0.044 , respectively ) . in contrast , there was no statistical difference observed for tnf--308 snps between cases and controls ( all p > 0.05 ) . abbreviations : cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; p1 , cad + nafld vs. cad nafld ; p2 , cad + nafld vs. control ; p3 , cad
( % ) . to assess whether these two tnf- polymorphisms associate with clinical parameters , we compared tnf- genotype with the clinical characteristics between nafld patients with or without cad and the control group ( table 3 ) . in nafld patients with cad ,
serum tg and ldl levels were significantly higher in the ga genotype carriers than those with tnf- gg genotype at position -238 ( p = 0.025 and 0.001 , respectively ) . the tnf--238 ga genotype of nafld patients with cad showed higher tc levels ( ga , 5.34 1.15 vs. gg , 5.18 0.90 ) and lower hdl levels ( ga , 1.18 0.41 vs. gg , 1.22 0.37 ) than those with the gg genotype . furthermore , cad - free nafld patients with the tnf--238 ga genotype exhibited higher tg levels than those with the gg genotype ( p = 0.017 ) . the tnf--308 ga genotype of nafld patients with cad was higher than in those of the gg genotype ( p = 0.030 ) . no statistically significant differences were observed between the controls and nafld patients without cad , with regards to presence of tnf--308 ( p > 0.05 ) . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; tg , triglyceride ; tc , total cholesterol . healthy controls were matched for sex and age with the nafld patient group , with or without cad ( p > 0.05 ) . as expected , nafld patients had a high prevalence of traditional risk factors for fatty liver , including hypertension , increased body - mass index ( bmi ) , serum tg , tc , and ldl levels and decreased hdl levels , compared to controls ( all p < 0.05 ) . moreover , serum alt and ast levels were also higher in patients compared to controls ( p < 0.05 ) . importantly , nafld patients with cad had a higher prevalence of tobacco use and hypertension , increased bmi , alt , ast , tg , tc , and ldl , and decreased hdl levels compared to healthy controls ( all p < 0.05 ) . in nafld patients without cad , we also found an increased prevalence of tobacco use , hypertension , tg , and ldl , and a lower hdl level compared to those with cad ( all p < 0.05 ) . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; p1 , cad + nafld vs. cad nafld ; p2 , cad + nafld vs. control ; p3 , cad nafld vs. control ; tc , total cholesterol ; tg , triglyceride . the genotype distributions of the two tnf- variants were in accordance with the hardy - weinberg equilibrium in both patients and controls ( all p > 0.05 ) . as shown in table 2 , at position tnf--238 , there was significant difference in genotypic and allelic distributions between the nafld patients and their control counterparts ( or : 3.007 , 95% ci : 1.8194.971 , p = 0.000 ; or : 2.760 , 95% ci : 1.7014.478 , p = 0.000 ; or : 1.824 , 95% ci : 1.0683.116 , p = 0.026 ; or : 1.759 , 95% ci : 1.0472.957 , p = 0.031 , respectively ) . moreover , a statistically significant difference was observed between nafld patients with and without cad ( or : 1.648 , 95% ci : 1.0362.624 , p = 0.034 ; or : 1.568 , 95% ci : 1.0092.439 , p = 0.044 , respectively ) . abbreviations : cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; p1 , cad + nafld vs. cad nafld ; p2 , cad + nafld vs. control ; p3 , cad
to assess whether these two tnf- polymorphisms associate with clinical parameters , we compared tnf- genotype with the clinical characteristics between nafld patients with or without cad and the control group ( table 3 ) . in nafld patients with cad ,
serum tg and ldl levels were significantly higher in the ga genotype carriers than those with tnf- gg genotype at position -238 ( p = 0.025 and 0.001 , respectively ) . the tnf--238 ga genotype of nafld patients with cad showed higher tc levels ( ga , 5.34 1.15 vs. gg , 5.18 0.90 ) and lower hdl levels ( ga , 1.18 0.41 vs. gg , 1.22 0.37 ) than those with the gg genotype . furthermore , cad - free nafld patients with the tnf--238 ga genotype exhibited higher tg levels than those with the gg genotype ( p = 0.017 ) . the tnf--308 ga genotype of nafld patients with cad was higher than in those of the gg genotype ( p = 0.030 ) . no statistically significant differences were observed between the controls and nafld patients without cad , with regards to presence of tnf--308 ( p > 0.05 ) . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; cad + nafld , non - alcoholic fatty liver disease patients with coronary artery disease ; cad nafld , nafld patients without cad ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; tg , triglyceride ; tc , total cholesterol . the current study is the first report to associate tnf- polymorphisms with risk of developing cad in nafld patients . we found that in these patients , presence of the tnf--238 ga genotype and the a allele increased the risk of cad compared with the gg homozygotes . future studies are required to address whether and how this polymorphism modulates tnf- expression , as well as the role of tnf- in the pathogenesis of cad in the setting of nafld . overexpression of tnf- may contribute to the development of nafld , atherosclerosis and cad by affecting major lipid metabolism , reducing insulin receptor signaling , and blocking insulin actions ( 38 - 40 ) . ( 41 ) showed that tnf--238 , and not -308 , had higher prevalence in italian patients with nafld compared to controls . and carriers of tnf--238 a allele had higher insulin resistance indices . we also found that compared to the control subjects , the frequencies of tnf--238 a allele , instead of -308 a allele were higher in subjects with nafld . moreover , tnf- promoter polymorphism at position -238 a allele increased tg and ldl levels in patients with nafld , indicating that a allele of tnf--238 may contribute to the development of nafld . studies examining the association between tnf- polymorphisms and the risk cad or other cardiovascular events have generated controversial results . specifically , these investigators found that the tnf--863 a allele may protect against the progression of cad ( 23 ) . ( 26 ) reported that tnf--308 polymorphism may increase the risk of developing cad in european women with type 2 diabetes mellitus . in agreement with previous studies
, korean researchers showed that tnf- g to a polymorphism at position -238 was significantly associated with cad and that carriers of the -238 a allele exhibited an increased risk of developing cad . in the present report ,
the frequency of tnf--238 ga heterozygote genotype and a allele in nafld patients with cad was substantially higher than in those without cad . also , nafld patients with cad who carried the tnf--238 a allele had an increase in serum tg and ldl levels than those who did not carry the a allele . however , we observed detrimental effects of tnf--308 a allele on lipid levels in nafld patients with cad , suggesting that tnf- a allele could contribute to insulin resistance in cad patients , which in turn , may exacerbate the risk of cardiovascular complications . in chronic heart failure patients , high tnf- levels
interestingly , anti - tnf- therapy significantly controlled the aortic stiffness , carotid atherosclerosis , and calprotectin in patients with inflammatory arthropathy , indicating that long - term anti - tnf- therapy reduced aortic stiffness and carotid intima media thickness progression in patients with inflammatory arthropathy ( 44 ) . secondly , we did not associate tnf- polymorphisms and the level of expression with insulin resistance or disease severity in nafld patients . in conclusion , this study provided preliminary evidence in favor of an association between presence of a tnf--238 polymorphism and the development of cad in nafld patients of chinese han origin . in addition , the tnf--308 ga heterozygote genotype was positively associated with increased levels of tg in nafld patients with cad , suggesting the potential role of tnf--308 ga genotype in the development of cad in these patients . however , mechanisms underlying the association between tnf- gene polymorphisms and the risk of cad in nafld patients will require further investigation . | [
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] |
hypertension is defined as elevated blood pressure ( bp ) above 140 mm hg systolic and 90 mm hg diastolic when measured under standardized conditions .
hypertension can be a separate chronic medical condition estimated to be affecting a quarter of the world 's adult population , as well as a risk factor for other chronic and nonchronic patient groups .
traditional high - risk patient groups include diabetics , pregnant women with gestational diabetes or preeclampsia , and kidney disease patients . for chronic hypertensive patients ,
persistent hypertension is one of the key risk factors for strokes , heart attacks , heart and kidney failure , and other heart and circulatory diseases and increased mortality .
preeclampsia is the most common cause of maternal and fetal death . for gestational diabetes and preeclampsia patients ,
the accurate measurement of bp during pregnancy is one of the most important aspects of prenatal care . for kidney disease patients and diabetics
, blood pressure should be kept below 130 mmhg systolic and 80 mm hg diastolic to protect the kidneys from bp - induced damage . as there are usually no symptoms , frequent blood pressure controls are highly relevant for these high - risk groups .
the level of the blood pressure is the main factor in the decision to start antihypertensive therapy and other interventions .
measurements can be performed either at the clinic or in the home setting . in the clinical setting
it is believed that this is due to the anxiety some people experience during a visit to the clinic .
this is known as the white coat effect and is reported to be affecting between 20% to 40% of all patients visiting a clinic [ 7 , 8 ] . as a consequence ,
the current international guideline on bp measurement , is to followup on measurements obtained in the clinic using bpsm to negate the white coat effect [ 59 ] .
bpsm is used to diagnose patients suspected of being hypertensive , as well as for long - term monitoring .
bpsm is considered a valid method for determining the blood pressure ( bp ) of patients with hypertension and other bp - related conditions , providing that the best - practice recommendations for obtaining the measurements are followed [ 1012 ] .
these recommendations are defined by a range of national and international clinical associations [ 1317 ] .
recommendations include the following : patient should be sufficiently rested and seated correctly before and during measurement ; patient should reside in a quiet environment and should not talk during measurement .
recommendations vary between the different countries and organizations , but in general they cover the same fundamental topics .
the aim of this study is to investigate currently used bpsm interventions and identify challenges that could influence the resulting data quality .
we planned an observational descriptive study consisting of a series of field studies at two medical clinics and two hospital departments combined with a questionnaire .
field studies were performed as a combination of observations [ 18 , 19 ] of patients getting instructions and performing bpsm , combined with open - ended and semistructured interviews [ 19 , 20 ] with healthcare providers , including doctors and nurses .
main objectives of the field studies were to investigate current state - of - the - art and usage of bpsm devices in the public danish healthcare sector , including how patients are instructed and bpsm data is handled .
a questionnaire targeting bpsm users was posted at the web site of the danish heart foundation to investigate user background and subjective understanding of the recommendations .
the primary qualitative findings of the study were analyzed , thematized , and triangulated with the literature [ 18 , 22 ] .
the department of internal medicine and cardiology , aarhus university hospital , covers a population of 200.000 people and has around .
primary age groups are the middle aged and seniors . as part of the department ,
the blood pressure clinic ( bpc ) specializes in severe hypertensive patients referred from other departments and the primary sector . during a two - day field study , we interviewed the consultant cardiologic physician in charge of the bpc in an open - ended interview on the relevance of bpsm , current clinical praxis , state - of - the - art , and other topics .
we also followed a specialist nurse while instructing hypertensive patients on how to perform both 24-hour ambulatory bpsm ( abpsm ) and 3 days of home bpsm ( hbpsm ) .
we observed how the nurse conducted interviews and training with five patients to establish various aspects of the patients ' overall health condition and use of medication , as well as handling of patients returning from bpsm in the home setting .
the department of obstetrics , aarhus university hospital , delivers around 5.000 babies each year and performs 20.000 scans .
also , at the department , the majority of eastern jutland 's pregnant women , covering a population of around 2.9 million suffering from complications in their pregnancy are received . in all 14.000 visits to the obstetric outpatient clinic
this includes pregnant women suffering from hypertension , diabetes , preeclampsia , and other complications .
some pregnant women need to visit the clinic up to 19 times during a pregnancy , for self - measuring bp , weight , protein levels , blood sugar , and fetal ctg , mainly in an unsupervised setting .
three nurses where followed in their daily routines spanning two days , first in a purely observational study observing five patients , then followed by a contextual inquiry into the routines with follow - up questions .
we interviewed a physician and the department nurses on the clinical praxis of bpsm , patient training , and data capture .
most hypertensive patients in denmark are treated by their general practitioner in a medical clinic .
we visited two medical clinics in the eastern jutland covering around 8.000 patients in total .
both clinics are typical examples of a modern general practioner 's ( gp ) clinic , with a community of four - to - six physicians and several support staff , including nurses and secretaries . at the two medical clinics over a period of three days , we interviewed two gp 's and two nurses on the topic of hbpsm , while also discussing medical adherence and related topics .
also , data delivery was discussed , including requirements from the staff on how data could arrive at the clinic and be sent automatically from the home of the patient and problems with staff reporting bias .
we observed how bp was measured while at the clinics and how patients were counseled on hbpsm and trained to follow the recommendations .
also , three videos were recorded of nurses instructing patients on hbpsm for future reference .
all the interviewed physicians and nurses reported that elevated bp levels up to 2060 mmhg were not uncommon when measured in the clinic , as compared with patients later self - monitoring their actual bp at home confirming the daily presence of the white coat effect .
thus , bpsm is considered an important and relevant diagnostic intervention for uncovering the actual and unbiased bp of the patients by all healthcare providers involved in the study .
we found that all four clinics use either abpsm devices for obtaining a 24-hour profile of patient bp with 15-minute interval , or three days of point measurements with a hbpsm device .
abpsm is used as the primary method in the bpc clinic and is considered the gold standard in all clinics .
patients who do not respond well to abpsm are instead sent home with a hbpsm device .
abpsm is used rarely in the two gp clinics , with hbpsm being the predominant intervention .
the department of obstetrics do not use abpsm but rely on patients using hbpsm devices unsupervised , while attending the outpatient clinic as part of their weekly visit .
bpc is the only clinic that has automatic data capture of hbpsm data , the other three relying on the patient keeping a manual paper log of measurements .
we found that all the studied clinics follow the recommendations from the danish hypertension association ( dha ) providing training for their patients on how to perform reliable hbpsm or abpsm .
with hbpsm we witnessed a total of ten patients getting instructed by nurses at the four clinics on how to perform reliable hbpsm following the established recommendations over the next three days .
these instructions varied slightly in scope , but all followed the overall guidelines as defined by the dha . also , we observed the patients performing a trial measurement . to the hbpsm patients , the healthcare providers would reiterate the importance of following the recommendations as handed out on paper , including taking the measurement on the same time of day , not lending the device to other people as the device can not differentiate between users . besides the recommendations from the danish hypertension association , the nurse at the bpc clinic also instructed the patient not to look at the first of the three measurements in a series , in order not to build up anxiety levels . in all
studied clinics we found that the hbpsm healthcare process is initiated with an interview , after which the patient is instructed in correct hbpsm usage including a supervised trial self - measurement .
next , the healthcare provider arranges for a new appointment for the patients to return with the device after three days of measurement except at the department of obstetrics where the patients self - measure at the clinic only .
the healthcare process ends with a nurse or physician collecting the data , either manually transferring the data from the paper log or using a data cable connecting the clinic computer to the hbpsm device , before handing the dataset over to the treating physician for further diagnosis .
this process is typically repeated in a half - yearly or quarterly cycle as hypertension is considered a life - long chronic condition . at the bpc clinic ,
the patients are referred to their gp for future reference , once they are considered well treated .
likewise , at the department of obstetrics the pregnant women are only followed during their pregnancy and then referred to their gp for followup . during our interviews with gps and cardiologists , we found that the hbpsm and abpsm devices used by healthcare professionals are required to be on a limited list of approved devices [ 16 , 24 ] .
all devices we observed being used during the field studies were later confirmed to be on this list . a review of the various device - manufactures product pages revealed that none of these bp devices supported verifying patient compliance to the recommendations .
we found that the healthcare personnel in general trusted in their patient 's abilities to perform correct home measurements .
however , the two interviewed gps also stated that they had no way to ascertain the actual level of patient adherence to the recommendations .
both interviewed gp 's reported that , when a patient 's self - reported data indicated that the patient had done a procedural mistake , the gp would ask the patient to demonstrate a sample measurement for verification of patient understanding of the procedure . however , both interviewed gp 's stated to have limited time to deal with such suspicions in daily praxis and did not keep records of the problem . as such , the level of user error could not be quantified .
the field studies only provided limited observations useful for evaluating patient awareness and understanding of the recommendations . at the bpc clinic
, one patient had taken off his abpsm device during the night as it annoyed him . during the field studies at the obstetric outpatient clinic we witnessed five pregnant patients self - monitoring . here , we observed user errors in four out of five measurements that could indicate limited patient understanding of the recommendations .
user errors included one patient having crossed legs and back unsupported during measurement , another patient talking with the nurse during measurement , while the last two patients did not rest for the required amount of time prior to measurement .
the field studies did not provide any direct observations of patient - induced reporting bias .
however , the interviewed nurses and physicians relayed their suspicions on this occurring as bp data developments would sometimes not appear realistic .
this could either be due to lack of patient understanding of proper protocol or due to reporting errors . as there are currently no means available for the healthcare personnel at the two gp clinics and the obstetric outpatient clinic to reliably detect such patient - induced reporting bias , the interviewed personnel could not account for the level of the problem . at the bpc clinic ,
patient - induced reporting bias is not conceivable , as data are recorded and transferred electronically . at the two gp clinics , both nurses and
gp 's stated that measurements could be subject to nurse - induced reporting bias when entering the individual measurements into the gp electronic patient record system .
several examples were given including difficulties in reading the patient 's hand - written diaries , transition from paper to screen errors , calculation errors while getting the average bp levels , and other sources of bias .
it was not possible to quantify the rate or severity of errors , but physicians and nurses from the two gp clinics deemed the problem to be relatively frequent .
the personnel at the bpc clinic suspected that some of the bpsm measurements that would automatically be recorded and transferred to the clinical computer system might stem from other persons than the intended patients .
as patients could easily lend out their devices to other persons , for example , relatives or friends , either knowingly or unknowingly , there was a potential risk of unintended persons ' bp measurements ending up as part of the originally intended patients bp data set .
at the two gp clinics and the obstetric outpatient clinic , the employed bpsm procedure relies on patients self - reporting data , transferring them manually from device screen to paper . here , this did not appear to be an issue due to the manual nature of the procedure .
a questionnaire was posted at the website of the danish heart foundation advertising for participants using hbpsm .
a total of 201 respondents in the age groups 2080 provided answers . of these , 130 ( 65% ) self - reported to suffer from hypertension and 124 ( 62% ) reported taking hypertensive medication .
a total of 124 ( 62% ) reported having a bp measurement device in their home .
more than half of the respondents , 121 , measure their bp at least once every third month at home , 35 ( 17% ) once a week , 24 ( 14% ) biyearly , 6 ( 3% ) yearly , while 24 ( 12% ) do not self - measure but get their bp measured at the clinic .
only 3 ( 1.5% ) respondents reported not measuring their bp at all . when asked about knowledge of the selected recommendations , 140 ( 70% ) of respondents knew that they had to rest five minutes prior to measurement .
another 86 ( 43% ) knew both of these recommendations , while none of the respondents had knowledge of all of the recommendations .
see figure 1 for an overview of respondent 's detailed knowledge of the recommendations . focusing the result set to those 130 respondents who self - reported to suffer from chronic hypertension
, a total of 104 ( 80% ) of respondents knew that they had to rest five minutes prior to measurement .
the recommendation not to talk during measurement was known by 74 ( 57% ) of the respondents .
another 65 ( 50% ) knew both of these recommendations , while none of the self - reported hypertensive respondents had knowledge of all of the recommendations .
further narrowing the scope to those measuring their bp once a week limits the number of respondents to 28 .
the majority of these , 24 ( 86% ) , knew that they had to rest 5 minutes prior to measurement , while 57% knew that they should not talk during measurement .
from both the field studies and the literature , it appears that hbpsm interventions are considered useful for avoiding white - coat - induced bias and obtaining the actual blood pressure of the patients [ 8 , 12 , 14 , 25 , 26 ] .
results indicate that the studied nurses and doctors all recognize the importance of following the recommendations for hbpsm interventions in the unsupervised setting and that they are instructing their patients on how to perform correct measurements in accordance with this .
however , several potential challenges that could affect the data quality of hbpsm measurements stand out from the study findings . in the following we provide a list of such potential challenges triangulated with findings from the literature .
findings from the field studies indicate that existing state - of - the - art bp devices do not automatically ensure that the recommendations are followed .
some devices are able to store bp readings along with date and time meta - data .
such devices have previously been used to determine the level of compliance with reporting self - measured data [ 2730 ] . however , the majorities of clinics visited ( 75% ) does not use the storage features of the bp devices but rather relies on paper - based log books for data capture and exchange .
furthermore , in the biomedical bp devices that are currently being used in the studied clinics , it is only date - and - time contextual meta - data that is captured and not factors related to the remaining recommendations .
a survey of all bhs - approved devices indicate that no state - of - the - art devices are available that can ensure that any of the recommendations as presented in figure 1 are followed [ 16 , 24 ] . as observed in the field studies , the healthcare providers relied on the individual patient 's ability to perform a correct measurement in the unsupervised setting , either the home or the outpatient clinic , being dependent on the patients training and willingness to adhere to the recommendations .
healthcare providers were not able to quantify adherence levels of the patients , beside time and date of measurements , and the bp measurements themselves .
patients are instructed by the healthcare professionals to use the home bp device for self - measurement , either in the home setting or at the outpatient clinic , and to follow the recommendations provided by the healthcare professionals .
findings from the field studies and the questionnaire study indicate that patients are not always aware of the recommendations and the importance of following them . as a consequence patients may only be following them in part or not at all .
the questionnaire study indicated that none of the respondents were aware of all the recommendations , while 91% were aware of one or more .
this was further supported by our observations in the field study , where we observed users not following the recommendations during self - measurements at the obstetric outpatient clinic .
patients who are keeping manual log of their bp self - measurement data could risk introducing errors during the transfer of data from the device display to the paper log .
this could occur not only through rounding errors or misreading of digits but also through accidental or deliberate deletion of individual measurements .
findings from the field studies indicate that not all healthcare professionals , including the staff at aarhus university hospital , skejby and at the two gp clinics , use automated data transfer from the biomedical devices . as 75% of the studied clinics rely on manual paper logs , this could indicate a potential risk of patient - induced reporting bias .
previous studies have found reporting bias to affect up to 50% of all bp self - measurements [ 2730 ] .
reporting bias , both healthcare provider and patient induced , is however easy to avoid by using state - of - the - art equipment with the ability to transfer data automatically from a bp device to electronic healthcare records .
findings from the field studies indicate , that patients keeping manual logbook of their self - measurement of bp data require the nurse ( or other healthcare provider ) to transfer data from the logbook to the computer - based records . in this process
the healthcare provider could risk inducing errors during the transfer . in the literature , this type of error has not been investigated thoroughly , and the level of data - transfer bias has not been quantified in the field studies , though it was indicated at both gp clinics as a potential risk . in the case of another user
than the intended patient using an automatic bp device for performing a measurement , the data will automatically , but erroneously , become part of the data set of the patient it is registered to .
the existing and currently employed automatic bp devices have no means of differentiating between different users , and the data can not be easily removed from the system by the user [ 16 , 24 ] . this risk was explicitly pointed out at the bpc clinic to patients during their training sessions . in the three other clinics , this
was not viewed as a likely problem , as the patient keeps a manual paper log that most likely would mitigate this challenge .
results indicate that bpsm as a clinical method could be subject to bias to such an extent that results are unfit for diagnostic , monitoring , or scientific use .
a user talking during self - measurement could bias the measured bp data levels with as much as 720 mm hg [ 6 , 13 ] , which would suffice to change patient diagnosis from normotensive ( healthy ) to hypertensive , thus requiring medication .
other bias could be introduced from other activities while underreporting could have the opposite effect for some . in the literature
, underreporting has been reported to occur frequently and on a significant scale [ 2830 ] .
this could affect the data quality of previous clinical studies , such as those reviewed by abudagga et al . where study methods rely on such unsupervised self - measured data sets .
furthermore , unsupervised healthcare self - measurement techniques could even be perceived as misleading or harmful , resulting in misdiagnosis and potential over- or under - medication of patients . however , as observed in the field study and known from the literature , the prevalence of the white coat effect necessitates hbpsm in the home of the patient as it is the only method to provide the actual bp of patients suffering from this syndrome . as
such , bpsm in the unsupervised setting is likely to remain a commonly used clinical method .
other incentives for utilizing bpsm include improved data material through more frequent measurements as well as for the convenience of the patients as they do not need to visit the clinic for having their measurements taken [ 30 , 31 ] .
the findings from the four reported field studies indicate that the problems of potentially reduced measurement - adherence reported in the literature could be relevant as it can not be verified whether patients self - measuring at home are always following the recommendations as intended .
a patient not following the recommendations in full leads to biased data and may thus result in potential misdiagnosis of patients .
the extent of the problem has not been quantified , as this requires further studies .
however , previous work has indicated that reporting bias alone may be affecting a large proportion of all self - measurements ( up to 50% ) and that this could be a major problem [ 2830 ] . as reporting bias
primarily consists of reading digits on the display and transferring them to paper , other problems which can not be measured with existing technology could be a much greater problem .
we have identified a range of potential challenges related to bpsm in the unsupervised setting . however , our findings are based on a limited qualitative empirical data material and should be treated as indicative . in order to investigate these challenges further , we need methods and tools that will allow us to gain a better understanding of how the patient is acting in situ in the home setting or at the outpatient clinic while self - monitoring .
traditional methods useful for this kind of research activities include observations in the home setting by researchers , either directly or through video capture [ 19 , 20 ] .
this requires the researchers to be present in the home and may be highly time consuming and privacy invading . as such
there is also the risk of inducing bias on patient behavior during measurements , while being observed .
the presence of researchers might prompt patients to follow recommendations to a larger extent when being observed .
however , they still require a fairly large technical setup as well as extensive reviewing work . also , the ability to correctly capture all context information might also be limited when using a single camera only
. it might for example be difficult to capture video for verifying both whether the user is correctly seated while also monitoring placement of the feet and the handling of the bp device .
as an alternative to direct or indirect visual observations , we suggest that it may be feasible to develop research prototypes that can discretely measure the desired use - context during a healthcare session .
for example , we may be able to develop a system with sensors to detect whether the user is correctly seated and measure noise levels during bpsm to quantify adherence levels . rather than by direct observations or video reviews , the researchers will gain a formalized and discrete data set of relevant user behavior that can automatically be data - processed and correlated with the measurements . in this way
, it may arguably be faster to perform longitudinal studies on a larger population , leading to a more comprehensive understanding than could be achieved with manual observations and data processing .
depending on the problem under investigation , there is a need for a data acquisition platform , consisting of a computer with relevant processing capabilities , sensors for data capture , a sufficient infrastructure , relevant user interface hardware , video and audio recording , patient identification , and other relevant features .
we find that context - aware technologies and concepts appear promising to meet these requirements .
context - aware technology is an established area within the combined fields of ubiquitous and pervasive computing [ 3234 ] .
dey has previously introduced the concept of context - tagging of information , where pervasive systems have the ability to attach relevant context meta - data to the primary data set . applying these concepts to the identified challenges of
this could include attaching relevant context information to a bp measurement data set , such as whether the user was sufficiently rested when performing the measurement , seated correctly when medication was taken , as well as any other relevant contextual factors which are known to impact bp measurements . to this purpose , a range of context - aware and extendable middleware technologies intended for developing context - aware pervasive healthcare [ 36 , 37 ] and telemedicine prototypes are available .
this includes soprano , openaal , jcaf , amica , hydra , spine , and opencare [ 3843 ] . building a platform for evaluating the reliability of bpsm and other relevant telemedicine topics by acquiring relevant context information
we suggest developing systems using existing context - aware technologies , for learning more on user behavior in the unsupervised setting .
an improved understanding of the disease domain and the related challenges could also be used to suggest novel strategies based on technological solutions for improving measurement - compliance . through the of use context - aware technology
, we may be able to better quantify the level of compliance with the recommendations , thus providing a marker on the quality of the data obtained in the home setting or during self - measurement in general .
lessons learned from experimental research prototypes may also contribute to designing more advanced biomedical devices for clinical use enabling bp devices to better sense the measurement context and possibly provide better user guidance relying on context - aware technologies .
this could include sensing noise levels and user activity levels , as well as whether the user is correctly seated .
if all bias sources and their relative effect on bp can be sufficiently determined , then it might be possible to automatically compensate and filter out such bias of the resulting bp , providing a more valid data set to the healthcare provider .
findings could also be applied to state - of - the - art pervasive healthcare telemedicine solutions , such as the intel health guide psh6000 and the tunstall mymedic telehealth monitor .
these healthcare platforms have been used in several clinical trials , including in the telekat project .
refitting such systems with context - aware technology for sensing user behavior is possible through the continua alliance open standards and would allow us to quantify bias during daily use .
this class of systems also provides enhanced communication features for easier user guidance , as well as more processing power than current stand - alone biomedical devices feature today .
finally , results could be useful for inspiring future smart home infrastructure technology design [ 48 , 49 ] . here ,
a built - in infrastructure for context awareness and user interaction is contemplated as being an integral element of every future smart home allowing us to track noncompliant user behavior [ 50 , 51 ] .
we found that the studied healthcare personnel perceived bpsm as a relevant and useful method for obtaining the unbiased bp of patients , providing that the clinical recommendations are followed .
contrasting this , the questionnaire respondents appeared to have an incomplete understanding of the recommendations and the need to follow them during bpsm .
these challenges indicated frequent and severe user - induced bias during bpsm which could result in an indeterminable data quality of the measurements .
biased and unreliable data leads to potential misdiagnosis of patients , possibly affecting large patient groups in current clinical praxis .
also , the challenges and resulting bias may impact the validity of results from previous clinical trials on bpsm , as patient compliance with the guidelines has not been investigated sufficiently in these studies .
in order to gain a better understanding of the six challenges and their consequences , we have proposed developing novel methods and context - aware technological support tools to better detect and quantify such user errors . these tools could also allow us to develop and investigate experimental assistive measures to help users overcome the challenges themselves or automatically compensate for any measurement bias .
we suggest that context - aware methodology and technology could be useful for such purposes building on the existing body of work within the pervasive healthcare community , including software frameworks and sensor technology . in the future our research could be used to develop better biomedical bp devices , as well as improve existing telemedicine and telemonitoring platforms and inspire future smart home technology to overcome the six identified challenges . | blood pressure self - measurement ( bpsm ) requires patients to follow a range of recommendations in order to be considered reliable for diagnostic use .
we investigated currently used bpsm interventions at four medical clinics combined with an online questionnaire targeting bpsm users .
we found that the participating healthcare personnel perceived bpsm as a relevant and useful intervention method providing that the recommendations are followed .
a total of six challenges were identified : ( 1 ) existing devices do not guarantee that the recommendations are followed , ( 2 ) healthcare providers can not verify whether self - monitoring patients follow the recommendations , ( 3 ) patients are not aware of all recommendations and the need to follow them , ( 4 ) risk of patient induced reporting bias , ( 5 ) risk of healthcare provider induced data - transfer bias , and ( 6 ) risk of data being registered as belonging to the wrong patient .
we conclude that existing bpsm interventions could be significantly affected by user - induced bias resulting in an indeterminable quality of the measurement data .
therefore , we suggest applying context - aware technological support tools to better detect and quantify user errors . this may allow us to develop solutions that could overcome or compensate for such errors in the future . | 1. Background
2. Methods
3. Results
4. Discussion
5. Conclusion | hypertension can be a separate chronic medical condition estimated to be affecting a quarter of the world 's adult population , as well as a risk factor for other chronic and nonchronic patient groups . for chronic hypertensive patients ,
persistent hypertension is one of the key risk factors for strokes , heart attacks , heart and kidney failure , and other heart and circulatory diseases and increased mortality . for gestational diabetes and preeclampsia patients ,
the accurate measurement of bp during pregnancy is one of the most important aspects of prenatal care . for kidney disease patients and diabetics
, blood pressure should be kept below 130 mmhg systolic and 80 mm hg diastolic to protect the kidneys from bp - induced damage . the level of the blood pressure is the main factor in the decision to start antihypertensive therapy and other interventions . in the clinical setting
it is believed that this is due to the anxiety some people experience during a visit to the clinic . this is known as the white coat effect and is reported to be affecting between 20% to 40% of all patients visiting a clinic [ 7 , 8 ] . as a consequence ,
the current international guideline on bp measurement , is to followup on measurements obtained in the clinic using bpsm to negate the white coat effect [ 59 ] . bpsm is considered a valid method for determining the blood pressure ( bp ) of patients with hypertension and other bp - related conditions , providing that the best - practice recommendations for obtaining the measurements are followed [ 1012 ] . these recommendations are defined by a range of national and international clinical associations [ 1317 ] . the aim of this study is to investigate currently used bpsm interventions and identify challenges that could influence the resulting data quality . we planned an observational descriptive study consisting of a series of field studies at two medical clinics and two hospital departments combined with a questionnaire . field studies were performed as a combination of observations [ 18 , 19 ] of patients getting instructions and performing bpsm , combined with open - ended and semistructured interviews [ 19 , 20 ] with healthcare providers , including doctors and nurses . main objectives of the field studies were to investigate current state - of - the - art and usage of bpsm devices in the public danish healthcare sector , including how patients are instructed and bpsm data is handled . a questionnaire targeting bpsm users was posted at the web site of the danish heart foundation to investigate user background and subjective understanding of the recommendations . the primary qualitative findings of the study were analyzed , thematized , and triangulated with the literature [ 18 , 22 ] . as part of the department ,
the blood pressure clinic ( bpc ) specializes in severe hypertensive patients referred from other departments and the primary sector . during a two - day field study , we interviewed the consultant cardiologic physician in charge of the bpc in an open - ended interview on the relevance of bpsm , current clinical praxis , state - of - the - art , and other topics . we observed how the nurse conducted interviews and training with five patients to establish various aspects of the patients ' overall health condition and use of medication , as well as handling of patients returning from bpsm in the home setting . in all 14.000 visits to the obstetric outpatient clinic
this includes pregnant women suffering from hypertension , diabetes , preeclampsia , and other complications . some pregnant women need to visit the clinic up to 19 times during a pregnancy , for self - measuring bp , weight , protein levels , blood sugar , and fetal ctg , mainly in an unsupervised setting . we interviewed a physician and the department nurses on the clinical praxis of bpsm , patient training , and data capture . we visited two medical clinics in the eastern jutland covering around 8.000 patients in total . at the two medical clinics over a period of three days , we interviewed two gp 's and two nurses on the topic of hbpsm , while also discussing medical adherence and related topics . also , data delivery was discussed , including requirements from the staff on how data could arrive at the clinic and be sent automatically from the home of the patient and problems with staff reporting bias . we observed how bp was measured while at the clinics and how patients were counseled on hbpsm and trained to follow the recommendations . all the interviewed physicians and nurses reported that elevated bp levels up to 2060 mmhg were not uncommon when measured in the clinic , as compared with patients later self - monitoring their actual bp at home confirming the daily presence of the white coat effect . thus , bpsm is considered an important and relevant diagnostic intervention for uncovering the actual and unbiased bp of the patients by all healthcare providers involved in the study . we found that all four clinics use either abpsm devices for obtaining a 24-hour profile of patient bp with 15-minute interval , or three days of point measurements with a hbpsm device . we found that all the studied clinics follow the recommendations from the danish hypertension association ( dha ) providing training for their patients on how to perform reliable hbpsm or abpsm . with hbpsm we witnessed a total of ten patients getting instructed by nurses at the four clinics on how to perform reliable hbpsm following the established recommendations over the next three days . to the hbpsm patients , the healthcare providers would reiterate the importance of following the recommendations as handed out on paper , including taking the measurement on the same time of day , not lending the device to other people as the device can not differentiate between users . besides the recommendations from the danish hypertension association , the nurse at the bpc clinic also instructed the patient not to look at the first of the three measurements in a series , in order not to build up anxiety levels . in all
studied clinics we found that the hbpsm healthcare process is initiated with an interview , after which the patient is instructed in correct hbpsm usage including a supervised trial self - measurement . next , the healthcare provider arranges for a new appointment for the patients to return with the device after three days of measurement except at the department of obstetrics where the patients self - measure at the clinic only . at the bpc clinic ,
the patients are referred to their gp for future reference , once they are considered well treated . during our interviews with gps and cardiologists , we found that the hbpsm and abpsm devices used by healthcare professionals are required to be on a limited list of approved devices [ 16 , 24 ] . a review of the various device - manufactures product pages revealed that none of these bp devices supported verifying patient compliance to the recommendations . we found that the healthcare personnel in general trusted in their patient 's abilities to perform correct home measurements . however , the two interviewed gps also stated that they had no way to ascertain the actual level of patient adherence to the recommendations . both interviewed gp 's reported that , when a patient 's self - reported data indicated that the patient had done a procedural mistake , the gp would ask the patient to demonstrate a sample measurement for verification of patient understanding of the procedure . the field studies only provided limited observations useful for evaluating patient awareness and understanding of the recommendations . during the field studies at the obstetric outpatient clinic we witnessed five pregnant patients self - monitoring . here , we observed user errors in four out of five measurements that could indicate limited patient understanding of the recommendations . the field studies did not provide any direct observations of patient - induced reporting bias . as there are currently no means available for the healthcare personnel at the two gp clinics and the obstetric outpatient clinic to reliably detect such patient - induced reporting bias , the interviewed personnel could not account for the level of the problem . at the bpc clinic ,
patient - induced reporting bias is not conceivable , as data are recorded and transferred electronically . at the two gp clinics , both nurses and
gp 's stated that measurements could be subject to nurse - induced reporting bias when entering the individual measurements into the gp electronic patient record system . the personnel at the bpc clinic suspected that some of the bpsm measurements that would automatically be recorded and transferred to the clinical computer system might stem from other persons than the intended patients . as patients could easily lend out their devices to other persons , for example , relatives or friends , either knowingly or unknowingly , there was a potential risk of unintended persons ' bp measurements ending up as part of the originally intended patients bp data set . at the two gp clinics and the obstetric outpatient clinic , the employed bpsm procedure relies on patients self - reporting data , transferring them manually from device screen to paper . here , this did not appear to be an issue due to the manual nature of the procedure . a questionnaire was posted at the website of the danish heart foundation advertising for participants using hbpsm . a total of 201 respondents in the age groups 2080 provided answers . a total of 124 ( 62% ) reported having a bp measurement device in their home . more than half of the respondents , 121 , measure their bp at least once every third month at home , 35 ( 17% ) once a week , 24 ( 14% ) biyearly , 6 ( 3% ) yearly , while 24 ( 12% ) do not self - measure but get their bp measured at the clinic . when asked about knowledge of the selected recommendations , 140 ( 70% ) of respondents knew that they had to rest five minutes prior to measurement . another 86 ( 43% ) knew both of these recommendations , while none of the respondents had knowledge of all of the recommendations . see figure 1 for an overview of respondent 's detailed knowledge of the recommendations . focusing the result set to those 130 respondents who self - reported to suffer from chronic hypertension
, a total of 104 ( 80% ) of respondents knew that they had to rest five minutes prior to measurement . another 65 ( 50% ) knew both of these recommendations , while none of the self - reported hypertensive respondents had knowledge of all of the recommendations . from both the field studies and the literature , it appears that hbpsm interventions are considered useful for avoiding white - coat - induced bias and obtaining the actual blood pressure of the patients [ 8 , 12 , 14 , 25 , 26 ] . results indicate that the studied nurses and doctors all recognize the importance of following the recommendations for hbpsm interventions in the unsupervised setting and that they are instructing their patients on how to perform correct measurements in accordance with this . however , several potential challenges that could affect the data quality of hbpsm measurements stand out from the study findings . findings from the field studies indicate that existing state - of - the - art bp devices do not automatically ensure that the recommendations are followed . furthermore , in the biomedical bp devices that are currently being used in the studied clinics , it is only date - and - time contextual meta - data that is captured and not factors related to the remaining recommendations . a survey of all bhs - approved devices indicate that no state - of - the - art devices are available that can ensure that any of the recommendations as presented in figure 1 are followed [ 16 , 24 ] . as observed in the field studies , the healthcare providers relied on the individual patient 's ability to perform a correct measurement in the unsupervised setting , either the home or the outpatient clinic , being dependent on the patients training and willingness to adhere to the recommendations . healthcare providers were not able to quantify adherence levels of the patients , beside time and date of measurements , and the bp measurements themselves . patients are instructed by the healthcare professionals to use the home bp device for self - measurement , either in the home setting or at the outpatient clinic , and to follow the recommendations provided by the healthcare professionals . findings from the field studies and the questionnaire study indicate that patients are not always aware of the recommendations and the importance of following them . the questionnaire study indicated that none of the respondents were aware of all the recommendations , while 91% were aware of one or more . this was further supported by our observations in the field study , where we observed users not following the recommendations during self - measurements at the obstetric outpatient clinic . patients who are keeping manual log of their bp self - measurement data could risk introducing errors during the transfer of data from the device display to the paper log . as 75% of the studied clinics rely on manual paper logs , this could indicate a potential risk of patient - induced reporting bias . previous studies have found reporting bias to affect up to 50% of all bp self - measurements [ 2730 ] . reporting bias , both healthcare provider and patient induced , is however easy to avoid by using state - of - the - art equipment with the ability to transfer data automatically from a bp device to electronic healthcare records . findings from the field studies indicate , that patients keeping manual logbook of their self - measurement of bp data require the nurse ( or other healthcare provider ) to transfer data from the logbook to the computer - based records . in the literature , this type of error has not been investigated thoroughly , and the level of data - transfer bias has not been quantified in the field studies , though it was indicated at both gp clinics as a potential risk . in the case of another user
than the intended patient using an automatic bp device for performing a measurement , the data will automatically , but erroneously , become part of the data set of the patient it is registered to . the existing and currently employed automatic bp devices have no means of differentiating between different users , and the data can not be easily removed from the system by the user [ 16 , 24 ] . in the three other clinics , this
was not viewed as a likely problem , as the patient keeps a manual paper log that most likely would mitigate this challenge . results indicate that bpsm as a clinical method could be subject to bias to such an extent that results are unfit for diagnostic , monitoring , or scientific use . a user talking during self - measurement could bias the measured bp data levels with as much as 720 mm hg [ 6 , 13 ] , which would suffice to change patient diagnosis from normotensive ( healthy ) to hypertensive , thus requiring medication . furthermore , unsupervised healthcare self - measurement techniques could even be perceived as misleading or harmful , resulting in misdiagnosis and potential over- or under - medication of patients . however , as observed in the field study and known from the literature , the prevalence of the white coat effect necessitates hbpsm in the home of the patient as it is the only method to provide the actual bp of patients suffering from this syndrome . other incentives for utilizing bpsm include improved data material through more frequent measurements as well as for the convenience of the patients as they do not need to visit the clinic for having their measurements taken [ 30 , 31 ] . the findings from the four reported field studies indicate that the problems of potentially reduced measurement - adherence reported in the literature could be relevant as it can not be verified whether patients self - measuring at home are always following the recommendations as intended . a patient not following the recommendations in full leads to biased data and may thus result in potential misdiagnosis of patients . however , previous work has indicated that reporting bias alone may be affecting a large proportion of all self - measurements ( up to 50% ) and that this could be a major problem [ 2830 ] . as reporting bias
primarily consists of reading digits on the display and transferring them to paper , other problems which can not be measured with existing technology could be a much greater problem . we have identified a range of potential challenges related to bpsm in the unsupervised setting . in order to investigate these challenges further , we need methods and tools that will allow us to gain a better understanding of how the patient is acting in situ in the home setting or at the outpatient clinic while self - monitoring . this requires the researchers to be present in the home and may be highly time consuming and privacy invading . the presence of researchers might prompt patients to follow recommendations to a larger extent when being observed . it might for example be difficult to capture video for verifying both whether the user is correctly seated while also monitoring placement of the feet and the handling of the bp device . as an alternative to direct or indirect visual observations , we suggest that it may be feasible to develop research prototypes that can discretely measure the desired use - context during a healthcare session . for example , we may be able to develop a system with sensors to detect whether the user is correctly seated and measure noise levels during bpsm to quantify adherence levels . we find that context - aware technologies and concepts appear promising to meet these requirements . context - aware technology is an established area within the combined fields of ubiquitous and pervasive computing [ 3234 ] . dey has previously introduced the concept of context - tagging of information , where pervasive systems have the ability to attach relevant context meta - data to the primary data set . applying these concepts to the identified challenges of
this could include attaching relevant context information to a bp measurement data set , such as whether the user was sufficiently rested when performing the measurement , seated correctly when medication was taken , as well as any other relevant contextual factors which are known to impact bp measurements . to this purpose , a range of context - aware and extendable middleware technologies intended for developing context - aware pervasive healthcare [ 36 , 37 ] and telemedicine prototypes are available . building a platform for evaluating the reliability of bpsm and other relevant telemedicine topics by acquiring relevant context information
we suggest developing systems using existing context - aware technologies , for learning more on user behavior in the unsupervised setting . an improved understanding of the disease domain and the related challenges could also be used to suggest novel strategies based on technological solutions for improving measurement - compliance . through the of use context - aware technology
, we may be able to better quantify the level of compliance with the recommendations , thus providing a marker on the quality of the data obtained in the home setting or during self - measurement in general . lessons learned from experimental research prototypes may also contribute to designing more advanced biomedical devices for clinical use enabling bp devices to better sense the measurement context and possibly provide better user guidance relying on context - aware technologies . if all bias sources and their relative effect on bp can be sufficiently determined , then it might be possible to automatically compensate and filter out such bias of the resulting bp , providing a more valid data set to the healthcare provider . findings could also be applied to state - of - the - art pervasive healthcare telemedicine solutions , such as the intel health guide psh6000 and the tunstall mymedic telehealth monitor . refitting such systems with context - aware technology for sensing user behavior is possible through the continua alliance open standards and would allow us to quantify bias during daily use . we found that the studied healthcare personnel perceived bpsm as a relevant and useful method for obtaining the unbiased bp of patients , providing that the clinical recommendations are followed . contrasting this , the questionnaire respondents appeared to have an incomplete understanding of the recommendations and the need to follow them during bpsm . these challenges indicated frequent and severe user - induced bias during bpsm which could result in an indeterminable data quality of the measurements . in order to gain a better understanding of the six challenges and their consequences , we have proposed developing novel methods and context - aware technological support tools to better detect and quantify such user errors . these tools could also allow us to develop and investigate experimental assistive measures to help users overcome the challenges themselves or automatically compensate for any measurement bias . we suggest that context - aware methodology and technology could be useful for such purposes building on the existing body of work within the pervasive healthcare community , including software frameworks and sensor technology . in the future our research could be used to develop better biomedical bp devices , as well as improve existing telemedicine and telemonitoring platforms and inspire future smart home technology to overcome the six identified challenges . | [
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chronic periodontitis , the commonest type of periodontal disease , is defined by the american academy of periodontology as an infectious disease resulting in inflammation within of the tissues supporting the teeth , progressive loss of attachment and bone loss .
this process leads to pocket formation around the tooth and/or gum recession.1 this definition is based on a model of infection and host response which , despite the extent of current knowledge on periodontal diseases , is still inadequate for a diagnosis leading to a full classification grounded in the etiology of the disease.2 in chronic periodontitis , the bacterial biofilm is assumed to be the etiological agent , implying that this disease is polymicrobial . in the community of bacteria beneath the gums ,
the gram - negative bacteria are recognized as periodontal pathogens3 and studies that take into account variables linked to the genetics of both patients and these potential pathogens are likely to give a better insight into the etiology of periodontal disease.4 there have been signs that periodontitis may contribute to chronic systemic disease .
thus , some results have revealed an association between periodontitis and atherosclerotic vascular disease.58 the explanation offered for this connection is that periodontal pathogens could circulate in the bloodstream and promote damage to blood vessel endothelium and atherosclerosis .
it is plausible that such damage occurs not only in the endothelia of the heart and brain but also in kidney endothelium .
certainly , it is known that various chronic and acute infections are able to incite an inflammatory response in the kidney called glomerulonephritis.9 this is a relevant and timely topic because the prevalence of kidney disease is increasing and the majority of patients suffering from kidney disease also suffer from periodontitis.10 one united states population - based study considers multiple risk factors , including periodontal status , in the identification of individuals at high risk of chronic kidney disease.11 with this in mind , some researchers have turned their attention to the relation between periodontitis and renal insufficiency and the results of some work carried out on dogs12 and on humans9 confirmed this relation . on the other hand , yamalik et al13 observed that bacterial accumulation was similar in systemically healthy individuals with periodontitis and patients with chronic renal failure and periodontitis . also , no significant differences were observed in mean gingival - periodontal index , probing pocket depth or bleeding index between individuals with and without chronic renal failure.14 fisher et al15 showed that edentulism and periodontitis were independently associated with chronic kidney disease .
thus , in light of the above controversy and given that the nature of this association is not fully understood , we believe that further investigation is necessary
. one little - used approach to studying this association is to test the hypothesis that periodontitis is correlated with the start of the development of kidney disease . to this end , this study was designed to assess i ) the correlation between periodontitis and the levels of kidney function markers in systemically healthy subjects and ii ) the distribution of these levels relative to their reference intervals .
the classic markers for kidney function1619 used in this study were the levels of albumin and non - protein nitrogen compounds in the serum and urine , albumin / urinary creatinine ratio , creatinine clearance , the abbreviated modification of diet in renal disease formula for predictive estimation of glomerular filtration rates and urinary volume .
this cross - sectional study ( case - control ) was conducted from november 2005 to july 2007 , after the protocol had been approved ( res.196/05 ) by the human subject ethics committee of the un - esp school of dentistry at araraquara , sp , brazil , and written informed consent was obtained prior to subject enrollment .
the sample size was calculated for =5% , with 1.0 mm significant difference in clinical measurements between groups , and a standard deviation of 0.8 mm .
therefore , with a sample size of 27 patients , the power of the significance test was calculated to be 85% .
a total of 60 subjects were thus included in the study and divided into two groups of 30 participants each .
thirty - six chronic periodontitis subjects ( 14 teeth in the mouth ( including third molars ) , of which 4 had at least one site with probing pocket depth ( ppd ) of 3 to 5 mm , and 4 others at least one site with 6 to 10 mm ppd1 ) were recruited from the periodontal clinic of the so paulo state university ( unesp ) school of dentistry at araraquara , sp , brazil to form the test group . six subjects had to be excluded from the group because they did not appear for the collection of biological material , hence , a final of thirty subjects were formed the group .
thirty periodontally healthy volunteers ( 14 teeth in the mouth including third molars , bleeding on probing ( bop ) at < 30% of periodontal sites with ppd of 1 to 3 mm , only 2 isolated sites with ppd of 4 mm and without bop , and attachment loss ( al ) of 3 mm or more at < 30% of all the sites in the mouth1,20,21 ) from araraquara city ( blood donors and others ) not barred by the exclusion criteria were identified and recruited to the control group .
exclusion criteria for both groups were ; unfavorable systemic conditions ( rheumatic fever or heart problems requiring prophylactic antibiotic treatment and kidney disease ) , pregnant women , women on hormone replacement therapy or hormonal contraceptives , patients taking steroidal or non - steroidal anti - inflammatory drugs ( previous 3 months ) or antibiotics ( previous 6 months ) , smokers and subjects showing high - sensitivity c - reactive protein ( crp ) above 10 mg / l , these being powerful conditions that could have affected the progression of periodontitis.22,23 both the control and test groups were instructed on how to perform appropriate oral hygiene and were given preventive or curative oral treatment .
the full - mouth periodontal examination ( six sites per tooth : mesio - buccal , mid - buccal , disto - buccal , mesio - lingual , mid - lingual , disto - lingual ) was performed by one calibrated examiner using williams probe ( hu - friedy manufacturing co. , chicago , usa ) of circular cross - section .
the following parameters were assessed at the 6 sites around each tooth : ( i ) ppd , ( ii ) al , ( iii ) bop and ( iv ) plaque index ( pi ) .
al was determined from the distance between the cementoenamel junction and the most apical part of the periodontal pocket .
bop indicates the presence ( + ) or absence ( - ) of bleeding after probing the pocket .
the presence ( + ) or absence ( - ) of plaque was observed on the mesial , buccal , distal and lingual faces of each tooth and the pi was calculated .
the extent and severity index for periodontitis was calculated from clinical variables ( ppd and al ) used to diagnose periodontitis ; measurements indicating periodontitis were considered to be al > 2 mm and ppd > 2 mm .
the extent of the disease was taken as the proportion of all sites examined that showed pathological symptoms , both for al and ppd .
severity was calculated from the scores assigned to al and ppd at each site : for each mm beyond 2 mm ( normal ) , the score was incremented by 1 and the sum of scores was divided by the number of abnormal sites.20,21,24 venous blood samples were taken ( 8 ml in evacuated tube ) from subjects after fasting for 12 h overnight .
the serum and urinary levels of renal function markers ( urea , creatinine , uric acid and albumin ) were measured16 and the glomerular filtration rate was estimated from creatinine clearance,17 from the abbreviated modification of diet in renal disease formula18 and from the albumin ( mg)/creatinine ( g ) ratio in the 24-hour urine sample.19 serum and urinary urea were determined by enzyme assay25 in which urea was hydrolyzed by urease to ammonia , which was assayed by measuring the uv absorbance of nadh consumed in the presence of glutamate dehydrogenase .
creatinine was analyzed by the lustosa - basques colorimetric method25 based on the absorbance of colored complexes formed by picrate with creatinine and with interfering compounds , at alkaline ph ; when acetic acid is added , the creatinine - picrate complex is destroyed and the difference between the two absorbances is proportional to the amount of creatinine .
the uricase assay25 was used to measure uric acid ; the latter is first oxidized while oxygen is converted to hydrogen peroxide , which is then assayed by reaction with peroxidase and a chromogen .
serum albumin was determined by reaction with bromocresol green,26 which forms a complex with albumin that absorbs at a specific wavelength in the visible .
albuminuria was assessed quantitatively by an immunonephelometric method ( n antiserum to human albumin ) , following the nephelometer kit manufacturer s instructions ( dade - behring gmbh , marburg , germany ) .
serum and urinary determinations of urea , uric acid , creatinine and albumin were performed in a technicon ra - xt autoanalyser ( dublin , ireland ) .
the reagents for quantitation of serum and urinary uric acid were from kovalent do brasil ltda .
( so gonalo , rio de janeiro ) , while those for serum albumin and serum and urinary urea and creatinine were from the labtest diagnostics s.a .
the chi - square test was used to determine whether a group lay outside or within the reference interval for a given variable , according to the frequency of subjects in each category . to compare means of quantitative variables in groups , students t - test was used , in conjunction with either a post - hoc mann - whitney non - parametric test or a prior log transformation of the data prior to testing . in all statistical tests ,
the data for the controls and those for the test group were also separately subjected to multivariate canonical correlation analysis , employing sas software , in order to reveal correlations between their canonical variables for the periodontal diagnosis variables ( number of teeth and values of ppd , al , bop and pi ) and the kidney function markers ( serum albumin and uric acid , serum and urinary creatinine and urea , urinary volume , creatinine clearance , abbreviated modification of diet in renal disease formula and albumin / creatinine ratio ) .
this cross - sectional study ( case - control ) was conducted from november 2005 to july 2007 , after the protocol had been approved ( res.196/05 ) by the human subject ethics committee of the un - esp school of dentistry at araraquara , sp , brazil , and written informed consent was obtained prior to subject enrollment .
the sample size was calculated for =5% , with 1.0 mm significant difference in clinical measurements between groups , and a standard deviation of 0.8 mm .
therefore , with a sample size of 27 patients , the power of the significance test was calculated to be 85% .
a total of 60 subjects were thus included in the study and divided into two groups of 30 participants each .
thirty - six chronic periodontitis subjects ( 14 teeth in the mouth ( including third molars ) , of which 4 had at least one site with probing pocket depth ( ppd ) of 3 to 5 mm , and 4 others at least one site with 6 to 10 mm ppd1 ) were recruited from the periodontal clinic of the so paulo state university ( unesp ) school of dentistry at araraquara , sp , brazil to form the test group . six subjects had to be excluded from the group because they did not appear for the collection of biological material , hence , a final of thirty subjects were formed the group .
thirty periodontally healthy volunteers ( 14 teeth in the mouth including third molars , bleeding on probing ( bop ) at < 30% of periodontal sites with ppd of 1 to 3 mm , only 2 isolated sites with ppd of 4 mm and without bop , and attachment loss ( al ) of 3 mm or more at < 30% of all the sites in the mouth1,20,21 ) from araraquara city ( blood donors and others ) not barred by the exclusion criteria were identified and recruited to the control group .
exclusion criteria for both groups were ; unfavorable systemic conditions ( rheumatic fever or heart problems requiring prophylactic antibiotic treatment and kidney disease ) , pregnant women , women on hormone replacement therapy or hormonal contraceptives , patients taking steroidal or non - steroidal anti - inflammatory drugs ( previous 3 months ) or antibiotics ( previous 6 months ) , smokers and subjects showing high - sensitivity c - reactive protein ( crp ) above 10 mg / l , these being powerful conditions that could have affected the progression of periodontitis.22,23 both the control and test groups were instructed on how to perform appropriate oral hygiene and were given preventive or curative oral treatment .
the full - mouth periodontal examination ( six sites per tooth : mesio - buccal , mid - buccal , disto - buccal , mesio - lingual , mid - lingual , disto - lingual ) was performed by one calibrated examiner using williams probe ( hu - friedy manufacturing co. , chicago , usa ) of circular cross - section .
the following parameters were assessed at the 6 sites around each tooth : ( i ) ppd , ( ii ) al , ( iii ) bop and ( iv ) plaque index ( pi ) .
al was determined from the distance between the cementoenamel junction and the most apical part of the periodontal pocket .
bop indicates the presence ( + ) or absence ( - ) of bleeding after probing the pocket .
the presence ( + ) or absence ( - ) of plaque was observed on the mesial , buccal , distal and lingual faces of each tooth and the pi was calculated .
the extent and severity index for periodontitis was calculated from clinical variables ( ppd and al ) used to diagnose periodontitis ; measurements indicating periodontitis were considered to be al > 2 mm and ppd > 2 mm .
the extent of the disease was taken as the proportion of all sites examined that showed pathological symptoms , both for al and ppd .
severity was calculated from the scores assigned to al and ppd at each site : for each mm beyond 2 mm ( normal ) , the score was incremented by 1 and the sum of scores was divided by the number of abnormal sites.20,21,24
venous blood samples were taken ( 8 ml in evacuated tube ) from subjects after fasting for 12 h overnight .
the serum and urinary levels of renal function markers ( urea , creatinine , uric acid and albumin ) were measured16 and the glomerular filtration rate was estimated from creatinine clearance,17 from the abbreviated modification of diet in renal disease formula18 and from the albumin ( mg)/creatinine ( g ) ratio in the 24-hour urine sample.19 serum and urinary urea were determined by enzyme assay25 in which urea was hydrolyzed by urease to ammonia , which was assayed by measuring the uv absorbance of nadh consumed in the presence of glutamate dehydrogenase .
creatinine was analyzed by the lustosa - basques colorimetric method25 based on the absorbance of colored complexes formed by picrate with creatinine and with interfering compounds , at alkaline ph ; when acetic acid is added , the creatinine - picrate complex is destroyed and the difference between the two absorbances is proportional to the amount of creatinine .
the uricase assay25 was used to measure uric acid ; the latter is first oxidized while oxygen is converted to hydrogen peroxide , which is then assayed by reaction with peroxidase and a chromogen . serum albumin was determined by reaction with bromocresol green,26 which forms a complex with albumin that absorbs at a specific wavelength in the visible .
albuminuria was assessed quantitatively by an immunonephelometric method ( n antiserum to human albumin ) , following the nephelometer kit manufacturer s instructions ( dade - behring gmbh , marburg , germany ) .
serum and urinary determinations of urea , uric acid , creatinine and albumin were performed in a technicon ra - xt autoanalyser ( dublin , ireland ) .
the reagents for quantitation of serum and urinary uric acid were from kovalent do brasil ltda .
( so gonalo , rio de janeiro ) , while those for serum albumin and serum and urinary urea and creatinine were from the labtest diagnostics s.a .
the chi - square test was used to determine whether a group lay outside or within the reference interval for a given variable , according to the frequency of subjects in each category . to compare means of quantitative variables in groups , students t - test was used , in conjunction with either a post - hoc mann - whitney non - parametric test or a prior log transformation of the data prior to testing . in all statistical tests ,
the data for the controls and those for the test group were also separately subjected to multivariate canonical correlation analysis , employing sas software , in order to reveal correlations between their canonical variables for the periodontal diagnosis variables ( number of teeth and values of ppd , al , bop and pi ) and the kidney function markers ( serum albumin and uric acid , serum and urinary creatinine and urea , urinary volume , creatinine clearance , abbreviated modification of diet in renal disease formula and albumin / creatinine ratio ) .
the distribution of sexes in the control and test groups was similar : 13 men and 17 women controls and 16 men and 14 women in the test group .
there was also no significant difference in age , as the means and standard deviations were 435 yrs and 466 yrs for the respective groups . in table 1
, it can be seen that the two groups had different mean numbers of teeth and also differed with respect to ppd , al , bop and pi .
the ppd and al results were classified as follows : <3 mm ( shallow ) , 35 mm ( medium ) and 6 mm ( deep ) , and the pattern of these classes differed appreciably between the groups ; thus , only the test group showed deep pockets .
the number of teeth and proportion of shallow pockets ( ppd and al ) among the sites were higher in the control group , while higher values of all other variables were found in the test group ( p=0.006 ) .
for the extent and severity index of both ppd and al , the test group had a centre of distribution ( median ) much higher than that of the control group ( p=0.001 ) .
the serum and urinary levels of the kidney function indicators in both groups are presented in table 2 .
no significant differences were revealed between the two groups in the mean values of any of these variables . the same was true of the glomerular filtration rate - related variables : creatinine clearance ( mean ) , abbreviated modification of diet in renal disease formula ( median ) and the urinary albumin / creatinine ratio ( median ) ( table 3 ) .
the absolute and percent frequencies of the renal function indicators found inside and outside their reference intervals in the two groups are displayed in table 4 .
whichever variable is analyzed , it can be seen that there is no evidence that the frequency of results within or outside the reference interval is different in the control and test groups .
multivariate canonical correlation analysis , applied to the control group , revealed that larger numbers of teeth and of sites with ppd < 3 mm and al < 3 mm , and low bop and pi , are associated with higher levels of serum uric acid , urinary urea and urinary volume , though these levels remained within the respective reference intervals .
in addition , a large number of sites with ppd between 3 and 5 mm correlated with higher rates of creatinine clearance . analyzing the test group , it was found that larger numbers of sites with ppd < 3 mm and low bop were both associated with higher levels of urinary creatinine and creatinine clearance and lower levels of serum albumin .
also in the test group , it was observed that small numbers of sites with ppd > 6 mm correlated with lower albumin/ creatinine ratios and large numbers of teeth with high serum urea levels . finally , in the test group , low pi was associated with higher levels of serum and urinary uric acid .
in this study , we investigated the possible association between periodontitis and renal insufficiency by assaying kidney disease markers in two groups of subjects , previously diagnosed with periodontitis ( test group ) and periodontally healthy ( control group ) . in the control group shallow pockets
were found at 90% of all sites , according to the al and ppd data .
the control group showed al at around 7.63% of sites and a median severity of 1.0 mm , while in the test group , al was recorded at 62.44% of sites , with a median severity of 2.58 mm .
thus , an 8 times greater proportion of sites within the mouth was affected by loss of attachment in the test group and it was 158% more severe in the test group than in the control group . analyzing the results for ppd , the control group had low values while the test group values were higher . with respect to extent , more than 50% of the sites
were affected in the test group , while the severity had a median value of 2.25 mm , against 5.23% and 1.00 mm for the control group .
therefore , the two groups were certainly quite distinct , proving that the test group members had severe periodontitis , while the controls did not .
the results demonstrated that patients with periodontitis suffered no alteration in the level of any of the blood or urinary indicators of renal dysfunction , relative to the control group , and furthermore these markers stayed within their normal ranges in both groups , suggesting that severe periodontitis does not affect renal function .
these findings were obtained from 8 direct and 3 indirect markers of renal function . moreover ,
microalbuminuria , which was similar in the control and test groups , was also within its reference interval in both groups.32 several studies have been published showing correlation between periodontitis and renal dysfunction , some of which involve specific bacterial species . according to socransky et al,33 the micro - organisms responsible for periodontitis belong to the red complex ( porphyromonas gingivalis , tanerella forsythia and treponema dentcola ) , while send et al34 report that there is considerable evidence that periodontitis - related microorganisms impair blood rheological parameters and thereby contribute significantly to accelerated systemic or local diseases that cause premature death in dialyzed patients . in the majority of published work on this association , the case group consists of patients with proved kidney disease . however , in this approach it is probable that patients have been included whose kidney disease is unrelated to periodontitis , since in most such patients , the illness stems from primary or hereditary syndromes such as goodpasture s syndrome , bergers disease , focal glomerulosclerosis and membranoproliferative glomerulonephritis , or more rarely alport s syndrome and autosomal dominant polycystic kidney disease , or from secondary processes such as diabetes mellitus , polyarteritis nodosa , scleroderma , myeloma kidney , sarcoidosis , amyloidosis , lupus nephritis , etc.35 thus , the constitution of the test group is critical , since the pathological conditions listed above may interfere with some markers , without necessarily being related to periodontitis .
in addition , for a number of years , reference values have been used for various diseases that do not yet signify the actual disease but are close to the upper limit of the normal range .
an example is impaired fasting plasma glucose.36 the same reasoning could be applied to laboratory assays used as markers for kidney damage , to allow preclinical signs to be detected before the onset of the disease .
thus , by analogy with diabetes , in this study the test and control groups are compared not only with each other but also with kidney dysfunction marker reference values .
hence , we would discover whether periodontitis could promote any detectable change in kidney function , even when the marker in question remained within its reference interval , albeit shifted nearer to the upper limit of this range . regarding the multivariate canonical correlation analysis , the results for both the control and test groups revealed no more than the other statistical tests , since the correlations found were not relevant .
for example , in the control group , the correlation between higher levels of uric acid , urinary urea , urine volume and creatinine clearance , and large numbers of teeth and a high proportion of sites with ppd<3 mm and between 3 and 5 mm , could be the result of better grinding of food during chewing , leading to enhanced digestion of proteins and nucleoproteins and improved absorption of aminoacids .
the same type of explanation could be given for all the apparent correlations in the test group .
our results are consistent with published data showing that mild , moderate or severe periodontitis was observed in dogs with normal renal function , assessed by serum levels of creatinine and urea and by urine density.37 leivadaros et al38 also found no correlation between the urinary albumin/ creatinine ratio and periodontitis , while taylor et al39 found no change in urea levels after extraction of all teeth from patients suffering from periodontitis . in another investigation into whether a link exists between periodontitis and renal function , which employed urinary markers of kidney function , such as volume of urine per 24 h and level of creatinine per 24 hours , and blood markers such as serum creatinine level and creatinine clearance per 24 hours , it was found that the increased incidence of chronic renal failure that occurs with age might enhance the probability of severe periodontitis in the target population.40 however , those patients were in a far higher age bracket than the present subjects , averaging 77 years of age .
other authors , who also worked with elderly patients , found an association between periodontitis and kidney disease.41 it is known that old age in itself enables the initiation of periodontitis6 and thus can be a significant confounding factor in the analysis of the periodontitis - kidney disease relation .
two methods were used to estimate the glomerular filtration rate , since neither is entirely satisfactory , though creatinine clearance is considered superior .
the drawback of creatinine clearance is its dependence on the rigorous collection of all urine for 24 h , while the disadvantage of the predictive abbreviated modification of diet in renal disease formula is that it is a mathematical model . for independent evidence that the whole urine sample was in fact collected
, the albumin / creatinine ratio was measured in a 24 h urine sample,19 even though the original technique for this determination was based on a single random sample.30 certainly , under these conditions , the ratio between the two concentrations should stay within the reference interval , and this was observed in 100% of the tests in both groups , indicating that the creatinine clearance results were reliable and thus excluding potentially incomplete urine collection .
the values of the glomerular filtration rate , estimated from both creatinine clearance and the abbreviated modification of diet in renal disease formula , revealed that a high percentage of subjects were within the reference interval in both the control and test groups . contradicting our findings , kshirsagar
et al9 reported an association between periodontitis and both raised serum creatinine levels and a glomerular filtration rate below 60 ml / min per 1.73 m , determined by the predictive equation .
an association was also observed by verterinen et al,42 however , out of all the clinical variables , radiographic results and analyses of saliva constituents , only the albumin level showed significant change .
taken together , these data indicate that the study model employed can have a considerable influence on the results .
an association between periodontitis and kidney disease is often encountered in studies using a population where kidney disease is already diagnosed.42,43 in these cases , the duration of end - stage renal failure and the type of systemic treatment given to patients affect the association significantly.43 a similar association has also been observed in a model used to analyze hemodialysis patients.44,45 however , even in the study model involving patients with end - stage kidney disease , there is some controversy about the association , since some cases have been reported where periodontal damage was no more severe in such patients.46 a recent study suggests that the central process leading to premature death lies primarily in an inability to eliminate bacteria and products from the blood , despite hemodialysis.34 shultis et al47 observed that periodontitis predicts development of overt nephropathy and end - stage kidney disease in individuals with type 2 diabetes . in groups as complex as hemodialysis patients and diabetics , such associations become hard to analyze or discuss .
it is our view that the model used in the present study to assess whether periodontitis is correlated with kidney disease suffers from fewer confounding factors ( tobacco , pregnancy , hormonal contraceptives , antibiotics and inflammatory treatment , cardiac and renal problems ) than many models found in the literature and it is more reliable than one involving diabetic patients , since the sequelae of diabetes are very complex and generate several kinds of interference , apart from the known tendency of diabetes per se to promote infectious processes.48 one limitation of this study is its cross - sectional design .
longitudinal studies are still necessary , either to consolidate the above negative results or perhaps to show that chronic periodontitis is , after all , a risk factor for kidney disease , by detecting subtle changes , within the reference intervals , following treatment for periodontitis .
another limitation of the present study is that , despite the clearcut statistical difference between the control and test groups , 10% of sites
6 mm might not have the severity of periodontitis necessary to trigger systemic reactions .
in other words , it is unknown how many deep pockets with bleeding on probing and for how long these pockets have to be there before we can measure a systemic reaction .
furthermore , there might be specific bacterial species that trigger stronger systemic responses than others .
such problems make it very difficult to evaluate the real effect of periodontitis on systemic health .
therefore , the absence of correlation observed between periodontitis and kidney disease in healthy adults leaves room for the possibility that severely impaired kidneys prepare the field for the progression of periodontitis or vice versa.34
within the limitations of this study and the methodology utilized , it may be suggested from the results that severe periodontitis was not correlated with renal dysfunction in the studied population .
further studies on therapeutic intervention ( tests before and after treatment ) would make an important contribution to the consolidation of these results . | objectives : the aim of this study was to assess a suggested association between periodontitis and renal insufficiency by assaying kidney disease markers.methods:variables used to diagnose periodontitis were : ( i ) probing pocket depth ( ppd ) , ( ii ) attachment loss ( al ) , ( iii ) bleeding on probing ( bop ) , ( iv ) plaque index ( pi ) and ( v ) extent and severity index . blood and urine were collected from 60 apparently healthy non - smokers ( men and women ) , consisting of a test group of 30 subjects with periodontitis ( age 466 yrs ) and a control group of 30 healthy subjects ( age 435 yrs ) .
kidney function markers ( urea , creatinine , uric acid and albumin contents ) were measured in the serum and urine . also , the glomerular filtration rate was estimated from creatinine clearance , from the abbreviated modification of diet in renal disease formula and from the albumin : creatinine ratio in a 24h sample of urine.results:it was found that the control group had a greater mean number of teeth than the test group and that the two groups also differed in ppd , al , bop and pi , all these variables being higher in the test group ( p=0.006 ) . for the extent and severity index of both ppd and al , the test group had much higher medians of both extent and severity than the control group ( p=0.001 ) . with regard to kidney function
, none of the markers revealed a significant difference between the control and test groups and all measured values fell within the reference intervals.conclusions:it is proposed that severe periodontitis is not associated with any alteration in kidney function . | INTRODUCTION
MATERIALS AND METHODS
Patient selection
Laboratory tests
Statistical analysis
RESULTS
DISCUSSION
CONCLUSIONS | this process leads to pocket formation around the tooth and/or gum recession.1 this definition is based on a model of infection and host response which , despite the extent of current knowledge on periodontal diseases , is still inadequate for a diagnosis leading to a full classification grounded in the etiology of the disease.2 in chronic periodontitis , the bacterial biofilm is assumed to be the etiological agent , implying that this disease is polymicrobial . thus , some results have revealed an association between periodontitis and atherosclerotic vascular disease.58 the explanation offered for this connection is that periodontal pathogens could circulate in the bloodstream and promote damage to blood vessel endothelium and atherosclerosis . certainly , it is known that various chronic and acute infections are able to incite an inflammatory response in the kidney called glomerulonephritis.9 this is a relevant and timely topic because the prevalence of kidney disease is increasing and the majority of patients suffering from kidney disease also suffer from periodontitis.10 one united states population - based study considers multiple risk factors , including periodontal status , in the identification of individuals at high risk of chronic kidney disease.11 with this in mind , some researchers have turned their attention to the relation between periodontitis and renal insufficiency and the results of some work carried out on dogs12 and on humans9 confirmed this relation . also , no significant differences were observed in mean gingival - periodontal index , probing pocket depth or bleeding index between individuals with and without chronic renal failure.14 fisher et al15 showed that edentulism and periodontitis were independently associated with chronic kidney disease . to this end , this study was designed to assess i ) the correlation between periodontitis and the levels of kidney function markers in systemically healthy subjects and ii ) the distribution of these levels relative to their reference intervals . the classic markers for kidney function1619 used in this study were the levels of albumin and non - protein nitrogen compounds in the serum and urine , albumin / urinary creatinine ratio , creatinine clearance , the abbreviated modification of diet in renal disease formula for predictive estimation of glomerular filtration rates and urinary volume . thirty - six chronic periodontitis subjects ( 14 teeth in the mouth ( including third molars ) , of which 4 had at least one site with probing pocket depth ( ppd ) of 3 to 5 mm , and 4 others at least one site with 6 to 10 mm ppd1 ) were recruited from the periodontal clinic of the so paulo state university ( unesp ) school of dentistry at araraquara , sp , brazil to form the test group . thirty periodontally healthy volunteers ( 14 teeth in the mouth including third molars , bleeding on probing ( bop ) at < 30% of periodontal sites with ppd of 1 to 3 mm , only 2 isolated sites with ppd of 4 mm and without bop , and attachment loss ( al ) of 3 mm or more at < 30% of all the sites in the mouth1,20,21 ) from araraquara city ( blood donors and others ) not barred by the exclusion criteria were identified and recruited to the control group . exclusion criteria for both groups were ; unfavorable systemic conditions ( rheumatic fever or heart problems requiring prophylactic antibiotic treatment and kidney disease ) , pregnant women , women on hormone replacement therapy or hormonal contraceptives , patients taking steroidal or non - steroidal anti - inflammatory drugs ( previous 3 months ) or antibiotics ( previous 6 months ) , smokers and subjects showing high - sensitivity c - reactive protein ( crp ) above 10 mg / l , these being powerful conditions that could have affected the progression of periodontitis.22,23 both the control and test groups were instructed on how to perform appropriate oral hygiene and were given preventive or curative oral treatment . the following parameters were assessed at the 6 sites around each tooth : ( i ) ppd , ( ii ) al , ( iii ) bop and ( iv ) plaque index ( pi ) . the extent and severity index for periodontitis was calculated from clinical variables ( ppd and al ) used to diagnose periodontitis ; measurements indicating periodontitis were considered to be al > 2 mm and ppd > 2 mm . the serum and urinary levels of renal function markers ( urea , creatinine , uric acid and albumin ) were measured16 and the glomerular filtration rate was estimated from creatinine clearance,17 from the abbreviated modification of diet in renal disease formula18 and from the albumin ( mg)/creatinine ( g ) ratio in the 24-hour urine sample.19 serum and urinary urea were determined by enzyme assay25 in which urea was hydrolyzed by urease to ammonia , which was assayed by measuring the uv absorbance of nadh consumed in the presence of glutamate dehydrogenase . serum and urinary determinations of urea , uric acid , creatinine and albumin were performed in a technicon ra - xt autoanalyser ( dublin , ireland ) . in all statistical tests ,
the data for the controls and those for the test group were also separately subjected to multivariate canonical correlation analysis , employing sas software , in order to reveal correlations between their canonical variables for the periodontal diagnosis variables ( number of teeth and values of ppd , al , bop and pi ) and the kidney function markers ( serum albumin and uric acid , serum and urinary creatinine and urea , urinary volume , creatinine clearance , abbreviated modification of diet in renal disease formula and albumin / creatinine ratio ) . thirty - six chronic periodontitis subjects ( 14 teeth in the mouth ( including third molars ) , of which 4 had at least one site with probing pocket depth ( ppd ) of 3 to 5 mm , and 4 others at least one site with 6 to 10 mm ppd1 ) were recruited from the periodontal clinic of the so paulo state university ( unesp ) school of dentistry at araraquara , sp , brazil to form the test group . thirty periodontally healthy volunteers ( 14 teeth in the mouth including third molars , bleeding on probing ( bop ) at < 30% of periodontal sites with ppd of 1 to 3 mm , only 2 isolated sites with ppd of 4 mm and without bop , and attachment loss ( al ) of 3 mm or more at < 30% of all the sites in the mouth1,20,21 ) from araraquara city ( blood donors and others ) not barred by the exclusion criteria were identified and recruited to the control group . exclusion criteria for both groups were ; unfavorable systemic conditions ( rheumatic fever or heart problems requiring prophylactic antibiotic treatment and kidney disease ) , pregnant women , women on hormone replacement therapy or hormonal contraceptives , patients taking steroidal or non - steroidal anti - inflammatory drugs ( previous 3 months ) or antibiotics ( previous 6 months ) , smokers and subjects showing high - sensitivity c - reactive protein ( crp ) above 10 mg / l , these being powerful conditions that could have affected the progression of periodontitis.22,23 both the control and test groups were instructed on how to perform appropriate oral hygiene and were given preventive or curative oral treatment . the following parameters were assessed at the 6 sites around each tooth : ( i ) ppd , ( ii ) al , ( iii ) bop and ( iv ) plaque index ( pi ) . the extent and severity index for periodontitis was calculated from clinical variables ( ppd and al ) used to diagnose periodontitis ; measurements indicating periodontitis were considered to be al > 2 mm and ppd > 2 mm . severity was calculated from the scores assigned to al and ppd at each site : for each mm beyond 2 mm ( normal ) , the score was incremented by 1 and the sum of scores was divided by the number of abnormal sites.20,21,24
venous blood samples were taken ( 8 ml in evacuated tube ) from subjects after fasting for 12 h overnight . the serum and urinary levels of renal function markers ( urea , creatinine , uric acid and albumin ) were measured16 and the glomerular filtration rate was estimated from creatinine clearance,17 from the abbreviated modification of diet in renal disease formula18 and from the albumin ( mg)/creatinine ( g ) ratio in the 24-hour urine sample.19 serum and urinary urea were determined by enzyme assay25 in which urea was hydrolyzed by urease to ammonia , which was assayed by measuring the uv absorbance of nadh consumed in the presence of glutamate dehydrogenase . creatinine was analyzed by the lustosa - basques colorimetric method25 based on the absorbance of colored complexes formed by picrate with creatinine and with interfering compounds , at alkaline ph ; when acetic acid is added , the creatinine - picrate complex is destroyed and the difference between the two absorbances is proportional to the amount of creatinine . serum and urinary determinations of urea , uric acid , creatinine and albumin were performed in a technicon ra - xt autoanalyser ( dublin , ireland ) . in all statistical tests ,
the data for the controls and those for the test group were also separately subjected to multivariate canonical correlation analysis , employing sas software , in order to reveal correlations between their canonical variables for the periodontal diagnosis variables ( number of teeth and values of ppd , al , bop and pi ) and the kidney function markers ( serum albumin and uric acid , serum and urinary creatinine and urea , urinary volume , creatinine clearance , abbreviated modification of diet in renal disease formula and albumin / creatinine ratio ) . the distribution of sexes in the control and test groups was similar : 13 men and 17 women controls and 16 men and 14 women in the test group . in table 1
, it can be seen that the two groups had different mean numbers of teeth and also differed with respect to ppd , al , bop and pi . the ppd and al results were classified as follows : <3 mm ( shallow ) , 35 mm ( medium ) and 6 mm ( deep ) , and the pattern of these classes differed appreciably between the groups ; thus , only the test group showed deep pockets . the number of teeth and proportion of shallow pockets ( ppd and al ) among the sites were higher in the control group , while higher values of all other variables were found in the test group ( p=0.006 ) . for the extent and severity index of both ppd and al , the test group had a centre of distribution ( median ) much higher than that of the control group ( p=0.001 ) . the serum and urinary levels of the kidney function indicators in both groups are presented in table 2 . no significant differences were revealed between the two groups in the mean values of any of these variables . the same was true of the glomerular filtration rate - related variables : creatinine clearance ( mean ) , abbreviated modification of diet in renal disease formula ( median ) and the urinary albumin / creatinine ratio ( median ) ( table 3 ) . the absolute and percent frequencies of the renal function indicators found inside and outside their reference intervals in the two groups are displayed in table 4 . whichever variable is analyzed , it can be seen that there is no evidence that the frequency of results within or outside the reference interval is different in the control and test groups . multivariate canonical correlation analysis , applied to the control group , revealed that larger numbers of teeth and of sites with ppd < 3 mm and al < 3 mm , and low bop and pi , are associated with higher levels of serum uric acid , urinary urea and urinary volume , though these levels remained within the respective reference intervals . analyzing the test group , it was found that larger numbers of sites with ppd < 3 mm and low bop were both associated with higher levels of urinary creatinine and creatinine clearance and lower levels of serum albumin . finally , in the test group , low pi was associated with higher levels of serum and urinary uric acid . in this study , we investigated the possible association between periodontitis and renal insufficiency by assaying kidney disease markers in two groups of subjects , previously diagnosed with periodontitis ( test group ) and periodontally healthy ( control group ) . the control group showed al at around 7.63% of sites and a median severity of 1.0 mm , while in the test group , al was recorded at 62.44% of sites , with a median severity of 2.58 mm . thus , an 8 times greater proportion of sites within the mouth was affected by loss of attachment in the test group and it was 158% more severe in the test group than in the control group . analyzing the results for ppd , the control group had low values while the test group values were higher . with respect to extent , more than 50% of the sites
were affected in the test group , while the severity had a median value of 2.25 mm , against 5.23% and 1.00 mm for the control group . therefore , the two groups were certainly quite distinct , proving that the test group members had severe periodontitis , while the controls did not . the results demonstrated that patients with periodontitis suffered no alteration in the level of any of the blood or urinary indicators of renal dysfunction , relative to the control group , and furthermore these markers stayed within their normal ranges in both groups , suggesting that severe periodontitis does not affect renal function . moreover ,
microalbuminuria , which was similar in the control and test groups , was also within its reference interval in both groups.32 several studies have been published showing correlation between periodontitis and renal dysfunction , some of which involve specific bacterial species . however , in this approach it is probable that patients have been included whose kidney disease is unrelated to periodontitis , since in most such patients , the illness stems from primary or hereditary syndromes such as goodpasture s syndrome , bergers disease , focal glomerulosclerosis and membranoproliferative glomerulonephritis , or more rarely alport s syndrome and autosomal dominant polycystic kidney disease , or from secondary processes such as diabetes mellitus , polyarteritis nodosa , scleroderma , myeloma kidney , sarcoidosis , amyloidosis , lupus nephritis , etc.35 thus , the constitution of the test group is critical , since the pathological conditions listed above may interfere with some markers , without necessarily being related to periodontitis . regarding the multivariate canonical correlation analysis , the results for both the control and test groups revealed no more than the other statistical tests , since the correlations found were not relevant . for example , in the control group , the correlation between higher levels of uric acid , urinary urea , urine volume and creatinine clearance , and large numbers of teeth and a high proportion of sites with ppd<3 mm and between 3 and 5 mm , could be the result of better grinding of food during chewing , leading to enhanced digestion of proteins and nucleoproteins and improved absorption of aminoacids . in another investigation into whether a link exists between periodontitis and renal function , which employed urinary markers of kidney function , such as volume of urine per 24 h and level of creatinine per 24 hours , and blood markers such as serum creatinine level and creatinine clearance per 24 hours , it was found that the increased incidence of chronic renal failure that occurs with age might enhance the probability of severe periodontitis in the target population.40 however , those patients were in a far higher age bracket than the present subjects , averaging 77 years of age . other authors , who also worked with elderly patients , found an association between periodontitis and kidney disease.41 it is known that old age in itself enables the initiation of periodontitis6 and thus can be a significant confounding factor in the analysis of the periodontitis - kidney disease relation . two methods were used to estimate the glomerular filtration rate , since neither is entirely satisfactory , though creatinine clearance is considered superior . the drawback of creatinine clearance is its dependence on the rigorous collection of all urine for 24 h , while the disadvantage of the predictive abbreviated modification of diet in renal disease formula is that it is a mathematical model . for independent evidence that the whole urine sample was in fact collected
, the albumin / creatinine ratio was measured in a 24 h urine sample,19 even though the original technique for this determination was based on a single random sample.30 certainly , under these conditions , the ratio between the two concentrations should stay within the reference interval , and this was observed in 100% of the tests in both groups , indicating that the creatinine clearance results were reliable and thus excluding potentially incomplete urine collection . the values of the glomerular filtration rate , estimated from both creatinine clearance and the abbreviated modification of diet in renal disease formula , revealed that a high percentage of subjects were within the reference interval in both the control and test groups . contradicting our findings , kshirsagar
et al9 reported an association between periodontitis and both raised serum creatinine levels and a glomerular filtration rate below 60 ml / min per 1.73 m , determined by the predictive equation . an association between periodontitis and kidney disease is often encountered in studies using a population where kidney disease is already diagnosed.42,43 in these cases , the duration of end - stage renal failure and the type of systemic treatment given to patients affect the association significantly.43 a similar association has also been observed in a model used to analyze hemodialysis patients.44,45 however , even in the study model involving patients with end - stage kidney disease , there is some controversy about the association , since some cases have been reported where periodontal damage was no more severe in such patients.46 a recent study suggests that the central process leading to premature death lies primarily in an inability to eliminate bacteria and products from the blood , despite hemodialysis.34 shultis et al47 observed that periodontitis predicts development of overt nephropathy and end - stage kidney disease in individuals with type 2 diabetes . it is our view that the model used in the present study to assess whether periodontitis is correlated with kidney disease suffers from fewer confounding factors ( tobacco , pregnancy , hormonal contraceptives , antibiotics and inflammatory treatment , cardiac and renal problems ) than many models found in the literature and it is more reliable than one involving diabetic patients , since the sequelae of diabetes are very complex and generate several kinds of interference , apart from the known tendency of diabetes per se to promote infectious processes.48 one limitation of this study is its cross - sectional design . longitudinal studies are still necessary , either to consolidate the above negative results or perhaps to show that chronic periodontitis is , after all , a risk factor for kidney disease , by detecting subtle changes , within the reference intervals , following treatment for periodontitis . another limitation of the present study is that , despite the clearcut statistical difference between the control and test groups , 10% of sites
6 mm might not have the severity of periodontitis necessary to trigger systemic reactions . therefore , the absence of correlation observed between periodontitis and kidney disease in healthy adults leaves room for the possibility that severely impaired kidneys prepare the field for the progression of periodontitis or vice versa.34
within the limitations of this study and the methodology utilized , it may be suggested from the results that severe periodontitis was not correlated with renal dysfunction in the studied population . | [
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pre - mrna splicing is a highly regulated process , the outcome of which is critical for homeostasis and disease .
the diversity of splicing variants positively correlates with evolutionary complexity , as it allows for an expansion of the possible protein isoforms that are derived from the same genetic information .
the regulation of splicing is still relatively poorly understood , which is caused by the many factors that can affect splicing outcome . at the level of rna sequences , splicing regulatory elements
the splice site junctions , as well as the polypyrimidine ( py ) tract and branch point , are relatively easy to identify .
splice regulatory elements exist , including the exonic and intronic splice enhancer and silencer elements ( eses , esss , ises , and isss , respectively ) , but these are more difficult to predict .
the expression levels of splicing regulatory proteins can also play an important role in splicing outcome .
furthermore , the speed of rna polymerase ii transcription is thought to affect splicing efficiency , because splicing mainly occurs co - transcriptionally , and the available splice sites that are present in the protruding pre - mrna within a certain time frame compete for the splicing machinery.4 , 5 additional levels of regulation are chromatin modifications and the composition of the utrs.6 , 7 all together , these diverse levels of regulation highlight the requirement for experimental testing of alternative splicing to validate predictions .
approximately 9% of pathogenic variants annotated in the human gene mutation database are linked to splicing defects ( http://www.hgmd.cf.ac.uk/ ) . in this database , variants that affect splicing predominantly concern those that are located at splice site junctions .
this is caused by the inherent detection bias for these variants , which are mostly identified using splice prediction algorithms .
however , variants can also alter splicing by the generation of new splice sites9 , 10 or by disturbing or generating other splice regulatory elements.11 , 12 in addition , variants in the coding region can have more than one effect ( i.e. , a missense variant can also have an effect on splicing ) .
furthermore , indirect effects on target genes are known ( e.g. , in response to mutations in master splicing factors such as the muscleblind family , sf3b1 , and u2af35).13 , 14 the consequences of such events can be diverse and may include ( partial ) exon skipping and/or ( partial ) intron retention
. the final mrna may be in frame , which in certain cases may result in a protein product that retains some biological activity .
a reading frameshift usually results in mrna decay and absence of a protein product , although the extent at which decay occurs may vary .
it is likely that many more pathogenic splicing variants exist that are located outside the region of canonical splice sites , and that the percentage of identified variants that affect splicing will increase in the future .
antisense oligonucleotides ( aons ) can modulate splicing by binding to the pre - mrna and blocking splicing regulatory sequences .
one type of aon has been modified from the naturally occurring u7 small nuclear rna ( snrna ) , which is normally utilized in histone pre - mrna processing but has been modified to target the pre - mrna of choice .
u7 snrna - based aons are stabilized by a stem loop and also contain an antisense sequence that is used for targeting .
another strategy is the use of chemically modified aons that are capable to bind to rna via watson - crick base pairing but are insensitive to rnase - mediated degradation due to their modified backbone chemistry .
various backbones with different properties are known , which include phosphorodiamidate morpholino oligomer ( pmo ) , 2-o - methyl phosphorothioate , 2-o - methoxyethyl phosphorothioate , and tricyclo - dna modifications . a straightforward approach to modulate splicing using aons
this will result in exon skipping , which can be advantageous , for example , to bypass a mutation hotspot and to restore the reading frame .
this will only work when the resulting truncated protein is at least partially active , which is the case for the dystrophin protein , providing a potential therapy for duchenne muscular dystrophy.18 , 19 this approach has been tested in multiple clinical trials and has moved forward toward conditional approval by the us food and drug administration ( fda ) .
many other examples have been published , in which a cryptic splice site is blocked with an aon to restore canonical splicing ( reviewed in havens and hastings ) .
we recently showed that this is possible in the case of gaa variants that cause pompe disease .
restoration of splicing becomes more challenging in cases where a splicing variant causes exon skipping and requires promotion of exon inclusion .
when there is no clear cryptic splice site involved that can be blocked by an aon , such cases require the identification of iss or ess elements that are critical for exon inclusion .
few examples for promotion of exon inclusion exist ; notably , for spinal muscular atrophy , caused by variants in the smn1 gene , aons have been designed that reactivate the normally silent sister gene smn2 .
smn2 contains an exonic variant that causes exon skipping , and aons have been identified that block ess or iss sequences and promote exon inclusion.21 , 22 one of these , which targets the iss , is currently being tested in clinical trials . because aons are sequence specific , they are usually only suitable for a subgroup of patients with similar gene variants . in pompe disease ,
a common splicing variant occurs in the majority of caucasian patients with the childhood / adult form .
this well - described variant , c.-32 - 13t > g ( also known as ivs1 , or r.-32 - 13u > g at the rna level ) , is located in the py tract of exon 2 of the acid -glucosidase ( gaa ) pre - mrna and causes weakening of the splice acceptor site , resulting in complete or partial skipping of exon 2.24 , 25 , 26 , 27 this exon contains the translation start codon , and its skipping results in mrna degradation and a subsequent reduction of gaa protein production .
approximately 10%15% of transcripts are spliced normally and produce a low level of wild - type gaa protein , which explains why these patients develop the childhood / adult form of pompe disease . a minimum gaa activity of 20% of the average healthy control values is required to prevent the disease
. the restoration of splicing from the ivs1 variant using aons would potentially provide a therapeutic option for the majority of caucasian patients with pompe disease .
this may be beneficial in addition to or instead of the current enzyme replacement therapy , which has a partial and heterogeneous response and is extremely expensive .
however , splicing correction from the ivs1 variant requires promotion of exon inclusion , and no negatively acting splicing regulatory sequences in the gaa gene that are amenable to inhibition using aons are currently known . here , we used a screen to modulate endogenous gaa splicing using u7 snrna - based aons expressed via lentiviral transduction in patient - derived primary fibroblasts .
this resulted in the identification of cis - acting splicing regulatory elements that were subsequently tested using pmo - based aons .
two overlapping aons were identified that promoted inclusion of exon 2 in cells from patients carrying the ivs1 variant . in cells from healthy controls ,
the aons did not change gaa splicing or expression , confirming that they acted on splicing rather than elevating total gaa transcript levels .
the aons increased gaa enzymatic activity in patient - derived cells to above the disease threshold of 20% .
these findings demonstrate the feasibility to correct aberrant splicing from the common ivs1 gaa variant by blocking a negative splicing element to promote exon inclusion .
previous work resulted in the characterization of aberrant splicing caused by the ivs1 gaa variant.24 , 25 , 26 , 27 splicing products include wild - type gaa mrna ( n ) caused by leaky normal splicing , partial skipping of exon 2 caused by utilization of a cryptic splice site in exon 2 ( sv3 ) , and full skipping of exon 2 ( sv2 ) .
these products can be identified by flanking exon rt - pcr in primary fibroblasts using primers that anneal to exons 1 and 3 ( figure 1a ) .
splicing is subject to regulation by additional cis - acting rna sequences besides the sequences surrounding the canonical splice sites .
these include esss , isss , eses , and ises and can be located distant from canonical splice sites ( hypothetical example given in figure 1b ) .
approximately 10%15% of the gaa pre - mrna is correctly spliced , indicating that the pathogenic effect of the ivs1 variant is not fully detrimental to splicing of gaa exon 2 .
we hypothesized that it may be possible to promote exon 2 inclusion by inhibition of a splicing negatively acting splicing regulatory element using an aon . to test this , in silico prediction
was performed using several algorithms , which resulted in a plethora of splicing regulatory elements ( figure 1c ) .
however , the results were algorithm dependent and many predicted enhancer and silencer sequences overlapped without indicating an obvious candidate silencer element that could be amenable to inhibition by an aon . to identify splicing regulatory elements experimentally , an unbiased screen was performed using a non - overlapping tiling array of aons that cover part of intron 1 and the complete exon 2 of the gaa pre - mrna ( figure 2a ) . because of the high costs involved when testing a large series of chemically modified aons , aons were expressed as u7 snrnas using a lentiviral vector .
the original u7 snrna vector was adapted to enable one - step cloning of an aon and intermediate throughput screening ( figure s1a ) . to validate the u7 snrna vector ,
a control experiment was performed on primary fibroblasts derived from a patient with adult pompe disease ( patient 1 ) , who carried the ivs1 variant on one allele and the c.525delt on the second allele .
a u7 snrna - based aon was targeted to the splice sites of exon 4 of cyclophilin a ( cypa ) pre - mrna ( figure s1b ) .
this aon was capable of inducing skipping of exon 4 , as shown by flanking exon rt - pcr ( figure s1c ) and exon internal qrt - pcr analysis ( figure s1d ) .
this confirmed previous reports and demonstrated that the modified u7 snrna construct can be used to modulate splicing .
subsequently , the u7 snrna screen using the aons indicated in figure 2a was performed on fibroblasts from patient 1 .
the screen resulted in the identification of a number of u7 snrna - based aons that modulated inclusion of exon 2 in the gaa mrna , as shown with qrt - pcr analysis ( figure 2b , black line ) .
importantly , only mrna from the allele carrying the ivs1 variant was detected due to the frameshift induced by the c.525delt variant on the second allele , which results in mrna degradation .
notably , u7 snrnas targeting two regions in intron 1 , at c.-32 - 179 and c.-32 - 219 , promoted inclusion of gaa exon 2 . exclusion of exon 2 was promoted by u7 snrnas that targeted regions in the 5 part of exon 2 .
similar results were obtained using flanking exon rt - pcr analysis ( figure 2c ) .
promotion and inhibition of exon 2 inclusion resulted in increased and decreased gaa enzymatic activity , respectively ( figure 2b , compare black and red lines ) .
next , the effect of the lentiviral amount was tested using p24 elisa ; 200 ng yielded an optimal ratio between biological effect and nonspecific reduction of cell viability and gaa expression seen at high viral amounts ( figure s2 ) . to fine - tune the optimal location of u7 snrnas targeting c.-32 - 179 and c.-32 - 219 , a microwalk was performed around these regions with u7 snrnas that shifted 2 nt ( figure 3a ) .
this showed that the locations identified using the initial u7 snrna screen were peak values .
in addition , the microwalk revealed two nearby locations at c.-32 - 183 and c.-32 - 185 whose inhibition promoted exon 2 inclusion ( figures 3b and 3c ) . taken together ,
the u7-based snrna screen of intron 1 and exon 2 identified regions potentially involved in repression of gaa exon 2 inclusion . to test whether morpholino - based aons can modulate aberrant splicing caused by the ivs1 variant
, aons were designed based on the results from the u7 snrna screen shown in figure 3 .
specific requirements of low g content on chemical synthesis posed constrains to the regions that could be targeted but it was possible to design aons that targeted the two major putative repressor sequences at c.-32 - 219 ( aons 1 and 2 ) and c.-32 - 179 ( aons 3 and 4 ) ( figures 4a and s3a ) . to test whether primary fibroblasts were amenable to splicing modulation using morpholino - based aons , aons targeting cypa were used as positive control .
two pmo - based aons that target the 3 donor splice site of exon 4 in the cypa pre - mrna ( figures s3a and s3b ) were able to promote skipping of exon 4 and exons 3 and 4 after transfection into primary fibroblasts , as shown by flanking exon rt - pcr and qrt - pcr analysis ( figures s3c and s3d ) , which is in agreement with our previous report .
next , aons 14 targeting the identified regions in the gaa pre - mrna were tested by transfecting primary fibroblasts from patient 1 .
qrt - pcr analysis of exon 2 inclusion showed that aons 3 and 4 promoted exon 2 inclusion more than 2-fold , whereas aons 1 and 2 were ineffective ( figure 4b ) .
concomitant effects on gaa enzymatic activity were found , with more than 2-fold enhancement by aons 3 and 4 , while aons 1 and 2 had no effect .
this showed that pmo - based aons that targeted the region of c.-32 - 179 promoted exon 2 inclusion , and that other pmo - based aons tested were ineffective .
next , the concentration - dependent effects of aons 3 and 4 were tested after transfection in fibroblasts from patient 1 ( figures 4c and 4d ) .
aons 3 and 4 induced a concentration - dependent increase in both the expression of full - length gaa transcript and gaa enzymatic activity .
effects were almost maximal at 2 m aon and reached a maximum at 520 m .
these results were confirmed in a fibroblast cell line from patient 2 ( figures 4e and 4f ) . this patient ( genotype ivs1/c.923a >
c ) contained a missense gaa variant on allele 2 , which was expressed at the mrna level and led to a high background
we conclude that aons 3 and 4 show in vitro activities that are sufficient to elevate canonical gaa pre - mrna splicing , and thus gaa enzymatic activity , to above the disease threshold of 20% of the average level present in healthy controls that has been established in the diagnostic department of our center .
to confirm the mechanism by which aon 3 and 4 enhanced gaa enzymatic activity ( i.e. , by modulating splicing rather than total gene expression ) , analysis of individual splicing products was performed . of note ,
full - skip ( sv2 ) and cryptic splicing transcripts ( sv3 ) lack the translation start codon and are subject to mrna degradation , hampering accurate relative quantification . the effect of aon 4 was tested in control fibroblasts and in fibroblast from patients 1 and 2 .
semiquantitative rt - pcr analysis using primers annealing to exon 1 and exon 3 showed that in patients with ivs1 , aon 4 treatment caused an increase in the amount of the full - length ( n ) transcript , while the amount of the full - skip ( sv2 ) transcript was reduced ( figure 4 g ) .
quantitative analysis by qrt - pcr utilized splicing product - specific primers , and this confirmed the semiquantitative analysis ( figure 4h ) .
these results suggest that aon 4 enhances expression of wild - type gaa mrna by promoting exon 2 inclusion during aberrant splicing caused by the ivs1 variant , rather than acting on enhancing total gaa gene expression .
this is further supported by the lack of effect on gaa expression in control cells .
the purpose of this work was to test whether aberrant gaa splicing caused by the common ivs1 variant could be restored using aons . to this end , we performed a lentiviral u7 snrna - based screen with the aim to identify splicing regulatory sequences that could be blocked to promote exon inclusion .
two aons were identified that promoted exon inclusion and gaa enzyme activity to above the disease threshold of 20% of average healthy control levels .
these results identify splicing regulatory elements that negatively regulate the ivs1 variant , and they provide proof of concept for the restoration of canonical splicing in cells from patients with pompe disease .
the ivs1 gaa variant represented a challenging case , because it is located in the py tract of exon 2 and weakens the recognition of the splice acceptor site of exon 2 .
it was not obvious whether it was possible to enhance exon inclusion using aons , as no negative splice regulatory elements in the gaa gene have been described that could be amenable to inhibition using an aon .
this contrasts with other , more rare , gaa variants that affect splicing by causing a major shift from canonical toward cryptic splicing . in these cases , aons that prevent cryptic
we were encouraged by the observation that the ivs1 variant also allows a low level of leaky wild - type splicing , indicating that canonical splicing was still possible and that the py tract was still partially functional .
we identified a splicing regulatory element in intron 1 that was located approximately 280 nt upstream of the canonical splice acceptor of gaa exon 2 . in an accompanying work ,
we show that this does not concern a classical iss motif , but that it is the py tract of a cryptic splice acceptor site that , together with a downstream splice donor , forms a pseudo exon .
current prediction programs for the identification of cis - acting elements that modulate splicing outcome vary widely in outcome , as illustrated in figure 1c .
we therefore performed an unbiased screen using u7 snrna - based aons modified from liu et al.16 , 29 the lentiviral vector employed here was modified to enable one - step cloning with a > 90% success rate .
lentiviral transduction showed near 100% infection of target cells , ensuring expression of the u7 snrnas in all target cells ( data not shown ) .
an alternative approach would be to order all desired aons and to transfect directly in target cells , but this is expensive and not feasible for most research laboratories .
the u7 snrna - based approach identified splicing regulatory motifs that both reduce ( intron 1 ) and enhance ( 5 part of exon 2 ) inclusion of gaa exon 2 ( figure 2b ) .
we were unable to identify these sequences using currently available splicing prediction programs , highlighting the need for experimental work .
this approach should be more generally applicable to unravel basic mechanisms of splicing and to identify therapeutic targets for splicing variants in human disease .
metabolic disorders such as pompe disease are good candidates for aon - mediated splicing correction .
often the enzyme deficiency only needs elevation to levels above a threshold to be fully functional.32 , 33 , 34 hits from the u7 snrna screen were tested using pmo - based aons , and these promoted exon inclusion and gaa enzyme activity to above the disease threshold of 20% .
this threshold was evident from the evaluation of fibroblast samples ( using 4-mu as substrate ) from 700 individuals in our center , 350 of which were diagnosed with pompe disease based on clinical examination , muscle pathology , and biochemical and dna analysis . while gaa enzyme activity levels in patients never exceeded 20% ,
we note that the gaa enzyme activity assay is very sensitive to cell culture and assay conditions , and different patient and normal ranges ( and therefore disease thresholds ) may apply depending on the diagnostic center .
usually the ivs1 variant is present in combination with a variant on the second allele with no residual enzymatic activity , causing childhood / adult pompe disease , although homozygosity of the ivs1 variant has been described in some patients with pompe disease.35 , 36 full restoration of the ivs1 allele in compound heterozygous patients would provide a maximum of 50% gaa activity relative to the average levels in healthy controls , and this activity is within the range of healthy controls .
these results form a proof of concept for the development of a potential alternative treatment of a large percentage of caucasian patients with pompe disease .
current enzyme replacement therapy has limitations and is based on intravenous delivery of rhgaa that is taken up by target cells via mannose-6-phosphate receptor - mediated endocytosis .
aon - mediated splicing correction presents a different strategy , as it enhances endogenous production of wild - type gaa enzyme .
further work is required to test the potential of the identified aons for clinical implementation .
the first step is to test aons in the relevant cell type , skeletal muscle cells , as splicing can be cell - type specific .
aon treatments are well tolerated without serious adverse events , as shown in phase i and ii clinical trials using 2-o - methoxyethyl phosphorothioate and pmo chemistries for spinal muscular atrophy and duchenne muscular dystrophy , respectively.23 , 38 this may accelerate testing of aons for the treatment of pompe disease in a clinical setting .
the ongoing development of improved methods for aon delivery , including tricyclo - dna - based backbones and cell - penetrating peptides like pip6a , is expected to be relevant for the future testing of aon - based drugs for the treatment of pompe disease .
dermal fibroblasts from one control ( control 1 ) and two patients ( patients 1 and 2 ) with pompe disease were obtained via skin biopsy with informed consent . the erasmus medical center ( mc ) institutional review board approved the study protocol .
all patient and control primary cell lines were negative for hiv , hepatitis b , and hepatitis c as tested by qpcr analysis at the diagnostic department of virology of the erasmus mc rotterdam .
the second allele was c.525delt for patient 1 and c.923a > c ( his > pro ) for patient 2 , which both are established pathogenic gaa variants ( www.pompecenter.nl ) .
indications to variants and/or locations on the cdna or ( pre-)mrna conform to human genome variation society ( hgvs ) standards ( http://www.hgvs.org/mutnomen/ ) .
the u7 snrna gene and promoter were amplified by pcr from female mouse genomic dna using fw - ms - u7 snrna - pst1 and rv - ms - u7 snrna - sali primers , which included psti and sali overhang restriction sites .
the pcr fragment ( 425 bp ) was cloned into a pcrii - topo vector according to the manufacturer s manual ( invitrogen ) .
smopt and nsii sites were generated by site - directed mutagenesis according to an inner and outer primer design with fw- and rv - u7 snrna - smopt or fw- and rv - u7 snrna - nsii as inner primers and with fw - m13 and rv - m13 as outer primers ( table s1 ) , and they were subcloned using the psti and sali sites upstream of the polypurine tract fragment of the lentiviral vector used for reprogramming , from which oskm and the spleen focus - forming virus ( sf ) promoter were removed .
aons were inserted via pcr amplification using a forward primer that contained the desired antisense sequence and the unique nsii restriction site and the reverse primer rv - ms- u7 snrna - sali .
the amplified pcr product was purified by agarose gel electrophorese , extracted using the gel extraction kit ( qiagen ) , digested with nsii and sali , purified with a pcr purification kit from ( qiagen ) , and cloned into the nsii and sali sites of the u7 snrna vector .
clones were verified by sequencing with the fw - ms - u7 snrna - psti ( table s1 ) and restriction enzyme digestion .
both hek293 t cells and human primary fibroblasts were cultured in high - glucose dmem ( gibco ) supplemented with 100 u / ml penicillin / streptomycin / glutamine ( gibco / thermo scientific ) and 10% fetal bovine serum ( hyclone / thermo scientific ) .
cells were passaged after reaching 80%/90% confluence with tryple ( gibco / thermo scientific ) .
all cell lines were routinely tested for mycoplasma infection using the mycoalert mycoplasma detection kit ( lonza ) and were negative .
lentiviruses were produced by co - transfecting hek293 t cells at 80% confluence in a 10-cm culture dish with the lentivirus transfer vector ( 3 g sf - u7 snrna vectors ) and packaging plasmids ( 2 g pspax2 and 1 g pvsv vectors ) using fugene 6 transfection according to the manufacturer s protocol ( promega ) .
lentiviruses were harvested from the medium after 72 hr of transfection and were filtered using a 0.45-m polyvinylidene fluoride ( pvdf ) filter ( millipore ) . after filtering ,
lentiviruses were concentrated by high - speed centrifugation for 2 hr at 20,000 rpm in a beckman coulter ultracentrifuge with a sw32 ti rotor at 4c . the supernatant was removed and the pellet was resuspended in 25 l low - glucose dmem ( invitrogen ) per plate .
viral titers were determined with the hiv-1 p24 antigen elisa kit ( retrotek ; zeptomix ) according to the manufacturer s manual .
each virus was diluted 1:400,000 and 1:1,000,000 and the optical density at 450 nm ( od450 ) was measured with the varioskan reader ( thermo scientific ) .
one day before infection , 6 10 cells per single well of a 12-well plate of primary fibroblasts derived from patient 1 were seeded .
one day later , the cells were infected with 200 ng virus containing the sf - u7 snrna constructs ; after 24 hr , cells were washed three times with pbs before fresh medium was added .
after 4 days , cells were washed with pbs and harvested with rlt buffer from the rnaeasy kit for rna isolation ( qiagen ) . for the gaa enzyme activity assay
morpholinos were dissolved in sterile water to a concentration of 1 mm and the appropriate volume was added to each culture well .
rna was extracted with the rneasy mini kit with dnase treatment ( qiagen ) and was stored at 80c in rnase - free water .
cdna was synthesized from 500 ng rna using the iscript cdna synthesis kit ( bio - rad ) .
cdna was diluted 5 , 10 , or 20 times and used with 7.5 l itaq universal sybr green supermix ( bio - rad ) with 10 pmol/l forward and reverse primers ( table s1 ) in a cfx96 real - time system ( bio - rad ) .
cycle threshold ( ct ) values were related to amounts using standard curves of four to six dilutions .
cdna ( diluted 10 times ) with gc gaa exon 13 fw and gc gaa exon 13 rv primers ( table s1 ) were used for rt - pcr with the advantage gc 2 pcr kit ( clontech / takara ) and a gc - melt concentration of 0.5 m according to the manufacturer s protocol .
the whole gc - pcr reaction was analyzed on a 1.5% agarose gel containing 0.5 g / ml ethidium bromide ( sigma - aldrich ) .
cells were harvested with ice - cold lysis buffer ( 50 mm tris , ph 7.5 , 100 mm nacl , 50 mm naf , 1% triton x-100 , and one tablet of roche complete protease inhibitor cocktail , with edta ) and incubated for 10 min on ice .
gaa enzyme activity was measured using 4-mu ( sigma - aldrich ) as substrate as described .
total protein concentration was determined using a bca protein assay kit ( pierce / thermo scientific ) . because gaa enzyme activity data strongly depend on assay conditions and so we could evaluate enzyme activities relative to the disease threshold , we normalized the data based on two criteria : ( 1 ) the enzyme activity of patient cells measured in a diagnostic setting rather than a research setting , and ( 2 ) the average enzyme activity present in healthy control fibroblasts .
these results , expressed as the percent of control , are presented in figure 4 .
all data represent means sd , and p values refer to two - sided student s t tests .
a.t.v.d.p . has provided consulting services for various industries in the field of pompe disease under an agreement between these industries and erasmus mc . | the most common variant causing pompe disease is c.-32 - 13t > g ( ivs1 ) in the acid -glucosidase ( gaa ) gene , which weakens the splice acceptor of gaa exon 2 and induces partial and complete exon 2 skipping .
it also allows a low level of leaky wild - type splicing , leading to a childhood / adult phenotype .
we hypothesized that cis - acting splicing motifs may exist that could be blocked using antisense oligonucleotides ( aons ) to promote exon inclusion . to test this ,
a screen was performed in patient - derived primary fibroblasts using a tiling array of u7 small nuclear rna ( snrna)-based aons .
this resulted in the identification of a splicing regulatory element in gaa intron 1 .
we designed phosphorodiamidate morpholino oligomer - based aons to this element , and these promoted exon 2 inclusion and enhanced gaa enzyme activity to levels above the disease threshold .
these results indicate that the common ivs1 gaa splicing variant in pompe disease is subject to negative regulation , and inhibition of a splicing regulatory element using aons is able to restore canonical gaa splicing and endogenous gaa enzyme activity . | Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest | the regulation of splicing is still relatively poorly understood , which is caused by the many factors that can affect splicing outcome . at the level of rna sequences , splicing regulatory elements
the splice site junctions , as well as the polypyrimidine ( py ) tract and branch point , are relatively easy to identify . splice regulatory elements exist , including the exonic and intronic splice enhancer and silencer elements ( eses , esss , ises , and isss , respectively ) , but these are more difficult to predict . the expression levels of splicing regulatory proteins can also play an important role in splicing outcome . furthermore , the speed of rna polymerase ii transcription is thought to affect splicing efficiency , because splicing mainly occurs co - transcriptionally , and the available splice sites that are present in the protruding pre - mrna within a certain time frame compete for the splicing machinery.4 , 5 additional levels of regulation are chromatin modifications and the composition of the utrs.6 , 7 all together , these diverse levels of regulation highlight the requirement for experimental testing of alternative splicing to validate predictions . this is caused by the inherent detection bias for these variants , which are mostly identified using splice prediction algorithms . however , variants can also alter splicing by the generation of new splice sites9 , 10 or by disturbing or generating other splice regulatory elements.11 , 12 in addition , variants in the coding region can have more than one effect ( i.e. , a missense variant can also have an effect on splicing ) . it is likely that many more pathogenic splicing variants exist that are located outside the region of canonical splice sites , and that the percentage of identified variants that affect splicing will increase in the future . antisense oligonucleotides ( aons ) can modulate splicing by binding to the pre - mrna and blocking splicing regulatory sequences . one type of aon has been modified from the naturally occurring u7 small nuclear rna ( snrna ) , which is normally utilized in histone pre - mrna processing but has been modified to target the pre - mrna of choice . u7 snrna - based aons are stabilized by a stem loop and also contain an antisense sequence that is used for targeting . various backbones with different properties are known , which include phosphorodiamidate morpholino oligomer ( pmo ) , 2-o - methyl phosphorothioate , 2-o - methoxyethyl phosphorothioate , and tricyclo - dna modifications . a straightforward approach to modulate splicing using aons
this will result in exon skipping , which can be advantageous , for example , to bypass a mutation hotspot and to restore the reading frame . many other examples have been published , in which a cryptic splice site is blocked with an aon to restore canonical splicing ( reviewed in havens and hastings ) . we recently showed that this is possible in the case of gaa variants that cause pompe disease . restoration of splicing becomes more challenging in cases where a splicing variant causes exon skipping and requires promotion of exon inclusion . when there is no clear cryptic splice site involved that can be blocked by an aon , such cases require the identification of iss or ess elements that are critical for exon inclusion . few examples for promotion of exon inclusion exist ; notably , for spinal muscular atrophy , caused by variants in the smn1 gene , aons have been designed that reactivate the normally silent sister gene smn2 . smn2 contains an exonic variant that causes exon skipping , and aons have been identified that block ess or iss sequences and promote exon inclusion.21 , 22 one of these , which targets the iss , is currently being tested in clinical trials . in pompe disease ,
a common splicing variant occurs in the majority of caucasian patients with the childhood / adult form . this well - described variant , c.-32 - 13t > g ( also known as ivs1 , or r.-32 - 13u > g at the rna level ) , is located in the py tract of exon 2 of the acid -glucosidase ( gaa ) pre - mrna and causes weakening of the splice acceptor site , resulting in complete or partial skipping of exon 2.24 , 25 , 26 , 27 this exon contains the translation start codon , and its skipping results in mrna degradation and a subsequent reduction of gaa protein production . approximately 10%15% of transcripts are spliced normally and produce a low level of wild - type gaa protein , which explains why these patients develop the childhood / adult form of pompe disease . the restoration of splicing from the ivs1 variant using aons would potentially provide a therapeutic option for the majority of caucasian patients with pompe disease . this may be beneficial in addition to or instead of the current enzyme replacement therapy , which has a partial and heterogeneous response and is extremely expensive . however , splicing correction from the ivs1 variant requires promotion of exon inclusion , and no negatively acting splicing regulatory sequences in the gaa gene that are amenable to inhibition using aons are currently known . here , we used a screen to modulate endogenous gaa splicing using u7 snrna - based aons expressed via lentiviral transduction in patient - derived primary fibroblasts . this resulted in the identification of cis - acting splicing regulatory elements that were subsequently tested using pmo - based aons . two overlapping aons were identified that promoted inclusion of exon 2 in cells from patients carrying the ivs1 variant . in cells from healthy controls ,
the aons did not change gaa splicing or expression , confirming that they acted on splicing rather than elevating total gaa transcript levels . the aons increased gaa enzymatic activity in patient - derived cells to above the disease threshold of 20% . these findings demonstrate the feasibility to correct aberrant splicing from the common ivs1 gaa variant by blocking a negative splicing element to promote exon inclusion . previous work resulted in the characterization of aberrant splicing caused by the ivs1 gaa variant.24 , 25 , 26 , 27 splicing products include wild - type gaa mrna ( n ) caused by leaky normal splicing , partial skipping of exon 2 caused by utilization of a cryptic splice site in exon 2 ( sv3 ) , and full skipping of exon 2 ( sv2 ) . these products can be identified by flanking exon rt - pcr in primary fibroblasts using primers that anneal to exons 1 and 3 ( figure 1a ) . splicing is subject to regulation by additional cis - acting rna sequences besides the sequences surrounding the canonical splice sites . these include esss , isss , eses , and ises and can be located distant from canonical splice sites ( hypothetical example given in figure 1b ) . approximately 10%15% of the gaa pre - mrna is correctly spliced , indicating that the pathogenic effect of the ivs1 variant is not fully detrimental to splicing of gaa exon 2 . we hypothesized that it may be possible to promote exon 2 inclusion by inhibition of a splicing negatively acting splicing regulatory element using an aon . to test this , in silico prediction
was performed using several algorithms , which resulted in a plethora of splicing regulatory elements ( figure 1c ) . however , the results were algorithm dependent and many predicted enhancer and silencer sequences overlapped without indicating an obvious candidate silencer element that could be amenable to inhibition by an aon . to identify splicing regulatory elements experimentally , an unbiased screen was performed using a non - overlapping tiling array of aons that cover part of intron 1 and the complete exon 2 of the gaa pre - mrna ( figure 2a ) . to validate the u7 snrna vector ,
a control experiment was performed on primary fibroblasts derived from a patient with adult pompe disease ( patient 1 ) , who carried the ivs1 variant on one allele and the c.525delt on the second allele . a u7 snrna - based aon was targeted to the splice sites of exon 4 of cyclophilin a ( cypa ) pre - mrna ( figure s1b ) . this confirmed previous reports and demonstrated that the modified u7 snrna construct can be used to modulate splicing . subsequently , the u7 snrna screen using the aons indicated in figure 2a was performed on fibroblasts from patient 1 . the screen resulted in the identification of a number of u7 snrna - based aons that modulated inclusion of exon 2 in the gaa mrna , as shown with qrt - pcr analysis ( figure 2b , black line ) . importantly , only mrna from the allele carrying the ivs1 variant was detected due to the frameshift induced by the c.525delt variant on the second allele , which results in mrna degradation . notably , u7 snrnas targeting two regions in intron 1 , at c.-32 - 179 and c.-32 - 219 , promoted inclusion of gaa exon 2 . exclusion of exon 2 was promoted by u7 snrnas that targeted regions in the 5 part of exon 2 . promotion and inhibition of exon 2 inclusion resulted in increased and decreased gaa enzymatic activity , respectively ( figure 2b , compare black and red lines ) . to fine - tune the optimal location of u7 snrnas targeting c.-32 - 179 and c.-32 - 219 , a microwalk was performed around these regions with u7 snrnas that shifted 2 nt ( figure 3a ) . this showed that the locations identified using the initial u7 snrna screen were peak values . in addition , the microwalk revealed two nearby locations at c.-32 - 183 and c.-32 - 185 whose inhibition promoted exon 2 inclusion ( figures 3b and 3c ) . taken together ,
the u7-based snrna screen of intron 1 and exon 2 identified regions potentially involved in repression of gaa exon 2 inclusion . to test whether morpholino - based aons can modulate aberrant splicing caused by the ivs1 variant
, aons were designed based on the results from the u7 snrna screen shown in figure 3 . specific requirements of low g content on chemical synthesis posed constrains to the regions that could be targeted but it was possible to design aons that targeted the two major putative repressor sequences at c.-32 - 219 ( aons 1 and 2 ) and c.-32 - 179 ( aons 3 and 4 ) ( figures 4a and s3a ) . to test whether primary fibroblasts were amenable to splicing modulation using morpholino - based aons , aons targeting cypa were used as positive control . two pmo - based aons that target the 3 donor splice site of exon 4 in the cypa pre - mrna ( figures s3a and s3b ) were able to promote skipping of exon 4 and exons 3 and 4 after transfection into primary fibroblasts , as shown by flanking exon rt - pcr and qrt - pcr analysis ( figures s3c and s3d ) , which is in agreement with our previous report . next , aons 14 targeting the identified regions in the gaa pre - mrna were tested by transfecting primary fibroblasts from patient 1 . qrt - pcr analysis of exon 2 inclusion showed that aons 3 and 4 promoted exon 2 inclusion more than 2-fold , whereas aons 1 and 2 were ineffective ( figure 4b ) . this showed that pmo - based aons that targeted the region of c.-32 - 179 promoted exon 2 inclusion , and that other pmo - based aons tested were ineffective . these results were confirmed in a fibroblast cell line from patient 2 ( figures 4e and 4f ) . this patient ( genotype ivs1/c.923a >
c ) contained a missense gaa variant on allele 2 , which was expressed at the mrna level and led to a high background
we conclude that aons 3 and 4 show in vitro activities that are sufficient to elevate canonical gaa pre - mrna splicing , and thus gaa enzymatic activity , to above the disease threshold of 20% of the average level present in healthy controls that has been established in the diagnostic department of our center . these results suggest that aon 4 enhances expression of wild - type gaa mrna by promoting exon 2 inclusion during aberrant splicing caused by the ivs1 variant , rather than acting on enhancing total gaa gene expression . the purpose of this work was to test whether aberrant gaa splicing caused by the common ivs1 variant could be restored using aons . to this end , we performed a lentiviral u7 snrna - based screen with the aim to identify splicing regulatory sequences that could be blocked to promote exon inclusion . two aons were identified that promoted exon inclusion and gaa enzyme activity to above the disease threshold of 20% of average healthy control levels . these results identify splicing regulatory elements that negatively regulate the ivs1 variant , and they provide proof of concept for the restoration of canonical splicing in cells from patients with pompe disease . the ivs1 gaa variant represented a challenging case , because it is located in the py tract of exon 2 and weakens the recognition of the splice acceptor site of exon 2 . it was not obvious whether it was possible to enhance exon inclusion using aons , as no negative splice regulatory elements in the gaa gene have been described that could be amenable to inhibition using an aon . in these cases , aons that prevent cryptic
we were encouraged by the observation that the ivs1 variant also allows a low level of leaky wild - type splicing , indicating that canonical splicing was still possible and that the py tract was still partially functional . we identified a splicing regulatory element in intron 1 that was located approximately 280 nt upstream of the canonical splice acceptor of gaa exon 2 . in an accompanying work ,
we show that this does not concern a classical iss motif , but that it is the py tract of a cryptic splice acceptor site that , together with a downstream splice donor , forms a pseudo exon . current prediction programs for the identification of cis - acting elements that modulate splicing outcome vary widely in outcome , as illustrated in figure 1c . we therefore performed an unbiased screen using u7 snrna - based aons modified from liu et al.16 , 29 the lentiviral vector employed here was modified to enable one - step cloning with a > 90% success rate . the u7 snrna - based approach identified splicing regulatory motifs that both reduce ( intron 1 ) and enhance ( 5 part of exon 2 ) inclusion of gaa exon 2 ( figure 2b ) . often the enzyme deficiency only needs elevation to levels above a threshold to be fully functional.32 , 33 , 34 hits from the u7 snrna screen were tested using pmo - based aons , and these promoted exon inclusion and gaa enzyme activity to above the disease threshold of 20% . this threshold was evident from the evaluation of fibroblast samples ( using 4-mu as substrate ) from 700 individuals in our center , 350 of which were diagnosed with pompe disease based on clinical examination , muscle pathology , and biochemical and dna analysis . while gaa enzyme activity levels in patients never exceeded 20% ,
we note that the gaa enzyme activity assay is very sensitive to cell culture and assay conditions , and different patient and normal ranges ( and therefore disease thresholds ) may apply depending on the diagnostic center . usually the ivs1 variant is present in combination with a variant on the second allele with no residual enzymatic activity , causing childhood / adult pompe disease , although homozygosity of the ivs1 variant has been described in some patients with pompe disease.35 , 36 full restoration of the ivs1 allele in compound heterozygous patients would provide a maximum of 50% gaa activity relative to the average levels in healthy controls , and this activity is within the range of healthy controls . these results form a proof of concept for the development of a potential alternative treatment of a large percentage of caucasian patients with pompe disease . aon - mediated splicing correction presents a different strategy , as it enhances endogenous production of wild - type gaa enzyme . the first step is to test aons in the relevant cell type , skeletal muscle cells , as splicing can be cell - type specific . the ongoing development of improved methods for aon delivery , including tricyclo - dna - based backbones and cell - penetrating peptides like pip6a , is expected to be relevant for the future testing of aon - based drugs for the treatment of pompe disease . the second allele was c.525delt for patient 1 and c.923a > c ( his > pro ) for patient 2 , which both are established pathogenic gaa variants ( www.pompecenter.nl ) . the u7 snrna gene and promoter were amplified by pcr from female mouse genomic dna using fw - ms - u7 snrna - pst1 and rv - ms - u7 snrna - sali primers , which included psti and sali overhang restriction sites . aons were inserted via pcr amplification using a forward primer that contained the desired antisense sequence and the unique nsii restriction site and the reverse primer rv - ms- u7 snrna - sali . the amplified pcr product was purified by agarose gel electrophorese , extracted using the gel extraction kit ( qiagen ) , digested with nsii and sali , purified with a pcr purification kit from ( qiagen ) , and cloned into the nsii and sali sites of the u7 snrna vector . both hek293 t cells and human primary fibroblasts were cultured in high - glucose dmem ( gibco ) supplemented with 100 u / ml penicillin / streptomycin / glutamine ( gibco / thermo scientific ) and 10% fetal bovine serum ( hyclone / thermo scientific ) . one day before infection , 6 10 cells per single well of a 12-well plate of primary fibroblasts derived from patient 1 were seeded . for the gaa enzyme activity assay
morpholinos were dissolved in sterile water to a concentration of 1 mm and the appropriate volume was added to each culture well . gaa enzyme activity was measured using 4-mu ( sigma - aldrich ) as substrate as described . because gaa enzyme activity data strongly depend on assay conditions and so we could evaluate enzyme activities relative to the disease threshold , we normalized the data based on two criteria : ( 1 ) the enzyme activity of patient cells measured in a diagnostic setting rather than a research setting , and ( 2 ) the average enzyme activity present in healthy control fibroblasts . all data represent means sd , and p values refer to two - sided student s t tests . has provided consulting services for various industries in the field of pompe disease under an agreement between these industries and erasmus mc . | [
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current dental implant research aims at developing of innovative surfaces able to promote a more favourable biological response to the implant material at the bone - implant interface and to accelerate osseointegration .
it has largely been demonstrated that rough surfaces present an increased bone fixation and bone - to - implant contact compared to smooth surfaces [ 24 ] .
in addition to surface topography , the chemical properties of implant surfaces also play an important role in promoting osseointegration .
modification of titanium implants using hydrogen fluoride at low concentrations results in the formation of nanostructures along the titanium surface as well as the incorporation of small amounts of fluoride into the crystal structure of the superficial layer of the implant [ 1 , 6 ] , thereby , modifying both , surface topography and surface chemistry . in vitro experiments have shown that fluoride - modified titanium implants stimulate osteoblast differentiation in different cell models [ 710 ] , enhance cell osteoblastic adhesion and expression of bone - specific mrna [ 8 , 11 ] , increase cell viability , improve the initial cell response to the implant , and augment the thrombogenic properties of titanium , promoting fibrinogen activation and rapid coagulation . in vivo ,
fluoride - modified titanium implants enhance interfacial bone formation , create a firmer bone anchorage , augment the amount of new bone formation in the voids and bone - to - implant contact , improve biomechanical properties due to a more mature and mineralized interfacial bone matrix , and increase implant osseointegration in osteoporotic bone .
the aim of the present study was to examine the in vitro bone response of mouse preosteoblast mc3t3-e1 cells to two commercial ti surfaces , osseospeed and tioblast , and to validate the claimed higher bone response of the new generation surface ( osseospeed ) compared to its respective predecessor ( tioblast ) .
the osteoblast response to these ti surfaces was also compared with tissue culture plastic ( tcp ) , which is normally considered the gold standard for tissue culture .
osseospeed is a further development of the moderately roughened ( grit blasted with titanium dioxide particles ) titanium surface tioblast .
osseospeed has been reported to gain its additional surface characteristics via a chemical ( fluoride ) treatment and a slight topographic modification of the tioblast surface [ 6 , 14 ] .
test implants used were all made of grade 2 titanium , with a diameter of 6.25 mm and a height of 1.95 mm .
the test surface was blasted with titanium dioxide ( tio2 ) particles ( tioblast ) to create a microrough surface . according to the manufacturer ,
fluoride modified implants ( osseospeed ) went through an additional cleaning process including diluted hf .
implants were premounted on the carriers , inserted individually in sealed containers , and sterilized by -irradiation ( astratech ab , mlndal , sweden ) . after surface treatments ,
the surface roughness of one sample per group was measured with a blue light profilometer ( pl 2300 , sensofar , terassa , spain ) .
three areas were imaged per surface at 50x magnification ( 254 191 m ) , and surface parameters were calculated after levelling the images by rotation with the program sensomap plus 4.1 ( sarl digital surf , besanon , france ) and application of a gaussian filter ( 50 50 m ) to remove underlying waviness .
topographical changes were as follows : average height deviation from the mean plan ( sa ) , surface skewness ( ssk ) , surface kurtosis ( sku ) , and core fluid retention index ( sci ) were recorded to quantify surface differences among the tioblast and the osseospeed groups .
the murine osteoblast cell line mc3t3-e1 ( dsmz , braunschweig , germany ) was used as in vitro model .
cells were routinely cultured at 37c in a humidified atmosphere of 5% co2 and maintained in alpha - mem ( paa laboratories gmbh , austria ) supplemented with 10% fetal calf serum ( paa laboratories gmbh , austria ) and 50 iu penicillin / ml and 50 g streptomycin / ml ( sigma , st .
cells were subcultured 1 : 4 before reaching confluence using pbs ( paa laboratories gmbh , austria ) and trypsin / edta ( sigma , st .
louis , mo , usa ) . to test the different surface modification of titanium implants ( tioblast and osseospeed ) , coins were placed in a 96-well plate ( with a diameter size per well of 6.5 mm ) , and 10 cells were seeded on each coin . the same number of cells was cultured in parallel in plastic ( tcp ) in all the experiments .
after 48 h , 7 , and 14 days , the culture media was collected , centrifuged at 500 g for 5 min at 4c , and the supernatant was stored at 4c .
ldh activity was determined spectrophotometrically according to the manufacturer 's kit instructions ( cytotoxicity detection kit , roche diagnostics , mannheim , germany ) and presented relative to the ldh activity in the medium of cells cultured on tcp for 2 days , which was set as 100% .
the mc3t3-e1-cell layers were washed twice with pbs after the respective culture time and fixed in a 4% formaldehyde solution in pbs for 30 min .
subsequently , the samples were washed again with pbs and mounted with a dapi - containing mounting medium ( prolong gold antifade reagent with dapi , invitrogen ltd , paisley , uk ) according to their protocol .
the samples were stored in dark at 20c until analysis by fluorescence microscopy ( leica dm rbe , leica microsystems , wetzlar , germany ) with connected digital camera ( olympus dm50 , olympus europe , hamburg , germany ) .
four implants were used per treatment and time point , and in each implant three different fields were analysed , giving the number of cells per m .
sem analyses were performed to study the morphology of mc3t3-e1 cells grown on the surface of both ti surfaces . for this purpose ,
cells were first fixed in a formaldehyde solution at 4% in pbs for 30 min .
cell layers were dried in increasing concentrations of ethanol followed by critical point drying ( e3000 , quorum tech , ashford , uk ) and sputter coated with a thin layer of carbon ( cressington carbon coater 108/carbon a , cressington scientific intruments ltd . ,
sem scans were taken ( philips xl 30 esem , fei electron optics , eindhoven , the netherlands ) to image the morphology of the cells attaching to the different surfaces .
pictures at 400x of magnification were taken after 2 , 7 , and 14 days and also at 2000x of magnification after 2 days .
cell culture supernatants were analysed for bmp-2 release , secreted to the culture medium after 2 days of cell culture , using an enzyme - linked immunosorbent assay ( elisa ) .
aliquots from the culture media were centrifuged at 1800 rpm for 5 minutes at 4c , and supernatants were used for bmp-2 determination following instructions described by the manufacturer ( quantikine immunoassay , r&d systems , minneapolis , mn , usa ) .
total rna was isolated using a monophasic solution of phenol and guanidine isothiocyanate ( trizol , invitrogen life technologies , carlsbad , ca , usa ) , following the instructions of the manufacturer .
rna was quantified at 260 nm using a nanodrop spectrophotometer ( nanodrop technologies , wilmington , de , usa ) . the same amount of total rna ( 2 g ) from each sample was reverse transcribed to cdna at 42c for 60 min in a final volume of 40 l , using iscript cdna synthesis kit ( biorad ) that contains both oligo(dt ) and random hexamers .
each cdna was diluted 1/5 , and aliquots were frozen ( 20c ) until the pcr reactions were carried out .
real - time pcr was performed for two housekeeping genes : 18s ribosomal rna ( 18s rrna ) , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , and thirteen target genes : alkaline phosphatase ( alp ) , bone sialoprotein ( bsp ) , cd44 , collagen type i ( coll - i ) , distal - less homeobox 2 ( dlx2 ) , hairy and enhancer of split 1 ( hes1 ) , insulin growth factor - i ( igf - i ) , interleukin-6 ( il-6 ) , osteoprotegerin ( opg ) , osterix ( osx ) , receptor activator of nfkappab ligand ( rankl ) , smad1 , and smad5 .
oligonucleotide primer sequences used for the real - time rt - pcr , the length of the resulting amplicons and the genebank accession number , are shown in table 1 .
real - time pcr was performed in the icycler ( biorad ) using sybr green detection .
each reaction contained 5 l of cdna , 500 nm of the sense and antisense specific primers ( for all , except for collagen - i which was 300 nm ) , and 12.5 l of 2x iq sybr green supermix in a final volume of 25 l .
the amplification program consisted of a preincubation step for denaturation of the template cdna ( 3 min 95c ) , followed by 40 cycles consisting of a denaturation step ( 15 s 95c ) , an annealing step ( 15 s 60c ; for all , except for alp which was 65c ) , and an extension step ( 30 s 72c ) .
real - time efficiencies were calculated from the given slopes in the icycler software using serial dilutions , showing all the investigated transcripts high real - time pcr efficiency rates , and high linearity ( r > 0.99 ) when different concentrations were used .
pcr products were subjected to a melting curve analysis on the icycler and subsequently 2% agarose / tae gel electrophoresis to confirm amplification specificity .
an aliquot of 25 l of culture media was assayed in duplicate for alkaline phosphatase activity by measuring the cleavage of p - nitrophenyl phosphate ( pnpp ) ( sigma , st .
louis , mo , usa ) in a soluble yellow end product which absorbs at 405 nm . a volume of 100 l of this substrate was used .
the reaction was stopped after 30 min in dark with the addition of 50 l of 3 m sodium hydroxide .
the absorbance of the stopped reaction was read at 405 nm . in parallel to the samples , a standard curve with calf intestinal alkaline phosphatase ( ciap , 1 u/l ) ( promega , madison , wi , usa ) was constructed , by mixing 1 l from the stock ciap with
5 ml of alkaline phosphatase buffer ( 1 : 5000 dilution ) and then making 1 : 5 serial dilutions .
cell culture supernatants were analysed for opg , osteocalcin , il-6 , tnf- , and rankl using the solid phase sandwich multiplex bead immunoassays ( mouse bone panel 1b lincoplex kit and mouse rankl single - plex kit , cat#mbn1b-41k and cat#mbn-41k-1rankl , linco research , st .
multianalyte profiling was performed on the luminex-100 analyser ( luminex corporation , austin , tx , usa ) .
after 14 days of cell culture , the entire surface of tioblast and osseospeed titanium coins ( n = 2 ) were examined for calcium deposition with a tabletop scanning electron microscope ( sem ) ( tm-1000 , hitachi , tokyo , japan ) .
cell layers were dried in increasing concentrations of ethanol followed by critical point drying ( e3000 , quorum tech , ashford , uk ) and sputter coated with a thin layer of carbon ( cressington carbon coater 108/carbon a , cressington scientific intruments ltd , watford , uk ) .
a 250x magnification was used to examine the shape , size , and number of calcium crystals .
the composition of the crystals was analysed by energy dispersive x - ray spectroscopy ( eds unit , tm-1000 , hitachi , tokyo , japan ) .
differences between groups were assessed by student 's t - test , using the program spss for windows , version 17.0 .
topographical analyses by blue - light profilometry ( table 2 ) revealed a significant increase of sa for the osseospeed surfaces compared to tioblast .
surface skewness was negative for both groups , and they can be imagined as bearing surfaces with holes . the skewness was significantly increased for osseospeed .
surface kurtosis was significantly higher for tioblast , indicating a more rounded appearance of the tioblast surfaces .
the core fluid retention index was significantly higher for osseospeed , in accordance with the sa values and pointing to a better fluid retention for osseospeed surfaces .
observation of the cell monolayer by sem microscopy ( figures 1 and 2 ) and by fluorescence microscopy ( figure 3 ) confirmed that mc3t3-e1 cells attached well to both ti surfaces , resulting in similar cell proliferation at the different time points investigated .
on osseospeed surface , cells exhibited a more branched shape morphology compared to tioblast . in order to determine the effect of the different ti surfaces and tcp on the cell viability after short- and long - term cell culture on the titanium implants ,
the ldh activity in the culture media was measured after 2 , 7 , and 14 days ( figure 4(a ) ) .
no differences were found between the osseospeed and tioblast in the ldh activity at any of the days analysed .
however , after 14 days of culture , cells cultured onto tcp showed significantly higher levels of ldh activity compared to cells cultured onto ti implants .
counting of cells at the different time points using dapi staining ( figure 4(b ) ) , revealed no differences in cell proliferation between the two ti surfaces .
rna content was quantified from cell monolayer after 2 , 7 , and 14 days of culture ( figure 4(c ) ) .
no differences were observed among the different groups , although rna content increased from 2 to 7 days of cell culture .
the potential to initiate osteogenic differentiation after 2 days of culture was investigated by measuring the bmp-2 release to the culture media using a bmp-2 immunoassay ( figure 5 ) .
the differentiation of mc3t3-e1 cells cultured on the different implant surfaces and onto plastic was examined by analysing the gene expression after 2 , 7 , and 14 days of cell culture .
as seen in figures 6 , 7 , and 8 , gene expression levels of fifteen different genes related to osteogenic differentiation like transcriptional factors and regulators , extracellular matrix molecules , cytokines , growth factors , and functional markers were analysed . regarding the osteogenic markers ( figure 6 ) , coll - i mrna expression was significantly higher in cells cultured onto both titanium surfaces compared to those cultured in plastic after 14 days .
bsp mrna expression was upregulated in cells cultured on plastic compared to those cultured on both titanium surfaces .
after 14 days of culture , the osseospeed group displayed higher bsp mrna levels compared to the tioblast one .
alp mrna levels were significantly higher in cells cultured onto plastic compared to those cultured on both titanium surfaces after 2 and 7 days of culture , while after 14 days , alp levels of cells cultured on both titanium surfaces were higher than those cultured on plastic , although just the osseospeed group reached statistical significance .
cd44 mrna levels were significantly higher in cells cultured on plastic compared to those cultured on both titanium surfaces ( day 7 ) and compared to the tioblast group ( day 2 ) .
after 14 days of culture , no differences were observed between groups . as regards transcriptional factors and regulators ( figure 7 ) , no significant differences were found for dlx2 and hes1 among the groups at the different time points , although their gene expression decreased likewise over the time period studied .
osterix mrna levels were higher in cells cultured on both titanium surfaces compared to those cultured on plastic after 2 days of culture .
after 14 days of culture , osseospeed implants induced an upregulation osterix mrna compared to tioblast .
after 14 days of culture , the osseospeed group displayed higher smad1 mrna levels compared to tcp .
smad5 mrna expression was significantly up - regulated in the tcp group compared to the tioblast one after 2 days of culture , and no differences were observed afterwards .
regarding cytokines and growth factors ( figure 8) , igf - i mrna expression was significantly higher in cells cultured on plastic compared to those cultured on both titanium surfaces after 2 and 7 days of culture .
however , after 14 days , igf - i levels of cells cultured on the osseospeed group were significantly higher than for the other groups .
no significant differences were found for il-6 or opg mrna levels among the groups at the different time points and their expression decreased likewise over the time period studied .
rankl mrna levels ( day 7 ) were significantly higher in the osseospeed group compared to the tcp one .
no differences were seen for the other groups or time points for this gene .
osteocalcin , opg , and il-6 release to the culture media was determined by a luminex bioassay after 2 , 7 , and 14 days of culture in the tioblast , the osseospeed and tcp groups ( figure 9 ) .
tnf- and rankl levels were under detection levels in all cell culture supernatants and are thus , not presented .
osteocalcin and opg release was higher in both implant groups compared to tcp after 7 and 14 days of culture . after 2 days , cells cultured on tioblast surfaces released higher amounts of opg than those cultured on osseospeed surfaces .
il-6 release was higher in the tioblast group than in the tcp one after 7 days of culture .
no statistical differences were found for alp activity in the culture medium after 2 , 7 , and 14 days of cell culture ( figure 10 ) . however , alp activity in the osseospeed group tended to be higher than in the tioblast one .
after 14 days of culture , cells cultured on osseospeed titanium surfaces displayed a higher number of calcium crystals compared to those cultured on tioblast surfaces ( 16.5 1.5 versus 9.5 0.5 , respectively ; p = 0.047 ) .
the present study provides a wide characterization of the in vitro osteogenic properties of two commercial surfaces , tioblast and osseospeed .
tioblast was the first moderately roughened implant surface with 10 years followup reported in the literature and the precursor of the osseospeed surface [ 1820 ] .
the osseospeed surface is a further development introduced in 2004 that incorporates small amounts of fluoride ions in the oxide layer , a slight increase on the micrometer scale in surface roughness , and the appearance of a nanoscale topography . in the present work ,
we showed the topographical differences between the two surfaces , mainly an increase in the sa value in the osseospeed surface , but also changes in surface skewness , kurtosis , and the core fluid retention index .
the obtained results confirm that osseospeed surface show an increased micrometer - scale surface roughness , together with the formation of nanostructures , as reported in earlier studies .
the presence of micro- and nanoscale topography in osseospeed compared to tioblast surface and the addition of fluoride , did not change the biocompatibility of the implants and the initial attachment and proliferation of the mc3t3-e1 cells . however , it was observed that osseospeed surfaces induced a more branched cell morphology .
it has been reported that this cell shape may increase the contractility of the cytoskeleton and lead to preferential osteoblastic differentiation , which has been found in the present study for the osseospeed surface compared to tioblast .
the increase found in the ldh activity on the tcp surface after 14 days is most probably due to the higher proliferation and/or cellular activity at this later time point on tcp .
mc3t3-e1 osteoblast - like cells undergo a developmental sequence of proliferation and differentiation similar to primary cells in culture .
osteoblast maturation in vitro is characterised by changes in gene expression at each developmental stage [ 2326 ] .
modulation of these expressed genes is subjected to a transcriptional control regulated by growth factors and cytokines [ 23 , 26 ] .
bmp-2 is a highly potent growth / differentiation factor that induces differentiation of progenitor cells into the osteoblast lineage , and exhibits this osteogenic action by activating smad signaling and by regulating transcription of osteogenic genes .
thus , the higher release of bmp-2 found in the osseospeed group could initiate osteogenic differentiation through the regulation of transcriptional factors .
runt - related transcription factor 2 ( runx2 ) is a master regulator of osteogenic gene expression that is necessary for the osteoblast lineage commitment and , as well , regulates osteoblast differentiation . here
, we did not determine runx2 mrna levels since , as we have previously reported that runx2 mrna expression is constant during osteoblast differentiation , probably due to the fact that mc3t3-e1 cells are already committed to the osteoblast lineage .
nevertheless , we have analysed the expression of different transcription factors that have been described to interact with runx2 : dlx2 , a downstream target of bmp-2 that is thought to directly activate runx2 and osterix genes , and hes1 that can stimulate the transactivating function of runx2 , although it negatively regulates bone phenotypic maturation and its expression decreases during osteoblast differentiation .
we have previously reported that hes1 and dlx2 are early responsive genes to roughness and fluoride treatment of titanium implants . in our previous report
, both genes were downregulated by fluoride treatment of rough titanium implants after one day in primary human osteoblasts . here
, no differential regulation was found for these two genes among the two surfaces analysed , neither when compared to tcp .
the difference between the results may have been caused by differences in the surfaces used for comparison and differences in the cell model .
the smad family of proteins has been identified as the downstream propagators of bmp signals .
bmp - activated smads induce runx2 gene expression and smads interact physically with the runx2 protein to induce osteoblast differentiation .
we found no important changes on smad1 and smad5 at the different time points and groups analysed ; only smad1 showed higher significant levels in osseospeed compared to tcp .
thus , although smad expression patterns are informative , future studies should investigate their phosphorylation stage to find out whether their activity is regulated in the different surfaces .
osterix is another transcription factor downstream of runx2 which is required for the ongoing differentiation within the osteogenic pathway , being involved in the differentiation step from preosteoblast to fully functional osteoblast .
here we found higher osx mrna levels in cells cultured onto osseospeed implants compared to tioblast , in agreement with earlier observations [ 8 , 10 ] .
type i collagen is expressed in high levels in the early proliferation stage , which is gradually decreased as the cell matures .
this downregulation was only observed in the tcp group , while tioblast and osseospeed showed higher coll-1 mrna levels . in agreement with these results ,
masaki et al . also found higher mrna levels of coll - i in human palatal mesenchymal stem cells cultured on tioblast and osseospeed than on tcp after 3 days , although these differences were not significant .
alkaline phosphatase increases during extracellular matrix maturation then decreases when mineralization is well progressed and bone sialoprotein is transiently expressed very early and then upregulated again in differentiated osteoblasts at the onset of mineralization [ 23 , 26 ] .
these two markers , that reflect a more advanced stage of osteoblast differentiation , showed higher mrna levels on tcp up to the first week , while osseospeed surfaces increased their levels greatly after 14 days , indicating that extracellular matrix of cells seeded on tcp was mature and competent for mineralization after 1 week and on osseospeed surfaces after 2 weeks .
this also indicates that differentiation of mc3t3-e1 cells on tioblast was delayed compared to osseospeed surface .
these results are in line with those obtained in the present study for the higher alp activity and number of crystals deposited in the cell monolayer of osseospeed surface . using the same in vitro model
, another study did not observe significant differences in bsp between the two titanium surfaces , although in this case the roughness was similar , and the titanium particles for grit - blastingwas smaller .
finally , cd44 was analysed as this marker has been indicated to be expressed in higher levels in osteocytes .
however , the analysis did not reveal important differences between the two titanium surfaces investigated , only for the tcp group .
besides these bone - specific markers , the effect of the different titanium implant surfaces on the expression of different growth factors and cytokines involved in bone formation was analysed .
il-6 is a cytokine produced by cells of the osteoblast and osteoclast lineages that not only has a role in inflammation but also increases bone resorption and possibly bone remodeling .
both the mrna levels and the secretion of il-6 decreased over the time in cell culture , but in a lesser extent for tioblast surface .
igf - i induces osteoblast proliferation , bone collagen , and matrix synthesis [ 40 , 41 ] and stimulates the activity of alkaline phosphatase .
similar to other osteogenic markers commented before , this growth factor was significantly upregulated in osseospeed surface compared to both tcp and tioblast .
similar results have been found in a previous study in vivo , suggesting that igf - i might play an important role stimulating bone formation when administered in combination with fluoride .
osteoblasts exert a crucial function in osteoclast activation and differentiation , through the production of specific biological mediators such as the activator of nuclear factor k b ligand ( rankl ) and its antagonist osteoprotegerin ( opg ) . in the present work ,
the total amount of rankl produced by mc3t3-e1 osteoblasts was below the detection limit of the luminex assay ( 3 pg / ml ) , while opg was detected in the supernatant during the whole culture period , was similar to the reported results by guida and coworkers using elisa .
however , opg production found in this study was similar between tioblast and osseospeed group in mc3t3-e1 osteoblasts , with higher levels on titanium when compared to tcp . as a trend
, tioblast surfaces showed higher levels than osseospeed , opposite to the results obtained in human bone marrow mesenchymal stem cells .
due to the lack of differences in opg / rankl mrna levels and the secretion of opg , we conclude that in mc3t3-e1 cells , the levels of opg / rankl are not regulated by the different surfaces used in the study .
other authors have reported increased opg levels in response to rough surfaces and different chemical composition . in conclusion
, the results from the present study demonstrate that the gain of using osseospeed surface is an improved osteoblast differentiation and mineralization , without additional effects on cell viability or proliferation .
the enhanced in vitro osteogenic properties are in line with the improved osseointegrating properties and clinical performance of fluoride - modified titanium implants [ 4749 ] . | current dental implant research aims at understanding the biological basis for successful implant therapy .
the aim of the study was to perform a full characterization of the effect of two commercial titanium ( ti ) surfaces , osseospeed and tioblast , on the behaviour of mouse preosteoblast mc3t3-e1 cells .
the effect of these ti surfaces was compared with tissue culture plastic ( tcp ) . in vitro experiments
were performed to evaluate cytotoxicity , cell morphology and proliferation , alkaline phosphatase activity , gene expression , and release of a wide array of osteoblast markers .
no differences were observed on cell viability and cell proliferation .
however , changes were observed in cell shape after 2 days , with a more branched morphology on osseospeed compared to tioblast .
moreover , osseospeed surface increased bmp-2 secretion after 2 days , and this was followed by increased igf - i , bsp , and osterix gene expression and mineralization compared to tioblast after 14 days . as compared to the gold standard tcp , both ti surfaces induced higher osteocalcin and opg release than tcp and differential temporal gene expression of osteogenic markers .
the results demonstrate that the gain of using osseospeed surface is an improved osteoblast differentiation and mineralization , without additional effects on cell viability or proliferation . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion | current dental implant research aims at developing of innovative surfaces able to promote a more favourable biological response to the implant material at the bone - implant interface and to accelerate osseointegration . in vitro experiments have shown that fluoride - modified titanium implants stimulate osteoblast differentiation in different cell models [ 710 ] , enhance cell osteoblastic adhesion and expression of bone - specific mrna [ 8 , 11 ] , increase cell viability , improve the initial cell response to the implant , and augment the thrombogenic properties of titanium , promoting fibrinogen activation and rapid coagulation . in vivo ,
fluoride - modified titanium implants enhance interfacial bone formation , create a firmer bone anchorage , augment the amount of new bone formation in the voids and bone - to - implant contact , improve biomechanical properties due to a more mature and mineralized interfacial bone matrix , and increase implant osseointegration in osteoporotic bone . the aim of the present study was to examine the in vitro bone response of mouse preosteoblast mc3t3-e1 cells to two commercial ti surfaces , osseospeed and tioblast , and to validate the claimed higher bone response of the new generation surface ( osseospeed ) compared to its respective predecessor ( tioblast ) . the osteoblast response to these ti surfaces was also compared with tissue culture plastic ( tcp ) , which is normally considered the gold standard for tissue culture . test implants used were all made of grade 2 titanium , with a diameter of 6.25 mm and a height of 1.95 mm . implants were premounted on the carriers , inserted individually in sealed containers , and sterilized by -irradiation ( astratech ab , mlndal , sweden ) . three areas were imaged per surface at 50x magnification ( 254 191 m ) , and surface parameters were calculated after levelling the images by rotation with the program sensomap plus 4.1 ( sarl digital surf , besanon , france ) and application of a gaussian filter ( 50 50 m ) to remove underlying waviness . topographical changes were as follows : average height deviation from the mean plan ( sa ) , surface skewness ( ssk ) , surface kurtosis ( sku ) , and core fluid retention index ( sci ) were recorded to quantify surface differences among the tioblast and the osseospeed groups . the murine osteoblast cell line mc3t3-e1 ( dsmz , braunschweig , germany ) was used as in vitro model . to test the different surface modification of titanium implants ( tioblast and osseospeed ) , coins were placed in a 96-well plate ( with a diameter size per well of 6.5 mm ) , and 10 cells were seeded on each coin . the same number of cells was cultured in parallel in plastic ( tcp ) in all the experiments . after 48 h , 7 , and 14 days , the culture media was collected , centrifuged at 500 g for 5 min at 4c , and the supernatant was stored at 4c . ldh activity was determined spectrophotometrically according to the manufacturer 's kit instructions ( cytotoxicity detection kit , roche diagnostics , mannheim , germany ) and presented relative to the ldh activity in the medium of cells cultured on tcp for 2 days , which was set as 100% . sem analyses were performed to study the morphology of mc3t3-e1 cells grown on the surface of both ti surfaces . cell layers were dried in increasing concentrations of ethanol followed by critical point drying ( e3000 , quorum tech , ashford , uk ) and sputter coated with a thin layer of carbon ( cressington carbon coater 108/carbon a , cressington scientific intruments ltd . ,
sem scans were taken ( philips xl 30 esem , fei electron optics , eindhoven , the netherlands ) to image the morphology of the cells attaching to the different surfaces . pictures at 400x of magnification were taken after 2 , 7 , and 14 days and also at 2000x of magnification after 2 days . cell culture supernatants were analysed for bmp-2 release , secreted to the culture medium after 2 days of cell culture , using an enzyme - linked immunosorbent assay ( elisa ) . total rna was isolated using a monophasic solution of phenol and guanidine isothiocyanate ( trizol , invitrogen life technologies , carlsbad , ca , usa ) , following the instructions of the manufacturer . real - time pcr was performed for two housekeeping genes : 18s ribosomal rna ( 18s rrna ) , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , and thirteen target genes : alkaline phosphatase ( alp ) , bone sialoprotein ( bsp ) , cd44 , collagen type i ( coll - i ) , distal - less homeobox 2 ( dlx2 ) , hairy and enhancer of split 1 ( hes1 ) , insulin growth factor - i ( igf - i ) , interleukin-6 ( il-6 ) , osteoprotegerin ( opg ) , osterix ( osx ) , receptor activator of nfkappab ligand ( rankl ) , smad1 , and smad5 . each reaction contained 5 l of cdna , 500 nm of the sense and antisense specific primers ( for all , except for collagen - i which was 300 nm ) , and 12.5 l of 2x iq sybr green supermix in a final volume of 25 l . the amplification program consisted of a preincubation step for denaturation of the template cdna ( 3 min 95c ) , followed by 40 cycles consisting of a denaturation step ( 15 s 95c ) , an annealing step ( 15 s 60c ; for all , except for alp which was 65c ) , and an extension step ( 30 s 72c ) . pcr products were subjected to a melting curve analysis on the icycler and subsequently 2% agarose / tae gel electrophoresis to confirm amplification specificity . an aliquot of 25 l of culture media was assayed in duplicate for alkaline phosphatase activity by measuring the cleavage of p - nitrophenyl phosphate ( pnpp ) ( sigma , st . in parallel to the samples , a standard curve with calf intestinal alkaline phosphatase ( ciap , 1 u/l ) ( promega , madison , wi , usa ) was constructed , by mixing 1 l from the stock ciap with
5 ml of alkaline phosphatase buffer ( 1 : 5000 dilution ) and then making 1 : 5 serial dilutions . after 14 days of cell culture , the entire surface of tioblast and osseospeed titanium coins ( n = 2 ) were examined for calcium deposition with a tabletop scanning electron microscope ( sem ) ( tm-1000 , hitachi , tokyo , japan ) . cell layers were dried in increasing concentrations of ethanol followed by critical point drying ( e3000 , quorum tech , ashford , uk ) and sputter coated with a thin layer of carbon ( cressington carbon coater 108/carbon a , cressington scientific intruments ltd , watford , uk ) . the composition of the crystals was analysed by energy dispersive x - ray spectroscopy ( eds unit , tm-1000 , hitachi , tokyo , japan ) . topographical analyses by blue - light profilometry ( table 2 ) revealed a significant increase of sa for the osseospeed surfaces compared to tioblast . surface kurtosis was significantly higher for tioblast , indicating a more rounded appearance of the tioblast surfaces . observation of the cell monolayer by sem microscopy ( figures 1 and 2 ) and by fluorescence microscopy ( figure 3 ) confirmed that mc3t3-e1 cells attached well to both ti surfaces , resulting in similar cell proliferation at the different time points investigated . on osseospeed surface , cells exhibited a more branched shape morphology compared to tioblast . in order to determine the effect of the different ti surfaces and tcp on the cell viability after short- and long - term cell culture on the titanium implants ,
the ldh activity in the culture media was measured after 2 , 7 , and 14 days ( figure 4(a ) ) . no differences were found between the osseospeed and tioblast in the ldh activity at any of the days analysed . however , after 14 days of culture , cells cultured onto tcp showed significantly higher levels of ldh activity compared to cells cultured onto ti implants . counting of cells at the different time points using dapi staining ( figure 4(b ) ) , revealed no differences in cell proliferation between the two ti surfaces . rna content was quantified from cell monolayer after 2 , 7 , and 14 days of culture ( figure 4(c ) ) . no differences were observed among the different groups , although rna content increased from 2 to 7 days of cell culture . the potential to initiate osteogenic differentiation after 2 days of culture was investigated by measuring the bmp-2 release to the culture media using a bmp-2 immunoassay ( figure 5 ) . the differentiation of mc3t3-e1 cells cultured on the different implant surfaces and onto plastic was examined by analysing the gene expression after 2 , 7 , and 14 days of cell culture . as seen in figures 6 , 7 , and 8 , gene expression levels of fifteen different genes related to osteogenic differentiation like transcriptional factors and regulators , extracellular matrix molecules , cytokines , growth factors , and functional markers were analysed . regarding the osteogenic markers ( figure 6 ) , coll - i mrna expression was significantly higher in cells cultured onto both titanium surfaces compared to those cultured in plastic after 14 days . after 14 days of culture , the osseospeed group displayed higher bsp mrna levels compared to the tioblast one . alp mrna levels were significantly higher in cells cultured onto plastic compared to those cultured on both titanium surfaces after 2 and 7 days of culture , while after 14 days , alp levels of cells cultured on both titanium surfaces were higher than those cultured on plastic , although just the osseospeed group reached statistical significance . cd44 mrna levels were significantly higher in cells cultured on plastic compared to those cultured on both titanium surfaces ( day 7 ) and compared to the tioblast group ( day 2 ) . after 14 days of culture , no differences were observed between groups . as regards transcriptional factors and regulators ( figure 7 ) , no significant differences were found for dlx2 and hes1 among the groups at the different time points , although their gene expression decreased likewise over the time period studied . osterix mrna levels were higher in cells cultured on both titanium surfaces compared to those cultured on plastic after 2 days of culture . after 14 days of culture , osseospeed implants induced an upregulation osterix mrna compared to tioblast . after 14 days of culture , the osseospeed group displayed higher smad1 mrna levels compared to tcp . smad5 mrna expression was significantly up - regulated in the tcp group compared to the tioblast one after 2 days of culture , and no differences were observed afterwards . regarding cytokines and growth factors ( figure 8) , igf - i mrna expression was significantly higher in cells cultured on plastic compared to those cultured on both titanium surfaces after 2 and 7 days of culture . however , after 14 days , igf - i levels of cells cultured on the osseospeed group were significantly higher than for the other groups . rankl mrna levels ( day 7 ) were significantly higher in the osseospeed group compared to the tcp one . no differences were seen for the other groups or time points for this gene . osteocalcin , opg , and il-6 release to the culture media was determined by a luminex bioassay after 2 , 7 , and 14 days of culture in the tioblast , the osseospeed and tcp groups ( figure 9 ) . osteocalcin and opg release was higher in both implant groups compared to tcp after 7 and 14 days of culture . after 2 days , cells cultured on tioblast surfaces released higher amounts of opg than those cultured on osseospeed surfaces . no statistical differences were found for alp activity in the culture medium after 2 , 7 , and 14 days of cell culture ( figure 10 ) . after 14 days of culture , cells cultured on osseospeed titanium surfaces displayed a higher number of calcium crystals compared to those cultured on tioblast surfaces ( 16.5 1.5 versus 9.5 0.5 , respectively ; p = 0.047 ) . the present study provides a wide characterization of the in vitro osteogenic properties of two commercial surfaces , tioblast and osseospeed . tioblast was the first moderately roughened implant surface with 10 years followup reported in the literature and the precursor of the osseospeed surface [ 1820 ] . the osseospeed surface is a further development introduced in 2004 that incorporates small amounts of fluoride ions in the oxide layer , a slight increase on the micrometer scale in surface roughness , and the appearance of a nanoscale topography . in the present work ,
we showed the topographical differences between the two surfaces , mainly an increase in the sa value in the osseospeed surface , but also changes in surface skewness , kurtosis , and the core fluid retention index . the obtained results confirm that osseospeed surface show an increased micrometer - scale surface roughness , together with the formation of nanostructures , as reported in earlier studies . the presence of micro- and nanoscale topography in osseospeed compared to tioblast surface and the addition of fluoride , did not change the biocompatibility of the implants and the initial attachment and proliferation of the mc3t3-e1 cells . however , it was observed that osseospeed surfaces induced a more branched cell morphology . it has been reported that this cell shape may increase the contractility of the cytoskeleton and lead to preferential osteoblastic differentiation , which has been found in the present study for the osseospeed surface compared to tioblast . the increase found in the ldh activity on the tcp surface after 14 days is most probably due to the higher proliferation and/or cellular activity at this later time point on tcp . osteoblast maturation in vitro is characterised by changes in gene expression at each developmental stage [ 2326 ] . bmp-2 is a highly potent growth / differentiation factor that induces differentiation of progenitor cells into the osteoblast lineage , and exhibits this osteogenic action by activating smad signaling and by regulating transcription of osteogenic genes . thus , the higher release of bmp-2 found in the osseospeed group could initiate osteogenic differentiation through the regulation of transcriptional factors . runt - related transcription factor 2 ( runx2 ) is a master regulator of osteogenic gene expression that is necessary for the osteoblast lineage commitment and , as well , regulates osteoblast differentiation . here
, we did not determine runx2 mrna levels since , as we have previously reported that runx2 mrna expression is constant during osteoblast differentiation , probably due to the fact that mc3t3-e1 cells are already committed to the osteoblast lineage . nevertheless , we have analysed the expression of different transcription factors that have been described to interact with runx2 : dlx2 , a downstream target of bmp-2 that is thought to directly activate runx2 and osterix genes , and hes1 that can stimulate the transactivating function of runx2 , although it negatively regulates bone phenotypic maturation and its expression decreases during osteoblast differentiation . bmp - activated smads induce runx2 gene expression and smads interact physically with the runx2 protein to induce osteoblast differentiation . we found no important changes on smad1 and smad5 at the different time points and groups analysed ; only smad1 showed higher significant levels in osseospeed compared to tcp . here we found higher osx mrna levels in cells cultured onto osseospeed implants compared to tioblast , in agreement with earlier observations [ 8 , 10 ] . also found higher mrna levels of coll - i in human palatal mesenchymal stem cells cultured on tioblast and osseospeed than on tcp after 3 days , although these differences were not significant . these two markers , that reflect a more advanced stage of osteoblast differentiation , showed higher mrna levels on tcp up to the first week , while osseospeed surfaces increased their levels greatly after 14 days , indicating that extracellular matrix of cells seeded on tcp was mature and competent for mineralization after 1 week and on osseospeed surfaces after 2 weeks . this also indicates that differentiation of mc3t3-e1 cells on tioblast was delayed compared to osseospeed surface . using the same in vitro model
, another study did not observe significant differences in bsp between the two titanium surfaces , although in this case the roughness was similar , and the titanium particles for grit - blastingwas smaller . however , the analysis did not reveal important differences between the two titanium surfaces investigated , only for the tcp group . besides these bone - specific markers , the effect of the different titanium implant surfaces on the expression of different growth factors and cytokines involved in bone formation was analysed . igf - i induces osteoblast proliferation , bone collagen , and matrix synthesis [ 40 , 41 ] and stimulates the activity of alkaline phosphatase . similar to other osteogenic markers commented before , this growth factor was significantly upregulated in osseospeed surface compared to both tcp and tioblast . similar results have been found in a previous study in vivo , suggesting that igf - i might play an important role stimulating bone formation when administered in combination with fluoride . in the present work ,
the total amount of rankl produced by mc3t3-e1 osteoblasts was below the detection limit of the luminex assay ( 3 pg / ml ) , while opg was detected in the supernatant during the whole culture period , was similar to the reported results by guida and coworkers using elisa . however , opg production found in this study was similar between tioblast and osseospeed group in mc3t3-e1 osteoblasts , with higher levels on titanium when compared to tcp . as a trend
, tioblast surfaces showed higher levels than osseospeed , opposite to the results obtained in human bone marrow mesenchymal stem cells . due to the lack of differences in opg / rankl mrna levels and the secretion of opg , we conclude that in mc3t3-e1 cells , the levels of opg / rankl are not regulated by the different surfaces used in the study . in conclusion
, the results from the present study demonstrate that the gain of using osseospeed surface is an improved osteoblast differentiation and mineralization , without additional effects on cell viability or proliferation . | [
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strawberry ( fragaria ananassa duch . ) is one of the most popular fruits with global economic importance . because of its appealing red coloration and abundant nutrition , strawberries are highly sought after by consumers [ 2 , 3 ] .
research suggests that anthocyanins have potential health benefits for a variety of conditions including cardiovascular disorders , advanced age - induced oxidative stress , inflammatory response , and diverse degenerative diseases [ 6 , 7 ] .
biosynthesis of anthocyanins is a complex biological process which is affected by genetic , developmental , and environmental factors . over the past few years
, most structural genes encoding enzymes in the anthocyanin biosynthetic pathways have been isolated and characterized in strawberries .
the first group of structural genes involved in these pathways includes phenylalanine ammonia lyase ( pal ) , chalcone synthase ( chs ) , flavanone 3-hydroxylase ( f3h ) , dihydroflavonol-4-reductase ( dfr ) , leucoanthocyanidin reductase ( lar)/anthocyanidin synthase ( ans ) , and udp - glucose flavonoid 3-o - glucosyltransferase ( ufgt ) .
these genes are regulated primarily by the myb / bhlh / wd40 ( mbw ) complex in many plants [ 10 , 11 ] .
it has been shown that anthocyanin synthesis increases rapidly after the white stage in strawberries .
phytohormones such as abscisic acid ( aba ) , cytokinin ( ctk ) , and ethylene and methyl jasmonate ( ja ) also play an important role in regulating the color development process in strawberries by increasing anthocyanin accumulation [ 12 , 13 ] .
auxin and gibberellins ( gas ) are known to reduce anthocyanin biosynthesis during the color development in fruit .
phytochromes , which act as photoreceptors , play an important role in light stimulation during the development of strawberries .
phytochromes are homodimeric chromoproteins where each holophytochrome is composed of a phytochrome protein covalently bound to a linear tetrapyrrole chromophore phytochromobilin ( pb ) .
pb is synthesized in the plastid from heme catalyzed by heme oxygenase ( ho ) and subsequently phytochromobilin synthase .
heme oxygenase 1 ( ho1 ) is crucial to this process and acts as a rate - limiting enzyme in the biosynthesis of pb .
ho1 catalyzes the oxygenation of heme to carbon monoxide , fe , and biliverdin ( bv ) in plants .
additionally , ho1 has been shown to play an important role in anthocyanin accumulation in plants .
for example , tomato [ 18 , 19 ] and arabidopsis [ 2022 ] ho1 deficient mutants , which can not synthesize the phytochrome chromophore , have a major reduction in anthocyanin accumulation .
hematin ( c34h33o5n4fe ) , a protoporphyrin complex , is an inducer and substrate of ho1 in animals and plants [ 17 , 23 ] .
exogenous hematin was shown to alleviate mercury - induced oxidative damage in the roots of medicago sativa , induce adventitious root numbers and root length of cucumbers , regulate brassica nigra seed germination under nanosilver stress , and relieve etiolation in the leaves of wheat seedlings under complete darkness [ 25 , 26 ] .
these effects might be derived from a hematin induced ho1 enzymatic reaction product . in this study ,
the effect of exogenous hematin on the white stage of strawberries was investigated by comparing the anthocyanin contents in the hematin - treated fruit and the control group . in order to gain insight into the underlying molecular mechanisms regulating fruit coloring in response to the hematin treatment
comparative transcriptome analyses may give us a better understanding of the mechanism of the coloring process in strawberry fruit .
benihoppe ) , an octoploid ( 2n = 8x = 56 ) species , was planted under standard culture conditions in a greenhouse ( 30/15c , 14/10 h day / night , relative humidity 5080% ) .
the developmental stage of the strawberry fruit was divided into seven visual stages : small green ( sg ) , big green ( bg ) , degreening ( dg ) , white ( wt ) , initial red ( ir ) , partial red ( pr ) , and full red ( fr ) .
strawberry plants at the white stage ( about 25 d after anthesis ) were chosen to study the effect of hematin on fruit coloration in the study . to study the effect of different concentration of hematin on fruit coloration ,
white stage strawberry plants ( n 50 for each treatment ) were sprayed with 0 , 1 , 10 , or 100 m hematin ( h3281 , sigma - aldrich , st .
strawberry fruits ( n 30 for each treatment ) were harvested when the fruit entered the pr stage ( 48 h after treatment ) .
the fruits were immediately frozen in liquid nitrogen and stored at 80c for further analysis .
we found the 10 m hematin - treated strawberries accumulated most anthocyanin among all treatments ( figure s1 , in supplementary material available online at http://dx.doi.org/10.1155/2016/6762731 ) . by using the same method to treat the fruits with 0 , 5 , 10 , or 15-m hematin
, we found the treatment by 5 m hematin was the optimal condition for increasing anthocyanin production in strawberry ( figure 1 ) ( p < 0.01 ) .
absorbance was recorded on a beckman du640b spectrophotometer ( fullerton , ca , usa ) at 510 and 700 nm for anthocyanin solutions in a ph 1.0 and ph 4.5 buffer , respectively .
the molar extinction coefficient is 26,900 as described in other studies ( e.g. , ) .
strawberry fruit ( 1 g ) was homogenized in a potter - elvehjem homogenizer using 1.2 ml ice - cold 0.25 m sucrose solution containing 1 mm phenylmethyl sulfonyl fluoride , 0.2 mm edta , and 50 mm potassium phosphate buffer ( ph 7.4 ) .
homogenates were centrifuged at 20,000 g for 20 min and chloroplasts were used for activity determination .
the concentration of bv was estimated using a molar absorption coefficient at 650 nm of 6.25 mm cm in 0.1 m hepes - naoh buffer ( ph 7.2 ) .
strawberry fruits were ground into powder and mixed at a ratio of 0.5 g powder to 20 ml extraction buffer ( 200 mm tris - hcl ( ph 8.2 ) , 100 mm licl , 50 mm eta , 1.5% sds , 2% pvp ( sigma , pcp40 ) , 2% bsa ( sigma ) , and 10 mm dtt ( sigma ) ) .
a total of 200 l 10 mg / ml proteinase k ( merck , darmstadt , germany ) was added to remove contaminating proteins .
total rna was extracted using phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) and precipitated in a sodium acetate and ethanol mixture .
the mixture was resuspended in an appropriate volume of depc - treated distilled water and then stored at 80c for the next step .
the quality and quantity of the total rna were measured using a nanodrop nd-1000 spectrophotometer ( nanodrop , wilmington , de , usa ) . only samples that met the criteria of 1.8 od260/280 d2.0 and od260/230 1.8 and concentration 200 ng/l were used for sequencing .
rna samples of 30 strawberry fruits harvested in the same treatment group were pooled together for subsequent experiments .
rna samples from two biological replicates were used for cdna library construction and rna - seq at the beijing genomics institute ( bgi , shenzhen , china ) .
total rna samples were treated with dnase i ( takara , dalian , china ) to remove any possible dna contamination .
the mrna was enriched by using oligo ( dt ) magnetic beads ( illumina , san diego , ca , usa ) and cut into short fragments ( about 200 bp ) .
the first - strand cdna was synthesized using a superscript preamplification system kit ( gibco - brl , grand island , ny , usa ) as described in the manufacturer 's instructions . the double stranded cdna was purified with the oligo ( dt ) magnetic beads following the manufacturer 's instructions .
the fragments were then enriched by pcr amplification with an initial denaturing step at 98c for 30 s , followed by 15 cycles of amplification ( 98c for 10 s , 65c for 30 s , and 72c for 5 min ) and a final extension at 72c for 5 min .
dna size , purity , and concentration were checked on an agilent 2100 bioanalyzer ( agilent , santa clara , ca , usa ) .
the original sequence data were filtered to obtain clean reads for further analyses by removing short reads ( less than 30 bp ) and trimming adapters .
adapter reads were trimmed by first calculating the average quality of the first 15 bases from 3-end until the average quality was larger than 10 and then removing the bases that were counted .
the high - quality clean reads were mapped against the strawberry reference genome ( http://strawberry-garden.kazusa.or.jp/ ) using ion torrent 's mapping program ( tmap , version 0.2.3 ; https://github.com/iontorrent/tmap ) .
quality assessment of reads , statistics of alignment , sequencing saturation analysis , and randomness assessments were carried out subsequently to assess the quality of sequencing .
raw counts were normalized to reads per kilobase of exon model per million mapped reads ( rpkm ) .
q value < 0.05 and |log2(fold change)| > 1 were set as the threshold to identify significant differentially expressed genes . in statistics ,
gene functions of strawberry were annotated according to the gene ontology ( go ) standardized terms for molecular function , cellular component , and biological process using blast2go ( https://www.blast2go.com/ ) .
gene function was annotated using the following databases : ncbi nonredundant protein sequences , ncbi nonredundant nucleotide sequences , protein family , clusters of orthologous groups of proteins , the manually annotated and reviewed protein sequence database , kegg ortholog database , and gene ontology .
after go annotation for degs , we performed go functional classification for degs by the wego software and analyzed the distribution of gene functions .
we used the go term finder tool ( http://www.yeastgenome.org/help/analyze/go-term-finder ) to search for significant shared go terms.(2)p=1i=0m1minmninn , where n is the number of all genes with go annotation ; n is the number of degs in n ; m is the number of all genes that are annotated to certain go terms ; and m is the number of degs in m. the calculated p value was subjected to bonferroni correction .
the pathway enrichment analyses using the kegg database ( http://www.genome.jp/kegg/ ) were conducted subsequently to study the functions of differentially expressed genes identified between the control and the hematin - treated strawberry groups .
n is the number of all genes with kegg annotation , n is the number of degs in n , m is the number of all genes annotated to specific pathways , and m is the number of degs in m. quantitative real - time pcr ( qrt - pcr ) experiments were conducted to assess the reliability of the rna - seq data .
eleven pairs of gene specific primers were designed to verify the degs in the strawberry fruit ( table s1 ) .
total rna was digested with dnase i for 30 min at 25c to remove dna contamination according to the manufacturer 's instructions .
the qrt - pcr was performed using a sybr pcr master mix ( takara , dalian , china ) on a bio - rad iq-5 thermal cycler ( bio - rad , philadelphia , pa , usa ) .
the relative expression level was calculated by the comparative 2 method [ 35 , 36 ] .
the significance was determined with the spss software ( spss 17.0 , ibm , chicago , il , usa ) ( p < 0.05 ) .
hematin is an inducer and substrate of ho1 in animals and plants [ 17 , 23 ] . in this study , strawberries at the wt stage were treated with 0 m ( control ) , 5 , 10 , or 15 m hematin .
the results indicated that the anthocyanin content in the hematin - treated strawberries was more than the control after 48 h. the treatment by 5 m hematin was found to be the optimal condition for increasing anthocyanin production ( figure 1 ) ( p < 0.01 ) . the anthocyanin content in the 5 m hematin - treated strawberries was 2.5 times higher than that in the control .
we measured the expression of faho-1 and the content of biliverdin ( bv ) which is the metabolite of heme oxygenase .
we found that hematin could also significantly increase the expression of faho-1 in strawberry fruit ( figure 4 ) and promote the accumulation of bv ( figure 2 ) .
high - throughput sequencing technology is utilized widely in analyzing gene expression in many organisms [ 37 , 38 ] . in this study ,
approximately 10.8 gbp raw tags were generated for each library . after eliminating adapters , ambiguous nucleotides , and low - quality sequences , a total of 34,618,832 and 45,464,123 clean reads between 150 and 200 nucleotides in length were obtained ( table 1 ) .
over 85% of the clean tags from each library mapped to reference genes with less than 2 bp mismatches .
less than 29% of the clean tags from each library could not be aligned to any reference genes because of incomplete sequences , and these tags were designated as unknown .
more than 67.3% of the clean tags in each library were mapped to a single gene , while less than 4.7% mapped to multiple reference genes .
unknown tags and tags mapped to multigenes were filtered out , and the unique clean tags that mapped to a single gene were retained for further deg analysis . in addition , the perfect match rates were 24.32% , 24.28% , 24.79% , and 24.35% in control 1 , control 2 , hematin 1 , and hematin 2 , respectively .
the number of the detected genes became saturated at about 2 million reads ( figure s2 ) .
the randomness of rna fragmentation in the four libraries was assessed for subsequent bioinformatics analysis .
the results showed that the reads in each position of the reference gene were distributed evenly and demonstrated highly similar tendencies in all libraries ( figure s3 ) .
based on sequencing results of the four mrna libraries , approximately 29,000 genes ( about 70% of the reference genes in the octoploid strawberry genome ) were detected in each library .
additionally , correlations among genes based on rpkm between the two biological replicates were analyzed ; the correlation coefficients ( r ) were high ( 0.93 and 0.95 for the control and hematin - treated group , resp . ) .
a total of 28,713 , 28,999 , 27,976 , and 28,211 genes ( table 1 ) were expressed in the control and hematin - treated samples . to reveal the molecular pathways regulating strawberry coloring in response to hematin , the degs between the control and hematin - treated fruit were analyzed .
we compared the gene expression profiles between the control and the treated samples in both biological replicates .
a total of 1,080 ( 402 up- and 678 downregulated ) genes were differentially expressed in the 5 m hematin - treated groups compared with the control groups .
more genes were downregulated in the hematin - treated group compared to the control group . to facilitate the global analysis of gene expression ,
a go analysis was performed by mapping each differentially expressed gene into the records of the go database .
gene ontology ( go ) enrichment analysis of the degs in the control and hematin - treated groups was performed to reveal the possible mechanisms under which hematin promotes anthocyanin biosynthesis .
go terms with p < 0.05 were represented among genes with significant changes in expression over a given interval . in this study , only three terms including anatomical structure arrangement , meristem structural organization , radial pattern formation were significantly enriched in biological process ( supplementary table s2 ) . although most go terms were not significantly enriched , they could provide a reference for further study . according to the go cellular components , a large number of the degs were classified into main cell organelles , such as vacuole , nucleolus , mitochondrion , apoplast , chloroplast , plastid , cytoplasm , and membrane . in particular , many degs were assigned to the plastid and chloroplast ( figure 3 ) , indicating that hematin has an important impact on the expression of plastid and chloroplast genes .
according to the go molecular function , a large number of the degs were classified into udp - glucosyltransferase , sucrose transmembrane transporter activity , nucleic acid binding , and dna binding terms which are closely related to anthocyanin biosynthesis .
many degs were classified into heme binding , peroxidase activity , and tetrapyrrole binding ( figure 3 ) .
it is probably because hematin can increase the activity of ho1 and promote the degradation of hemoglobin .
in addition , anthocyanin accumulation in fruit is closely related to hormone and carbohydrate biosynthesis . according to the go biological process
, a large number of the degs were classified into regulation of hormone levels , cytokinin metabolic process , response to hormone , response to abscisic acid , response to auxin , and response to ethylene terms , indicating that the hormone - related degs are involved in regulating the anthocyanin biosynthesis in the hematin - treated fruit .
many degs were classified into starch and sucrose biosynthetic and transport process and phenylpropanoid metabolic process terms , indicating that hematin can promote anthocyanin accumulation via the phenylpropanoid metabolic and carbohydrate metabolism .
in addition , many degs were classified into the process for response to light stimulus term , indicating that hematin may also participate in the light stimulus system .
pathway enrichment analysis was performed to understand the biological functions of the degs by identifying significantly enriched signal transduction pathways or metabolic pathways . in the study ,
a number of altered biological pathways associated with the hematin treatment were identified , and three pathways , including rna polymerase , pyrimidine metabolism , and purine metabolism , were significantly enriched ( q - value < 0.05 ) ( table 2 ) .
although the majority of the pathway terms were not significantly enriched , the pathway enrichment analysis helps us to further understand the biological functions of the degs and the molecular mechanisms that regulate fruit coloring in response to the hematin treatment .
these pathway terms included metabolic pathways , biosynthesis of secondary metabolites , isoflavonoid biosynthesis , phenylalanine metabolism , phenylalanine , tyrosine and tryptophan biosynthesis , starch and sucrose metabolism , plant hormone signal transduction , flavone and flavonol biosynthesis , and phenylpropanoid biosynthesis terms ( supplementary table s2 ) .
transcription factors ( tfs ) are proteins that regulate the expression of downstream target genes .
many tfs , such as v - myb avian myeloblastosis viral oncogene homolog ( myb ) , basic helix - loop - helix ( bhlh ) , wd40-repeats protein ( wd40 ) , and mads - box ( mads ) , directly regulating the expression of the structural genes in anthocyanin biosynthesis have been identified from many species .
therefore , the enhanced expression of key tfs might regulate the anthocyanin biosynthesis . in our study
, some anthocyanin biosynthesis - related tfs were found to be uniquely present in the deg profiling of the hematin - treated fruit , for example , myb , bhlh , pif3 , mads - box , ap2-erebp , and abi3/vp1 ( table 3 ) .
the anthocyanin biosynthesis genes are regulated primarily by the mbw transcription factor , which is a ternary transcription factor complex [ 10 , 11 ] .
however , mybs can act as repressors too , such as strawberry famyb1 and famyb9 and grapevine vvmyb4 , which can significantly suppress the biosynthesis of anthocyanins and flavonols . in this study , three unigenes belonging to myb transcription factors
of these , the unigene fanhyb_rscf00000146.1.g00007.1 from the myb family of r2r3 myb transcription factors was significantly upregulated .
moreover , increasing evidence indicates that the expression of the bhlhs promotes anthocyanin accumulation in fruit . in our study ,
all of the unigenes from the bhlh family were downregulated , among which the expression level of unigene fanhyb_rscf00001292.1.g00003.1 was significantly decreased 6250-fold ( table 3 ) .
fanhyb_rscf00002210.1.g00001.1 encodes a predicted mads - box transcription factor , and it was significantly downregulated .
there were three unigenes in the ap2-erebp family , with only one upregulated and the others downregulated .
these results suggest that exogenous hematin is involved in developmental transcriptional regulation of anthocyanin biosynthesis .
the anthocyanin biosynthetic pathway via the phenylpropanoid pathway is well known . in this study ,
the transcriptome data showed a significant increase in the expression of genes in the anthocyanin biosynthesis pathway when exposed to exogenous hematin .
the structural genes involved in anthocyanin biosynthesis , including dfr , lar , and udp - glycosyltransferase , exhibited significant differential expression in response to the hematin treatment ( table 4 ) .
for example , two dfr genes and one lar gene were significantly upregulated ( the expression increased 18- , 30- , and 3-fold in the hematin - treated fruit , resp . ) .
in addition , all six genes encoding udp - glycosyltransferase were significantly upregulated . among these , the expression of one gene ( fanhy_icon15742070o.1.g00001.1 ) was upregulated 18305-fold .
the results suggest that exogenous hematin may have an important role in promoting anthocyanin biosynthesis .
auxin has been shown to negatively regulate the expression of the anthocyanin biosynthesis genes . in this study , there were three unigenes involved in the auxin signaling pathway , and all were downregulated , including one gene encoding auxin efflux carrier component 1-like ( aux1-like ) and two genes encoding auxin response factors ( arf ) .
these results suggest that hematin promotes anthocyanin accumulation by regulating auxin in the coloration process of strawberries .
it has shown that auxin suppresses anthocyanin biosynthesis in the red - fleshed apple callus .
endogenous expression of auxin [ 44 , 45 ] has been found to impede anthocyanin accumulation in strawberries . in additional ,
it has shown to enhance anthocyanin accumulation in zea mays and regulate anthocyanin production and composition in suspension cultures of strawberry cells . in this study , two unigenes related to the cytokinin
one gene was identified as cytokinin dehydrogenase 5-like which catalyzes the irreversible degradation of cytokinins .
extensive researches have shown that aba plays an important role in the regulation of anthocyanin biosynthesis in nonclimacteric fruit . in this study ,
a total of five unigenes related to the aba signaling pathway were identified , among which two were upregulated and three were downregulated .
these two unigenes encode 9-cis - epoxycarotenoid dioxygenase 1 ( nced1 ) and aba overly sensitive 5 ( abo5 ) , respectively .
the rate - limiting step in these reactions is catalyzed by 9-cis - epoxycarotenoid dioxygenase 1 ( nced1 ) .
mutations in the fanced1 gene result in colorless strawberry fruit , which can become colored through the application of exogenous aba .
another unigene was identified to encode abscisic acid 8-hydroxylase 1-like , a key enzyme in the oxidative catabolism of abscisic acid , which was downregulated .
application of exogenous aba regulates phenylalanine ammonia lyase activity and increases the phenolic and anthocyanin content of strawberry fruit [ 12 , 49 ] .
our results suggest that hematin promotes aba biosynthesis and inhibits aba disintegration in the hematin - treated fruit . in arabidopsis
, jas can affect anthocyanin accumulation via the interaction of negative regulators with the mbw complex of transcription factors involved in anthocyanin biosynthesis .
jasmonate o - methyltransferase - like is also known to be a key enzyme for jasmonate - regulated plant responses to stimuli [ 53 , 54 ] .
these results suggest that hematin increases the activity of the ja - regulated anthocyanin biosynthesis .
phytochromes are homodimeric chromoproteins , where each holophytochrome is composed of a phytochrome protein ( apophytochrome ) covalently bound to a linear tetrapyrrole pb .
ho1 is crucial to this process and acts as a rate - limiting enzyme in the biosynthesis of pb .
exogenous hematin can also induce ho-1 expression in many plants [ 56 , 57 ] . in this study
, we found exogenous hematin significantly increased the expression of faho1 ( table 5 ) .
recently , it has been reported that hematin could induce the accumulation of far - red phytochrome and phytochrome a transcripts in the etiolated leaves of wheat seedling phytochromes .
it is probably because the hematin - treated strawberries were not shaded during the experiment , while the function of phytochrome a is light - dependent .
in addition , we found that a downstream gene of phytochrome , pif3 ( fanhyb_rscf00000669.1.g00002.1 ) , was significantly upregulated .
pif3 is thought to be a positive regulator of phytochrome b mediated by light - dependent signal transduction .
these results suggest that the phytochrome pathways may be involved in the anthocyanin biosynthesis promoted by hematin .
proteomic approaches have revealed that starch degradation contributes to anthocyanin accumulation in tuberous roots of the purple sweet potato variety .
starch and sucrose biosynthesis is important for anthocyanin accumulation in strawberry fruit . in this study
, we found the degs involved in starch and sucrose synthesis in the hematin - treated fruit were all downregulated ( table 5 ) .
for example , the expression of the unigenes encoding soluble starch synthase 3 , sucrose transport protein suc2-like , and a probable sucrose - phosphate synthase 4-like enzyme were decreased 2.3- , 4.6- , 3.2- , and 2-fold , respectively .
this suggests that exogenous hematin regulates the biosynthesis of starch and sucrose and hence affects fruit coloring .
calcium can also increase the transcription levels of key structural genes f3h , dfr , ant , and ufgt in the white stage of strawberries . in this study
, we found that three degs in calcium biosynthesis and transport were downregulated in the hematin - treated fruit ( table 5 ) .
the unigenes encoding a calpain - type cysteine protease and a predicted calcium - binding protein pbp1-like were downregulated , respectively .
exogenous application of calcium can promote apple coloring in addition to variation in anthocyanin content .
our results indicate that the biosynthesis and transport of calcium are involved in the development of coloring in hematin - treated strawberries . to validate the expression of the degs obtained from rna - seq
, 11 degs were selected for qrt - pcr , including structural genes ( dfr and udfgs ) , transcription factor genes ( myb and bhlh ) , and phytochrome chromophore - related gene ( ho-1 ) .
the qrt - pcr results were consistent with the rna - seq data ( figure 4 ) , except for fanhyb_rscf00000141.1.g00016.1 , which had a higher log2 ratio ( hematin - treated / control ) in the transcriptome data than in qrt - pcr .
these results indicate the rna - seq data from the strawberry transcriptome is reproducible and accurate .
in this study , the anthocyanin content in the strawberry fruit was elevated by the application of exogenous hematin .
furthermore , we explored the effects of the exogenous hematin on metabolic pathways using genome - wide transcriptome analysis .
the results indicate that the expression levels of many genes involved in anthocyanin biosynthesis were significantly altered with the hematin treatment .
this suggests that the physiological process of fruit color development is regulated through complex interactions among anthocyanin biosynthesis pathways , plant hormone signal transduction pathways , phytochrome signal transduction pathways , starch and sugar metabolic pathways , calcium pathways , and transcription factors ( figure 5 ) .
this study adds to the in - depth understanding of the fruit coloration process in strawberry . | anthocyanin in strawberries has a positive effect on fruit coloration . in this study ,
the role of exogenous hematin on anthocyanin biosynthesis was investigated .
our result showed that the white stage of strawberries treated with exogenous hematin had higher anthocyanin content , compared to the control group . among all treatments ,
5 m of hematin was the optimal condition to promote color development . in order to explore the molecular mechanism of fruit coloring regulated by hematin
, transcriptomes in the hematin- and non - hematin - treated fruit were analyzed .
a large number of differentially expressed genes ( degs ) were identified in regulating anthocyanin synthesis , including the degs involved in anthocyanin biosynthesis , hormone signaling transduction , phytochrome signaling , starch and sucrose degradation , and transcriptional pathways .
these regulatory networks may play an important role in regulating the color process of strawberries treated with hematin . in summary ,
exogenous hematin could promote fruit coloring by increasing anthocyanin content in the white stage of strawberries .
furthermore , transcriptome analysis suggests that hematin - promoted fruit coloring occurs through multiple related metabolic pathways , which provides valuable information for regulating fruit color via anthocyanin biosynthesis in strawberries . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions | research suggests that anthocyanins have potential health benefits for a variety of conditions including cardiovascular disorders , advanced age - induced oxidative stress , inflammatory response , and diverse degenerative diseases [ 6 , 7 ] . over the past few years
, most structural genes encoding enzymes in the anthocyanin biosynthetic pathways have been isolated and characterized in strawberries . the first group of structural genes involved in these pathways includes phenylalanine ammonia lyase ( pal ) , chalcone synthase ( chs ) , flavanone 3-hydroxylase ( f3h ) , dihydroflavonol-4-reductase ( dfr ) , leucoanthocyanidin reductase ( lar)/anthocyanidin synthase ( ans ) , and udp - glucose flavonoid 3-o - glucosyltransferase ( ufgt ) . it has been shown that anthocyanin synthesis increases rapidly after the white stage in strawberries . phytohormones such as abscisic acid ( aba ) , cytokinin ( ctk ) , and ethylene and methyl jasmonate ( ja ) also play an important role in regulating the color development process in strawberries by increasing anthocyanin accumulation [ 12 , 13 ] . auxin and gibberellins ( gas ) are known to reduce anthocyanin biosynthesis during the color development in fruit . phytochromes , which act as photoreceptors , play an important role in light stimulation during the development of strawberries . pb is synthesized in the plastid from heme catalyzed by heme oxygenase ( ho ) and subsequently phytochromobilin synthase . ho1 catalyzes the oxygenation of heme to carbon monoxide , fe , and biliverdin ( bv ) in plants . additionally , ho1 has been shown to play an important role in anthocyanin accumulation in plants . for example , tomato [ 18 , 19 ] and arabidopsis [ 2022 ] ho1 deficient mutants , which can not synthesize the phytochrome chromophore , have a major reduction in anthocyanin accumulation . exogenous hematin was shown to alleviate mercury - induced oxidative damage in the roots of medicago sativa , induce adventitious root numbers and root length of cucumbers , regulate brassica nigra seed germination under nanosilver stress , and relieve etiolation in the leaves of wheat seedlings under complete darkness [ 25 , 26 ] . in this study ,
the effect of exogenous hematin on the white stage of strawberries was investigated by comparing the anthocyanin contents in the hematin - treated fruit and the control group . in order to gain insight into the underlying molecular mechanisms regulating fruit coloring in response to the hematin treatment
comparative transcriptome analyses may give us a better understanding of the mechanism of the coloring process in strawberry fruit . the developmental stage of the strawberry fruit was divided into seven visual stages : small green ( sg ) , big green ( bg ) , degreening ( dg ) , white ( wt ) , initial red ( ir ) , partial red ( pr ) , and full red ( fr ) . strawberry plants at the white stage ( about 25 d after anthesis ) were chosen to study the effect of hematin on fruit coloration in the study . to study the effect of different concentration of hematin on fruit coloration ,
white stage strawberry plants ( n 50 for each treatment ) were sprayed with 0 , 1 , 10 , or 100 m hematin ( h3281 , sigma - aldrich , st . we found the 10 m hematin - treated strawberries accumulated most anthocyanin among all treatments ( figure s1 , in supplementary material available online at http://dx.doi.org/10.1155/2016/6762731 ) . by using the same method to treat the fruits with 0 , 5 , 10 , or 15-m hematin
, we found the treatment by 5 m hematin was the optimal condition for increasing anthocyanin production in strawberry ( figure 1 ) ( p < 0.01 ) . strawberry fruits were ground into powder and mixed at a ratio of 0.5 g powder to 20 ml extraction buffer ( 200 mm tris - hcl ( ph 8.2 ) , 100 mm licl , 50 mm eta , 1.5% sds , 2% pvp ( sigma , pcp40 ) , 2% bsa ( sigma ) , and 10 mm dtt ( sigma ) ) . the first - strand cdna was synthesized using a superscript preamplification system kit ( gibco - brl , grand island , ny , usa ) as described in the manufacturer 's instructions . the fragments were then enriched by pcr amplification with an initial denaturing step at 98c for 30 s , followed by 15 cycles of amplification ( 98c for 10 s , 65c for 30 s , and 72c for 5 min ) and a final extension at 72c for 5 min . q value < 0.05 and |log2(fold change)| > 1 were set as the threshold to identify significant differentially expressed genes . in statistics ,
gene functions of strawberry were annotated according to the gene ontology ( go ) standardized terms for molecular function , cellular component , and biological process using blast2go ( https://www.blast2go.com/ ) . gene function was annotated using the following databases : ncbi nonredundant protein sequences , ncbi nonredundant nucleotide sequences , protein family , clusters of orthologous groups of proteins , the manually annotated and reviewed protein sequence database , kegg ortholog database , and gene ontology . the pathway enrichment analyses using the kegg database ( http://www.genome.jp/kegg/ ) were conducted subsequently to study the functions of differentially expressed genes identified between the control and the hematin - treated strawberry groups . n is the number of all genes with kegg annotation , n is the number of degs in n , m is the number of all genes annotated to specific pathways , and m is the number of degs in m. quantitative real - time pcr ( qrt - pcr ) experiments were conducted to assess the reliability of the rna - seq data . eleven pairs of gene specific primers were designed to verify the degs in the strawberry fruit ( table s1 ) . in this study , strawberries at the wt stage were treated with 0 m ( control ) , 5 , 10 , or 15 m hematin . the results indicated that the anthocyanin content in the hematin - treated strawberries was more than the control after 48 h. the treatment by 5 m hematin was found to be the optimal condition for increasing anthocyanin production ( figure 1 ) ( p < 0.01 ) . the anthocyanin content in the 5 m hematin - treated strawberries was 2.5 times higher than that in the control . we found that hematin could also significantly increase the expression of faho-1 in strawberry fruit ( figure 4 ) and promote the accumulation of bv ( figure 2 ) . in this study ,
approximately 10.8 gbp raw tags were generated for each library . after eliminating adapters , ambiguous nucleotides , and low - quality sequences , a total of 34,618,832 and 45,464,123 clean reads between 150 and 200 nucleotides in length were obtained ( table 1 ) . in addition , the perfect match rates were 24.32% , 24.28% , 24.79% , and 24.35% in control 1 , control 2 , hematin 1 , and hematin 2 , respectively . the results showed that the reads in each position of the reference gene were distributed evenly and demonstrated highly similar tendencies in all libraries ( figure s3 ) . based on sequencing results of the four mrna libraries , approximately 29,000 genes ( about 70% of the reference genes in the octoploid strawberry genome ) were detected in each library . additionally , correlations among genes based on rpkm between the two biological replicates were analyzed ; the correlation coefficients ( r ) were high ( 0.93 and 0.95 for the control and hematin - treated group , resp . ) a total of 28,713 , 28,999 , 27,976 , and 28,211 genes ( table 1 ) were expressed in the control and hematin - treated samples . to reveal the molecular pathways regulating strawberry coloring in response to hematin , the degs between the control and hematin - treated fruit were analyzed . a total of 1,080 ( 402 up- and 678 downregulated ) genes were differentially expressed in the 5 m hematin - treated groups compared with the control groups . more genes were downregulated in the hematin - treated group compared to the control group . gene ontology ( go ) enrichment analysis of the degs in the control and hematin - treated groups was performed to reveal the possible mechanisms under which hematin promotes anthocyanin biosynthesis . in this study , only three terms including anatomical structure arrangement , meristem structural organization , radial pattern formation were significantly enriched in biological process ( supplementary table s2 ) . according to the go cellular components , a large number of the degs were classified into main cell organelles , such as vacuole , nucleolus , mitochondrion , apoplast , chloroplast , plastid , cytoplasm , and membrane . in particular , many degs were assigned to the plastid and chloroplast ( figure 3 ) , indicating that hematin has an important impact on the expression of plastid and chloroplast genes . according to the go molecular function , a large number of the degs were classified into udp - glucosyltransferase , sucrose transmembrane transporter activity , nucleic acid binding , and dna binding terms which are closely related to anthocyanin biosynthesis . many degs were classified into heme binding , peroxidase activity , and tetrapyrrole binding ( figure 3 ) . according to the go biological process
, a large number of the degs were classified into regulation of hormone levels , cytokinin metabolic process , response to hormone , response to abscisic acid , response to auxin , and response to ethylene terms , indicating that the hormone - related degs are involved in regulating the anthocyanin biosynthesis in the hematin - treated fruit . many degs were classified into starch and sucrose biosynthetic and transport process and phenylpropanoid metabolic process terms , indicating that hematin can promote anthocyanin accumulation via the phenylpropanoid metabolic and carbohydrate metabolism . in addition , many degs were classified into the process for response to light stimulus term , indicating that hematin may also participate in the light stimulus system . pathway enrichment analysis was performed to understand the biological functions of the degs by identifying significantly enriched signal transduction pathways or metabolic pathways . in the study ,
a number of altered biological pathways associated with the hematin treatment were identified , and three pathways , including rna polymerase , pyrimidine metabolism , and purine metabolism , were significantly enriched ( q - value < 0.05 ) ( table 2 ) . although the majority of the pathway terms were not significantly enriched , the pathway enrichment analysis helps us to further understand the biological functions of the degs and the molecular mechanisms that regulate fruit coloring in response to the hematin treatment . these pathway terms included metabolic pathways , biosynthesis of secondary metabolites , isoflavonoid biosynthesis , phenylalanine metabolism , phenylalanine , tyrosine and tryptophan biosynthesis , starch and sucrose metabolism , plant hormone signal transduction , flavone and flavonol biosynthesis , and phenylpropanoid biosynthesis terms ( supplementary table s2 ) . many tfs , such as v - myb avian myeloblastosis viral oncogene homolog ( myb ) , basic helix - loop - helix ( bhlh ) , wd40-repeats protein ( wd40 ) , and mads - box ( mads ) , directly regulating the expression of the structural genes in anthocyanin biosynthesis have been identified from many species . therefore , the enhanced expression of key tfs might regulate the anthocyanin biosynthesis . in our study
, some anthocyanin biosynthesis - related tfs were found to be uniquely present in the deg profiling of the hematin - treated fruit , for example , myb , bhlh , pif3 , mads - box , ap2-erebp , and abi3/vp1 ( table 3 ) . the anthocyanin biosynthesis genes are regulated primarily by the mbw transcription factor , which is a ternary transcription factor complex [ 10 , 11 ] . in this study , three unigenes belonging to myb transcription factors
of these , the unigene fanhyb_rscf00000146.1.g00007.1 from the myb family of r2r3 myb transcription factors was significantly upregulated . moreover , increasing evidence indicates that the expression of the bhlhs promotes anthocyanin accumulation in fruit . these results suggest that exogenous hematin is involved in developmental transcriptional regulation of anthocyanin biosynthesis . in this study ,
the transcriptome data showed a significant increase in the expression of genes in the anthocyanin biosynthesis pathway when exposed to exogenous hematin . the structural genes involved in anthocyanin biosynthesis , including dfr , lar , and udp - glycosyltransferase , exhibited significant differential expression in response to the hematin treatment ( table 4 ) . for example , two dfr genes and one lar gene were significantly upregulated ( the expression increased 18- , 30- , and 3-fold in the hematin - treated fruit , resp . ) the results suggest that exogenous hematin may have an important role in promoting anthocyanin biosynthesis . in this study , there were three unigenes involved in the auxin signaling pathway , and all were downregulated , including one gene encoding auxin efflux carrier component 1-like ( aux1-like ) and two genes encoding auxin response factors ( arf ) . these results suggest that hematin promotes anthocyanin accumulation by regulating auxin in the coloration process of strawberries . it has shown that auxin suppresses anthocyanin biosynthesis in the red - fleshed apple callus . in this study , two unigenes related to the cytokinin
one gene was identified as cytokinin dehydrogenase 5-like which catalyzes the irreversible degradation of cytokinins . extensive researches have shown that aba plays an important role in the regulation of anthocyanin biosynthesis in nonclimacteric fruit . in this study ,
a total of five unigenes related to the aba signaling pathway were identified , among which two were upregulated and three were downregulated . mutations in the fanced1 gene result in colorless strawberry fruit , which can become colored through the application of exogenous aba . another unigene was identified to encode abscisic acid 8-hydroxylase 1-like , a key enzyme in the oxidative catabolism of abscisic acid , which was downregulated . application of exogenous aba regulates phenylalanine ammonia lyase activity and increases the phenolic and anthocyanin content of strawberry fruit [ 12 , 49 ] . our results suggest that hematin promotes aba biosynthesis and inhibits aba disintegration in the hematin - treated fruit . in arabidopsis
, jas can affect anthocyanin accumulation via the interaction of negative regulators with the mbw complex of transcription factors involved in anthocyanin biosynthesis . these results suggest that hematin increases the activity of the ja - regulated anthocyanin biosynthesis . exogenous hematin can also induce ho-1 expression in many plants [ 56 , 57 ] . in this study
, we found exogenous hematin significantly increased the expression of faho1 ( table 5 ) . recently , it has been reported that hematin could induce the accumulation of far - red phytochrome and phytochrome a transcripts in the etiolated leaves of wheat seedling phytochromes . it is probably because the hematin - treated strawberries were not shaded during the experiment , while the function of phytochrome a is light - dependent . these results suggest that the phytochrome pathways may be involved in the anthocyanin biosynthesis promoted by hematin . starch and sucrose biosynthesis is important for anthocyanin accumulation in strawberry fruit . in this study
, we found the degs involved in starch and sucrose synthesis in the hematin - treated fruit were all downregulated ( table 5 ) . for example , the expression of the unigenes encoding soluble starch synthase 3 , sucrose transport protein suc2-like , and a probable sucrose - phosphate synthase 4-like enzyme were decreased 2.3- , 4.6- , 3.2- , and 2-fold , respectively . this suggests that exogenous hematin regulates the biosynthesis of starch and sucrose and hence affects fruit coloring . calcium can also increase the transcription levels of key structural genes f3h , dfr , ant , and ufgt in the white stage of strawberries . in this study
, we found that three degs in calcium biosynthesis and transport were downregulated in the hematin - treated fruit ( table 5 ) . exogenous application of calcium can promote apple coloring in addition to variation in anthocyanin content . our results indicate that the biosynthesis and transport of calcium are involved in the development of coloring in hematin - treated strawberries . to validate the expression of the degs obtained from rna - seq
, 11 degs were selected for qrt - pcr , including structural genes ( dfr and udfgs ) , transcription factor genes ( myb and bhlh ) , and phytochrome chromophore - related gene ( ho-1 ) . the qrt - pcr results were consistent with the rna - seq data ( figure 4 ) , except for fanhyb_rscf00000141.1.g00016.1 , which had a higher log2 ratio ( hematin - treated / control ) in the transcriptome data than in qrt - pcr . in this study , the anthocyanin content in the strawberry fruit was elevated by the application of exogenous hematin . furthermore , we explored the effects of the exogenous hematin on metabolic pathways using genome - wide transcriptome analysis . the results indicate that the expression levels of many genes involved in anthocyanin biosynthesis were significantly altered with the hematin treatment . this suggests that the physiological process of fruit color development is regulated through complex interactions among anthocyanin biosynthesis pathways , plant hormone signal transduction pathways , phytochrome signal transduction pathways , starch and sugar metabolic pathways , calcium pathways , and transcription factors ( figure 5 ) . this study adds to the in - depth understanding of the fruit coloration process in strawberry . | [
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in many countries in eastern europe large scale social and economic transition over the past 15 years has been associated with widespread poverty and increasing levels of unemployment and population migration , particularly among women.1,2 such transition has also been associated with shifts in patterns of sexual behaviour and sexual relationships , including increased commercial sex work , the growth of informal sex work economies and associated human trafficking.3 additionally , a number of countries in the former soviet union have experienced a rapid or explosive spread of hiv associated with injecting drug use.47 these epidemics are potentially exacerbated by close links between sex work and injecting drug use.2,811 this has caused concerns that high levels of sexual mixing between injecting drug users ( idus ) and their sexual partners in settings of high hiv and sexual transmitted infection ( sti ) prevalence may transform hiv infection from concentrated epidemics among risk populations to generalised epidemics.9,10 it is therefore essential to assess the diffusion of hiv and related risk behaviour in hidden vulnerable populations , such as idus and sex workers ( sws ) , as accurately as possible . there has been increased and recent interest in an approach to sampling hidden populations known as respondent driven sampling ( rds).12,13 one of the claimed advantages of rds over other methods of sampling hidden populations , such as time location sampling , is that it requires little in - depth formative research among study populations.13,14 rds begins with a set number of non - randomly selected seeds ( members of the target population ) .
seeds recruit their peers ( other target population members who make up their social network ) who in turn recruit their peers into the study .
this occurs through successive waves of recruits which , it is argued , become increasingly more representative of the underlying population as the recruitment progresses .
since rds claims to generate a representative sample from non - randomly selected seeds , many researchers have assumed that sampling can proceed without detailed prior knowledge about the local target population.13,14 rds has proved feasible and successful in recruiting hidden populations of idus in a variety of settings , resulting in the rapid acquisition of long and varied recruitment chains.11,1419 rds has also been used in several countries to collect both behavioural and biological data from sws ( e.g. , vietnam , india ) and men who have sex with men ( e.g. , bangladesh , cambodia , uganda , united states).2024 however , little is known about the feasibility of rds as a method of recruiting hidden populations of sws in settings where the organisation of sex work and access to sws is highly controlled and where there is little or no contact between the target population and local services .
drawing upon three case studies in eastern europe targeting sws and idus involved in sw ( idu / sws ) , we explore the feasibility of rds , identifying some barriers to successful recruitment .
these findings have implications for the direction of future research exploring the feasibility of rds methods .
using rds , data on hiv prevalence and associated risk factors were gathered from sws in serbia and montenegro and from idus involved in sex work in russia ( see table 1 ) .
sws in serbia and montenegro were given three uniquely numbered coupons with which to recruit other sws . in russia , although no rds study directly targeting sws was carried out , rds was used to sample sws / idus in an rds study among idus .
incentives were provided to participants completing the survey ( primary incentive ) and for recruiting other eligible participants ( secondary incentive ) .
participants in belgrade were also offered a rapid hiv test and pre- and post - test voluntary and confidential counselling ( vct ) .
those with preliminary - positive tests for hiv were referred to a local clinic for confirmatory testing .
the studies were undertaken by local fieldwork teams coordinated as part of larger internationally funded research initiatives .
table 1.three rds studies among sex workers and injection drug using sex workerscountrycity ( population size)formative researchrecruitment criteria for swsnumber of seedssample reached ( target)year , study durationserbiabelgrade ( about 2 million)literature search ; questions on network characteristics and rds study feasibility posed during individual interviews with sws in 2005 ( n=10)exchanged sex for drugs , money or other goods in last 30 days ( irrespective of gender or sexual orientation)six initially , six added later209 ( 400)2005 , 2 monthsmontenegropodgorica ( about 300,000)literature search ; questions on network characteristics and rds study feasibility posed during in - depth interviews with sws in 2005 ( n=4)exchanged sex for drugs , money or other goods in last 30 days ( irrespective of gender or sexual orientation)one to start with , one added later2 ( 150)2005 , 2 monthsrussian federationtogliatti ( 740,636)literature search [ relied on previous qualitative study of idus conducted in 2001 ( n=57 ) and epidemiological survey of idus including sws ( n=426)]exchanged sex for drugs , money or other goods and injected drugs in last 4 weeksone , 35 sws recruited by a female non - sw idu seed36 ( not set)2004 , 1.5 months three rds studies among sex workers and injection drug using sex workers all studies were approved by appropriate local ethical committees and international human subject review boards .
participants were not linked to the study with any personal identifiers , and all participant information was kept confidential and anonymous .
prior to data collection , formative research , comprising a combination of key informant interviews and a literature review , was undertaken to assess the local feasibility of the rds study identifying the optimal study set up ( table 1 , column 3 ) .
the formative research elicited data on size estimates of the sex work population ; description of how sex work is organised ( sectors of sex work , freedom of movement , average earnings and prices charged for services in each sector ) ; and the legal situation surrounding sex work . in serbia and montenegro , researchers also asked sws about the size of their social networks in order to fine - tune questions on which rds analysis would be based .
a social network was defined as the number of sws , 18 years and older , each sw knows and has seen in the past week , month , three months , six months , or year .
as shown in table 1 , a total of 209 sws were recruited in belgrade ( serbia ) , two in podgorica ( montenegro ) and 36 in togliatti ( russian federation ) .
rds was conducted in the autumn of 2005 in belgrade , serbia s capital , by a local fieldwork team with a limited access to sw networks .
estimates of the size of the sw population in the municipality , based on police records and anecdotal estimates , vary widely from 900 to 3,000 sws.2,24,25 sex work is illegal .
findings from in - depth interviews of sws suggest that there are approximately ten areas located near the main transit routes in and out of belgrade where street sws operate in small groups .
qualitative data suggests that the majority of sws operate independently , advertising their services in newspapers or through referrals via existing client networks , potentially making them harder to reach through rds .
formative interviews also found that there was little mixing of sws across different sex work sectors ( street , agency , and sws working independently ) , among street sws themselves ( transvestite , roma and serbian / montenegrin heterosexual sws ) and among sws working independently .
however , in addition to a lack of strong connection among sws and their independent working situations , the study team undertaking formative qualitative research associated sws reluctance to participate in the study to perceptions of inadequate incentive ( 10 euros for 1 hour of their time ) and a general mistrust associated with coming into contact with official agencies . however , the same interviews suggested that in principle sws would be interested in participating in a survey involving the collection of both behavioural and biological data through hiv testing .
interviewees indicated that they knew approximately 20 other sws , which again suggested that rds would be feasible .
the sample needed to be large enough for a statistically reliable analysis of risk factors and hiv prevalence , should rds analysis show that the sample is reasonably representative .
the interview site was on the premises of a non - governmental organisation ( ngo ) located in central belgrade easily accessible by public transport .
the site was open everyday from 3 to 9 p.m. and could accommodate three to four participants an hour because of the computer administered questionnaire .
after two months , the study ceased , having reached only 209 sws . in total ,
12 seeds were used during the study . of the initial six seeds only three produced any subsequent waves .
the rate of recruiting increased noticeably after an rds study of idus being implemented at the same site had ceased . due to the difficulties experienced by the research team in locating new seeds through other means ,
four participants with dense social networks were drawn from the seventh wave of two of the most productive recruitment chains and used as new seeds .
the average sw network size reported during the study was 12 . in response to the question ,
, the majority of sws replied that they solicited on the street ( 43% ) , followed by referral from an agency , pimps , friends or other sws ( 28.9% ) and by telephone ( 25.2% ) .
importantly , the take - up of vct provided as part of the rds study was high ( 71.8% ) , and sws indicated that they participated because of the hiv testing , rather than the monetary incentive .
there are no estimates of the number of sws in podgorica , the republic s capital .
no street - based sws were identified and the majority of sws were found to work independently from their homes .
key informant interviews ( idus , ex - idus , taxi drivers , public health officials , ngo representatives and journalists ) identified the only semi - public sex work sites as being approximately six illegal brothels , registered as lap dancing venues , in which most of the sws also lived .
at least one such brothel was found not to allow sws to leave the premises .
other links between brothels and public services included taxi drivers , beauty salons and hotels .
the intersection of the police and sex work networks , the small size of the city and the high number of implicated actors suggested that sws could be easily monitored and controlled .
previous attempts by the local ngos to conduct research among sws in podgorica had failed .
key informants , interviewed during formative rapid assessment , reported knowing at least one sw or their pimps but in almost all cases introductions were denied or never materialized .
those conducting the formative rapid assessment received strong advice from local experts and key informants including ngos , police , and taxi drivers against approaching brothel owners because of the highly controlled nature of sw venues in the city . after two weeks of trying to make a contact , four sws were accessed via their drug user networks , the only entry points into sw networks available to researchers .
these sws were interviewed at their place of work ( brothel and pimp s flat ) .
each of the four interviewed sws independently estimated that there were approximately 200 sws in the city , many of them also drug injectors , most of whom worked independently , advertising their services via newspapers and television and via client recommendation but who were not open about their sw status .
all four sws interviewed indicated that they would participate in a rds study and would invite others to do so , and each estimated they had personal regular contact with between five and ten sws .
the four sws participating in the formative rapid assessment were invited to participate in the rds study as seeds .
she reported having had contact with ten sws in 4 weeks , but no additional seeds were secured . despite recruiting an additional sw ( who required complete anonymity fearing being identified as a sw by others to his family and who had not seen other sws in the past month ) , the rds study failed to identify a sufficient pool of seeds willing to participate .
the rds study of idus and idu / sws was conducted in may 2004 in togliatti city .
togliatti city is the second city of samara region situated approximately 1,000 km south of moscow .
togliatti was selected as a study site because evidence suggests a recent increase in the number of idus and sws and an explosive spread of hiv associated with injecting drug use.6,11 approximately 2,000 sws are estimated to operate in togliatti , half of whom work from the street.6 despite ambiguous russian legislation towards sex work , it is not tolerated by the wider community or the police .
the city has been noted for its intense street policing of drug users and sws , often involving fines levied under administrative codes , such as possession of drugs , causing a public nuisance , or lack of official residency permit , rather than criminal codes.26,27 street - based sws in togliatti operate openly along the main roads and highways .
two popular locations are moskovsky prospect where approximately 70 women are working at any one time , and pobeda street where about 100 women work . in a study conducted in togliatti city in 2001 , 50% of female idus ( n=155 ) reported ever exchanging sex for goods or money , of whom 86% ( n=66 ) were currently sws.6
although no stand - alone rds studies of sws have been conducted in russia , attempts have been made to boost participation of idu / sws in rds studies of idus motivated by the evidence of high proportion of sws who also inject drugs in other cities.2830 a cross - sectional survey of idus and female idu / sws was undertaken in togliatti in 2004 as a follow up to the 2001 baseline survey .
a total of 476 idus were sampled during a 5-week period in may 2004 using rds . among these ,
55 ( 11% ) reported ever exchanging sex for money , drugs or goods , and of these , 36 in the past month , which was lower than expected . at the start of the field work ,
seven seeds were selected , of which one seed was female who had exchanged sex in the past month .
sws were explicitly asked to recruit other sws , and non - sws were encouraged to recruit either sws or non - sws ( table 1 , column 4 ) .
another female non - sw started a chain that recruited a total of 22 female idus who had ever exchanged sex , of whom 13 had exchanged sex in the past month .
this female non - sw seed produced a total of 135 recruits by the end of the study equivalent to 28% of the total sample , of which 15% ( 20 ) were sws and who reported working on the street . among the other seeds , only a handful of current sws were recruited ( n=4 ) . towards the end of the project
more coupons were distributed to female sws in an attempt to increase the sw sample ( n=5 ) , none of which produced any recruits . in order to increase the sample of sws in the survey ,
data on the number of female sws within an individual s idu network were unfortunately not provided .
the mean idu network size among sws was 19.5 ( sd 14.2 ) and among non - sws 27.1 ( sd 33.0 ) .
only one sw recruited through rds reported working from an apartment ; all others were street - based sws .
we expected that rds would enable us to recruit more hidden populations of sws such as those working from apartments or hotels .
the fact that sw seeds failed to produce any waves of sws or were lost to follow up , indicates that sw networks may not be as well connected .
field notes show that initial attempts to access street sws at moskovsky prospect and pobeda street had failed primary because of intensified police action in the area .
a local officer of the federal security service believed he had been recently infected with hiv by a sw from the area , and , in revenge , he and his colleagues enforced a repressive policy of fines and arrests among sws .
recruiting sws towards the end of the survey was more successful when policing practices returned to normal .
rds was conducted in the autumn of 2005 in belgrade , serbia s capital , by a local fieldwork team with a limited access to sw networks .
estimates of the size of the sw population in the municipality , based on police records and anecdotal estimates , vary widely from 900 to 3,000 sws.2,24,25 sex work is illegal .
findings from in - depth interviews of sws suggest that there are approximately ten areas located near the main transit routes in and out of belgrade where street sws operate in small groups .
qualitative data suggests that the majority of sws operate independently , advertising their services in newspapers or through referrals via existing client networks , potentially making them harder to reach through rds .
formative interviews also found that there was little mixing of sws across different sex work sectors ( street , agency , and sws working independently ) , among street sws themselves ( transvestite , roma and serbian / montenegrin heterosexual sws ) and among sws working independently .
however , in addition to a lack of strong connection among sws and their independent working situations , the study team undertaking formative qualitative research associated sws reluctance to participate in the study to perceptions of inadequate incentive ( 10 euros for 1 hour of their time ) and a general mistrust associated with coming into contact with official agencies .
however , the same interviews suggested that in principle sws would be interested in participating in a survey involving the collection of both behavioural and biological data through hiv testing .
interviewees indicated that they knew approximately 20 other sws , which again suggested that rds would be feasible .
the sample needed to be large enough for a statistically reliable analysis of risk factors and hiv prevalence , should rds analysis show that the sample is reasonably representative .
the interview site was on the premises of a non - governmental organisation ( ngo ) located in central belgrade easily accessible by public transport .
the site was open everyday from 3 to 9 p.m. and could accommodate three to four participants an hour because of the computer administered questionnaire .
after two months , the study ceased , having reached only 209 sws . in total ,
12 seeds were used during the study . of the initial six seeds only three produced any subsequent waves .
the rate of recruiting increased noticeably after an rds study of idus being implemented at the same site had ceased . due to the difficulties experienced by the research team in locating new seeds through other means ,
four participants with dense social networks were drawn from the seventh wave of two of the most productive recruitment chains and used as new seeds .
in response to the question , where do you most often solicit your clients ? , the majority of sws replied that they solicited on the street ( 43% ) , followed by referral from an agency , pimps , friends or other sws ( 28.9% ) and by telephone ( 25.2% ) .
importantly , the take - up of vct provided as part of the rds study was high ( 71.8% ) , and sws indicated that they participated because of the hiv testing , rather than the monetary incentive .
there are no estimates of the number of sws in podgorica , the republic s capital .
no street - based sws were identified and the majority of sws were found to work independently from their homes .
key informant interviews ( idus , ex - idus , taxi drivers , public health officials , ngo representatives and journalists ) identified the only semi - public sex work sites as being approximately six illegal brothels , registered as lap dancing venues , in which most of the sws also lived .
at least one such brothel was found not to allow sws to leave the premises .
other links between brothels and public services included taxi drivers , beauty salons and hotels .
the intersection of the police and sex work networks , the small size of the city and the high number of implicated actors suggested that sws could be easily monitored and controlled .
previous attempts by the local ngos to conduct research among sws in podgorica had failed .
key informants , interviewed during formative rapid assessment , reported knowing at least one sw or their pimps but in almost all cases introductions were denied or never materialized .
those conducting the formative rapid assessment received strong advice from local experts and key informants including ngos , police , and taxi drivers against approaching brothel owners because of the highly controlled nature of sw venues in the city . after two weeks of trying to make a contact , four sws were accessed via their drug user networks , the only entry points into sw networks available to researchers .
these sws were interviewed at their place of work ( brothel and pimp s flat ) .
each of the four interviewed sws independently estimated that there were approximately 200 sws in the city , many of them also drug injectors , most of whom worked independently , advertising their services via newspapers and television and via client recommendation but who were not open about their sw status .
all four sws interviewed indicated that they would participate in a rds study and would invite others to do so , and each estimated they had personal regular contact with between five and ten sws .
the four sws participating in the formative rapid assessment were invited to participate in the rds study as seeds .
she reported having had contact with ten sws in 4 weeks , but no additional seeds were secured . despite recruiting an additional sw ( who required complete anonymity fearing being identified as a sw by others to his family and who had not seen other sws in the past month ) , the rds study failed to identify a sufficient pool of seeds willing to participate .
the rds study of idus and idu / sws was conducted in may 2004 in togliatti city .
togliatti city is the second city of samara region situated approximately 1,000 km south of moscow .
togliatti was selected as a study site because evidence suggests a recent increase in the number of idus and sws and an explosive spread of hiv associated with injecting drug use.6,11 approximately 2,000 sws are estimated to operate in togliatti , half of whom work from the street.6 despite ambiguous russian legislation towards sex work , it is not tolerated by the wider community or the police . the city has been noted for its intense street policing of drug users and sws , often involving fines levied under administrative codes , such as possession of drugs , causing a public nuisance , or lack of official residency permit , rather than criminal codes.26,27 street - based sws in togliatti operate openly along the main roads and highways .
two popular locations are moskovsky prospect where approximately 70 women are working at any one time , and pobeda street where about 100 women work . in a study conducted in togliatti city in 2001 , 50% of female idus ( n=155 )
reported ever exchanging sex for goods or money , of whom 86% ( n=66 ) were currently sws.6 although no stand - alone rds studies of sws have been conducted in russia , attempts have been made to boost participation of idu / sws in rds studies of idus motivated by the evidence of high proportion of sws who also inject drugs in other cities.2830 a cross - sectional survey of idus and female idu / sws was undertaken in togliatti in 2004 as a follow up to the 2001 baseline survey .
a total of 476 idus were sampled during a 5-week period in may 2004 using rds . among these ,
55 ( 11% ) reported ever exchanging sex for money , drugs or goods , and of these , 36 in the past month , which was lower than expected . at the start of the field work ,
seven seeds were selected , of which one seed was female who had exchanged sex in the past month .
sws were explicitly asked to recruit other sws , and non - sws were encouraged to recruit either sws or non - sws ( table 1 , column 4 ) .
another female non - sw started a chain that recruited a total of 22 female idus who had ever exchanged sex , of whom 13 had exchanged sex in the past month .
this female non - sw seed produced a total of 135 recruits by the end of the study equivalent to 28% of the total sample , of which 15% ( 20 ) were sws and who reported working on the street . among the other seeds ,
only a handful of current sws were recruited ( n=4 ) . towards the end of the project
more coupons were distributed to female sws in an attempt to increase the sw sample ( n=5 ) , none of which produced any recruits . in order to increase the sample of sws in the survey ,
data on the number of female sws within an individual s idu network were unfortunately not provided .
the mean idu network size among sws was 19.5 ( sd 14.2 ) and among non - sws 27.1 ( sd 33.0 ) .
only one sw recruited through rds reported working from an apartment ; all others were street - based sws .
we expected that rds would enable us to recruit more hidden populations of sws such as those working from apartments or hotels .
the fact that sw seeds failed to produce any waves of sws or were lost to follow up , indicates that sw networks may not be as well connected .
field notes show that initial attempts to access street sws at moskovsky prospect and pobeda street had failed primary because of intensified police action in the area .
a local officer of the federal security service believed he had been recently infected with hiv by a sw from the area , and , in revenge , he and his colleagues enforced a repressive policy of fines and arrests among sws . recruiting
sws towards the end of the survey was more successful when policing practices returned to normal .
despite undertaking formative research in the planning of these rds studies , we found that rds methods were unable to attain sufficient samples of sws in serbia , montenegro , and russia .
a number of inter - related factors emerged as important to the success of rds : low motivation of sws to participate due to their perception of inadequate incentive ; low - density sw networks combined with high level of independence in the organisation of sex work ; a tightly controlled small number of sex work venues ; lack of contact between sws and local services in the context of high levels of mistrust of
official agencies ; and difficulties emerging in the organisation of the rds studies of sws at the same time as a parallel rds study of idus .
whereas in other settings ( e.g. , vietnam , north - eastern india , brazil ) sws have reacted favourably to nominal monetary incentives in rds studies , including incentives which fall below the rate charged for sex,1617 it is possible that the incentives offered were perceived as being too low . in order to earn primary and secondary incentives , which together in all three studies amounted to about a half - an - hour sw s rate , they had to leave their work and travel to and from the fixed interview site at least twice plus spend at least an hour involved in the study .
it is possible that sws in eastern europe may be less interested in the incentives offered since they have comparatively higher earnings .
indications are that free hiv / sti health assessments and referral for treatment should be explored in addition to the incentive .
the high acceptance of vct in belgrade , for example , is a good example of how provision of services can be more effective than monetary incentives in recruiting survey participants .
if incentives are purely monetary , they should be adequate to entice sws to participate ; yet this is often not feasible given the risk of high incentives also encouraging the attempted participation of non - sws .
unlike other rds studies conducted in vietnam,16 sws in belgrade felt uncomfortable being interviewed at a location where a concurrent study of idus was being conducted . based on anecdotal information and the fact that sws in belgrade started participating in the study
once the idu study had finished , we believe that sws were dissuaded from going to a location that was used by idus .
this problem can be assessed during formative research using focus groups with sws to assess whether they would feel comfortable sitting in a waiting room with a group of male and female idus .
perhaps the main reason for the poor success of rds recruitment among sws in these three settings is the characteristics of sw networks .
however , the information on the network sizes of a few sws gathered through our formative research are not easy to interpret since there is no known cut - off point for determining an appropriate network size or density for rds . based on
the results of our studies , sws in our three study areas often work independently , thus forming smaller , more isolated , networks .
those working on the street and in brothels are also under tight control by managers , pimps and police thus restricting their movement and weakening network density .
on the other hand , prevalent societal norms a form of social control prevent sws who work independently from revealing their sw status and participating . the low recruitment rate encountered in our studies
might be overcome by allowing studies to run for a much longer time perhaps as long as six months or a year allowing sws to gain trust in the investigators . in the studies described above ,
interview sites were rented and staff employed for a fixed amount of time . a preferable
setting is one with on - going services for sws that can be used as an interview site for a long period of time allowing recruits to come at a slow rate .
however , time was unlikely a major factor contributing to difficulties in rds recruitment in the cities where our studies were conducted .
the dearth of well - established projects targeting sws in eastern europe means that greater effort should be invested prior to rds studies to identify a greater number of seeds and engender sws trust . in some cases existence of a well - established service with established contacts with local sw communities
for example , this was the case in a rds study among sws in tbilisy , georgia ( january 2006 , written communication , johnston lg ) .
in addition to promoting trust , such service organisations can provide additional networking opportunities for loosely connected sws .
given the cost of conducting second - generation sero - surveillance among highly vulnerable populations , formative research is important for establishing the appropriate type of sampling and reward system .
we found the making available free and confidential hiv testing services was an effective incentive , and perhaps more so than monetary incentives alone .
although formative research would require some additional costs , it can increase efficiency in a more extensive and successful study . during formative research , we suggest that policy makers , service providers and target populations be consulted to determine the visibility of sex work , as low visibility can be associated with tight control of the sex work industry and weak networking among sws , indicating difficulties using rds .
if researchers find it difficult to recruit sws for formative study , the rds study is likely to fail .
depending on the timescale , funding and interest among existing service providers , additional services may be provided well ahead of a rds study in order to access sw respondents .
further investigation is needed into the ethnography of sw networks in this region , especially on network composition and service access . in the case of idus who exchange sex for money , goods , or drugs , it is important to understand their primary network linkage injecting drug use or sex work and how they represent their other identity within each network .
incentives need to be better identified in advance of rds studies , specifically as they relate to level of income , opportunity costs , and trust in the investigators and seeds . without this cultural and legal sensitivity , surveillance for hiv among this vulnerable population will not provide adequate information on epidemic spread or program effectiveness . | respondent driven sampling ( rds ) has been used in several counties to sample injecting drug users , sex workers ( sws ) and men who have sex with men and as a means of collecting behavioural and biological health data .
we report on the use of rds in three separate studies conducted among sws between 2004 and 2005 in the russian federation , serbia , and montenegro .
findings suggest that there are limitations associated with the use of rds in sw populations in these regions .
findings highlight three main factors that merit further investigation as a means of assessing the feasibility and appropriateness of rds in this high risk population : the network characteristics of sws ; the appropriate level of participant incentives ; and lack of service contact .
the highly controlled and hidden nature of sw organizations and weak sw social networks in the region can combine to undermine assumptions underpinning the feasibility of rds approaches and potentially severely limit recruitment .
we discuss the implications of these findings for recruitment and the use of monetary and non - monetary incentives in future rds studies of sw populations in eastern europe . | Introduction
Methods
Results
Case 1: Serbia
Case 2: Montenegro
Case 3: Russia
Discussion
Conclusion | in many countries in eastern europe large scale social and economic transition over the past 15 years has been associated with widespread poverty and increasing levels of unemployment and population migration , particularly among women.1,2 such transition has also been associated with shifts in patterns of sexual behaviour and sexual relationships , including increased commercial sex work , the growth of informal sex work economies and associated human trafficking.3 additionally , a number of countries in the former soviet union have experienced a rapid or explosive spread of hiv associated with injecting drug use.47 these epidemics are potentially exacerbated by close links between sex work and injecting drug use.2,811 this has caused concerns that high levels of sexual mixing between injecting drug users ( idus ) and their sexual partners in settings of high hiv and sexual transmitted infection ( sti ) prevalence may transform hiv infection from concentrated epidemics among risk populations to generalised epidemics.9,10 it is therefore essential to assess the diffusion of hiv and related risk behaviour in hidden vulnerable populations , such as idus and sex workers ( sws ) , as accurately as possible . there has been increased and recent interest in an approach to sampling hidden populations known as respondent driven sampling ( rds).12,13 one of the claimed advantages of rds over other methods of sampling hidden populations , such as time location sampling , is that it requires little in - depth formative research among study populations.13,14 rds begins with a set number of non - randomly selected seeds ( members of the target population ) . since rds claims to generate a representative sample from non - randomly selected seeds , many researchers have assumed that sampling can proceed without detailed prior knowledge about the local target population.13,14 rds has proved feasible and successful in recruiting hidden populations of idus in a variety of settings , resulting in the rapid acquisition of long and varied recruitment chains.11,1419 rds has also been used in several countries to collect both behavioural and biological data from sws ( e.g. , vietnam , india ) and men who have sex with men ( e.g. , bangladesh , cambodia , uganda , united states).2024 however , little is known about the feasibility of rds as a method of recruiting hidden populations of sws in settings where the organisation of sex work and access to sws is highly controlled and where there is little or no contact between the target population and local services . drawing upon three case studies in eastern europe targeting sws and idus involved in sw ( idu / sws ) , we explore the feasibility of rds , identifying some barriers to successful recruitment . these findings have implications for the direction of future research exploring the feasibility of rds methods . using rds , data on hiv prevalence and associated risk factors were gathered from sws in serbia and montenegro and from idus involved in sex work in russia ( see table 1 ) . table 1.three rds studies among sex workers and injection drug using sex workerscountrycity ( population size)formative researchrecruitment criteria for swsnumber of seedssample reached ( target)year , study durationserbiabelgrade ( about 2 million)literature search ; questions on network characteristics and rds study feasibility posed during individual interviews with sws in 2005 ( n=10)exchanged sex for drugs , money or other goods in last 30 days ( irrespective of gender or sexual orientation)six initially , six added later209 ( 400)2005 , 2 monthsmontenegropodgorica ( about 300,000)literature search ; questions on network characteristics and rds study feasibility posed during in - depth interviews with sws in 2005 ( n=4)exchanged sex for drugs , money or other goods in last 30 days ( irrespective of gender or sexual orientation)one to start with , one added later2 ( 150)2005 , 2 monthsrussian federationtogliatti ( 740,636)literature search [ relied on previous qualitative study of idus conducted in 2001 ( n=57 ) and epidemiological survey of idus including sws ( n=426)]exchanged sex for drugs , money or other goods and injected drugs in last 4 weeksone , 35 sws recruited by a female non - sw idu seed36 ( not set)2004 , 1.5 months three rds studies among sex workers and injection drug using sex workers all studies were approved by appropriate local ethical committees and international human subject review boards . the formative research elicited data on size estimates of the sex work population ; description of how sex work is organised ( sectors of sex work , freedom of movement , average earnings and prices charged for services in each sector ) ; and the legal situation surrounding sex work . in serbia and montenegro , researchers also asked sws about the size of their social networks in order to fine - tune questions on which rds analysis would be based . a social network was defined as the number of sws , 18 years and older , each sw knows and has seen in the past week , month , three months , six months , or year . as shown in table 1 , a total of 209 sws were recruited in belgrade ( serbia ) , two in podgorica ( montenegro ) and 36 in togliatti ( russian federation ) . rds was conducted in the autumn of 2005 in belgrade , serbia s capital , by a local fieldwork team with a limited access to sw networks . findings from in - depth interviews of sws suggest that there are approximately ten areas located near the main transit routes in and out of belgrade where street sws operate in small groups . formative interviews also found that there was little mixing of sws across different sex work sectors ( street , agency , and sws working independently ) , among street sws themselves ( transvestite , roma and serbian / montenegrin heterosexual sws ) and among sws working independently . however , in addition to a lack of strong connection among sws and their independent working situations , the study team undertaking formative qualitative research associated sws reluctance to participate in the study to perceptions of inadequate incentive ( 10 euros for 1 hour of their time ) and a general mistrust associated with coming into contact with official agencies . however , the same interviews suggested that in principle sws would be interested in participating in a survey involving the collection of both behavioural and biological data through hiv testing . the interview site was on the premises of a non - governmental organisation ( ngo ) located in central belgrade easily accessible by public transport . in response to the question ,
, the majority of sws replied that they solicited on the street ( 43% ) , followed by referral from an agency , pimps , friends or other sws ( 28.9% ) and by telephone ( 25.2% ) . there are no estimates of the number of sws in podgorica , the republic s capital . no street - based sws were identified and the majority of sws were found to work independently from their homes . the intersection of the police and sex work networks , the small size of the city and the high number of implicated actors suggested that sws could be easily monitored and controlled . those conducting the formative rapid assessment received strong advice from local experts and key informants including ngos , police , and taxi drivers against approaching brothel owners because of the highly controlled nature of sw venues in the city . each of the four interviewed sws independently estimated that there were approximately 200 sws in the city , many of them also drug injectors , most of whom worked independently , advertising their services via newspapers and television and via client recommendation but who were not open about their sw status . despite recruiting an additional sw ( who required complete anonymity fearing being identified as a sw by others to his family and who had not seen other sws in the past month ) , the rds study failed to identify a sufficient pool of seeds willing to participate . togliatti was selected as a study site because evidence suggests a recent increase in the number of idus and sws and an explosive spread of hiv associated with injecting drug use.6,11 approximately 2,000 sws are estimated to operate in togliatti , half of whom work from the street.6 despite ambiguous russian legislation towards sex work , it is not tolerated by the wider community or the police . the city has been noted for its intense street policing of drug users and sws , often involving fines levied under administrative codes , such as possession of drugs , causing a public nuisance , or lack of official residency permit , rather than criminal codes.26,27 street - based sws in togliatti operate openly along the main roads and highways . in a study conducted in togliatti city in 2001 , 50% of female idus ( n=155 ) reported ever exchanging sex for goods or money , of whom 86% ( n=66 ) were currently sws.6
although no stand - alone rds studies of sws have been conducted in russia , attempts have been made to boost participation of idu / sws in rds studies of idus motivated by the evidence of high proportion of sws who also inject drugs in other cities.2830 a cross - sectional survey of idus and female idu / sws was undertaken in togliatti in 2004 as a follow up to the 2001 baseline survey . among these ,
55 ( 11% ) reported ever exchanging sex for money , drugs or goods , and of these , 36 in the past month , which was lower than expected . sws were explicitly asked to recruit other sws , and non - sws were encouraged to recruit either sws or non - sws ( table 1 , column 4 ) . another female non - sw started a chain that recruited a total of 22 female idus who had ever exchanged sex , of whom 13 had exchanged sex in the past month . this female non - sw seed produced a total of 135 recruits by the end of the study equivalent to 28% of the total sample , of which 15% ( 20 ) were sws and who reported working on the street . in order to increase the sample of sws in the survey ,
data on the number of female sws within an individual s idu network were unfortunately not provided . the mean idu network size among sws was 19.5 ( sd 14.2 ) and among non - sws 27.1 ( sd 33.0 ) . a local officer of the federal security service believed he had been recently infected with hiv by a sw from the area , and , in revenge , he and his colleagues enforced a repressive policy of fines and arrests among sws . rds was conducted in the autumn of 2005 in belgrade , serbia s capital , by a local fieldwork team with a limited access to sw networks . findings from in - depth interviews of sws suggest that there are approximately ten areas located near the main transit routes in and out of belgrade where street sws operate in small groups . formative interviews also found that there was little mixing of sws across different sex work sectors ( street , agency , and sws working independently ) , among street sws themselves ( transvestite , roma and serbian / montenegrin heterosexual sws ) and among sws working independently . however , in addition to a lack of strong connection among sws and their independent working situations , the study team undertaking formative qualitative research associated sws reluctance to participate in the study to perceptions of inadequate incentive ( 10 euros for 1 hour of their time ) and a general mistrust associated with coming into contact with official agencies . however , the same interviews suggested that in principle sws would be interested in participating in a survey involving the collection of both behavioural and biological data through hiv testing . the interview site was on the premises of a non - governmental organisation ( ngo ) located in central belgrade easily accessible by public transport . due to the difficulties experienced by the research team in locating new seeds through other means ,
four participants with dense social networks were drawn from the seventh wave of two of the most productive recruitment chains and used as new seeds . , the majority of sws replied that they solicited on the street ( 43% ) , followed by referral from an agency , pimps , friends or other sws ( 28.9% ) and by telephone ( 25.2% ) . there are no estimates of the number of sws in podgorica , the republic s capital . no street - based sws were identified and the majority of sws were found to work independently from their homes . those conducting the formative rapid assessment received strong advice from local experts and key informants including ngos , police , and taxi drivers against approaching brothel owners because of the highly controlled nature of sw venues in the city . each of the four interviewed sws independently estimated that there were approximately 200 sws in the city , many of them also drug injectors , most of whom worked independently , advertising their services via newspapers and television and via client recommendation but who were not open about their sw status . despite recruiting an additional sw ( who required complete anonymity fearing being identified as a sw by others to his family and who had not seen other sws in the past month ) , the rds study failed to identify a sufficient pool of seeds willing to participate . togliatti was selected as a study site because evidence suggests a recent increase in the number of idus and sws and an explosive spread of hiv associated with injecting drug use.6,11 approximately 2,000 sws are estimated to operate in togliatti , half of whom work from the street.6 despite ambiguous russian legislation towards sex work , it is not tolerated by the wider community or the police . the city has been noted for its intense street policing of drug users and sws , often involving fines levied under administrative codes , such as possession of drugs , causing a public nuisance , or lack of official residency permit , rather than criminal codes.26,27 street - based sws in togliatti operate openly along the main roads and highways . in a study conducted in togliatti city in 2001 , 50% of female idus ( n=155 )
reported ever exchanging sex for goods or money , of whom 86% ( n=66 ) were currently sws.6 although no stand - alone rds studies of sws have been conducted in russia , attempts have been made to boost participation of idu / sws in rds studies of idus motivated by the evidence of high proportion of sws who also inject drugs in other cities.2830 a cross - sectional survey of idus and female idu / sws was undertaken in togliatti in 2004 as a follow up to the 2001 baseline survey . among these ,
55 ( 11% ) reported ever exchanging sex for money , drugs or goods , and of these , 36 in the past month , which was lower than expected . sws were explicitly asked to recruit other sws , and non - sws were encouraged to recruit either sws or non - sws ( table 1 , column 4 ) . another female non - sw started a chain that recruited a total of 22 female idus who had ever exchanged sex , of whom 13 had exchanged sex in the past month . this female non - sw seed produced a total of 135 recruits by the end of the study equivalent to 28% of the total sample , of which 15% ( 20 ) were sws and who reported working on the street . in order to increase the sample of sws in the survey ,
data on the number of female sws within an individual s idu network were unfortunately not provided . the mean idu network size among sws was 19.5 ( sd 14.2 ) and among non - sws 27.1 ( sd 33.0 ) . a local officer of the federal security service believed he had been recently infected with hiv by a sw from the area , and , in revenge , he and his colleagues enforced a repressive policy of fines and arrests among sws . despite undertaking formative research in the planning of these rds studies , we found that rds methods were unable to attain sufficient samples of sws in serbia , montenegro , and russia . a number of inter - related factors emerged as important to the success of rds : low motivation of sws to participate due to their perception of inadequate incentive ; low - density sw networks combined with high level of independence in the organisation of sex work ; a tightly controlled small number of sex work venues ; lack of contact between sws and local services in the context of high levels of mistrust of
official agencies ; and difficulties emerging in the organisation of the rds studies of sws at the same time as a parallel rds study of idus . , vietnam , north - eastern india , brazil ) sws have reacted favourably to nominal monetary incentives in rds studies , including incentives which fall below the rate charged for sex,1617 it is possible that the incentives offered were perceived as being too low . it is possible that sws in eastern europe may be less interested in the incentives offered since they have comparatively higher earnings . the high acceptance of vct in belgrade , for example , is a good example of how provision of services can be more effective than monetary incentives in recruiting survey participants . unlike other rds studies conducted in vietnam,16 sws in belgrade felt uncomfortable being interviewed at a location where a concurrent study of idus was being conducted . based on anecdotal information and the fact that sws in belgrade started participating in the study
once the idu study had finished , we believe that sws were dissuaded from going to a location that was used by idus . perhaps the main reason for the poor success of rds recruitment among sws in these three settings is the characteristics of sw networks . however , the information on the network sizes of a few sws gathered through our formative research are not easy to interpret since there is no known cut - off point for determining an appropriate network size or density for rds . those working on the street and in brothels are also under tight control by managers , pimps and police thus restricting their movement and weakening network density . the dearth of well - established projects targeting sws in eastern europe means that greater effort should be invested prior to rds studies to identify a greater number of seeds and engender sws trust . given the cost of conducting second - generation sero - surveillance among highly vulnerable populations , formative research is important for establishing the appropriate type of sampling and reward system . we found the making available free and confidential hiv testing services was an effective incentive , and perhaps more so than monetary incentives alone . during formative research , we suggest that policy makers , service providers and target populations be consulted to determine the visibility of sex work , as low visibility can be associated with tight control of the sex work industry and weak networking among sws , indicating difficulties using rds . further investigation is needed into the ethnography of sw networks in this region , especially on network composition and service access . in the case of idus who exchange sex for money , goods , or drugs , it is important to understand their primary network linkage injecting drug use or sex work and how they represent their other identity within each network . incentives need to be better identified in advance of rds studies , specifically as they relate to level of income , opportunity costs , and trust in the investigators and seeds . | [
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vi nanoscale semiconductor compounds have attracted much attention over the past few years due to their fundamental properties and applications , mostly as tunable emitters for biomedical labeling , light emitting diodes ( led ) , lasers , and sensors [ 3 - 5 ] .
however , wide band gap semiconductor nanocrystals , such as zinc chalcogenide ones doped with transition metal ions [ 6,7,9 - 11 ] , may overcome this concern and yet maintain the advantages of the nanocrystal emitters . in 1994 , bhargava et al .
initiatively proposed luminescence properties of mn - doped zns nanocrystals that were prepared by a room temperature chemical process .
then , nanocrystalline zns has been widely and deeply investigated since nanosized zns can be easily prepared . as a typical ii
vi semiconductor , zns , especially doped with divalent manganese ions , has been commercially used as a phosphor as well as in thin film electroluminescent devices . in the bulk form
vi semiconductor with a large band gap 3.68 ev ( 340 nm ) . at the nanometer scale ,
zns has attracted more attention due to its interesting optical , electric properties , and large quantum efficiencies depending on its size .
for instance , it can be used as a higher band gap material to passivate other quantum semiconductor heterostructure to increase their quantum yields .
the properties of the photoluminescent and electroluminescent materials could be greatly affected by doping concentration of mn .
the mn ion , used as a dopant in many luminescent materials , has a d configuration that exhibits a broad emission peak , and its position strongly depends on the host lattice , which lies on the change in strength of crystal field with host .
the emission color can vary from green to deep red , corresponding to a t1a1 transition . to obtain nanometer - sized particles ,
a variety of methods have been proposed , including microemulsion method , sol gel processing , competitive reaction chemistry method , and aqueous chemical method . since dixit et al
water(a / w ) solution , there have been few reports on generating of nano - crystallined ceo2 and silicon in a / w mixed solvents or in ethylalcohol liquid bridges . in this paper , we focus on preparation of stabilized mn - doped zns nanocrystals by soft template method and coated zn(oh)2 shells through precipitation reaction .
photoenhanced luminescence and higher quantum efficiency have been observed in zns : mn / zn(oh)2 nanoparticle .
zn(ch3coo)22h2o(a.r . ) , na2s9h2o(a.r . ) , and mn(ch3coo)24h2o(a.r . ) were employed as raw materials and c2h5oh and h2o mixed solution as soft template .
first , na2s and dopant precursor mn(ch3coo)2 were ground in mortar to form mns crystal nucleus , and c2h5oh and h2o solutions were added as soft template in grinding process .
then , zn(ch3coo)2 was added and ground in soft template for the regrowth of zns on the surface of mns layer .
the concentration of the dopant in the nuclei can be regulated varying the precursor ratio .
the products were collected by centrifugal sedimentation and first washed by ionized water then by alcohol .
finally , the products were ultrasonically dispersed in alcohol and dried at 40c in vacuum .
zn(oh)2 shell , coating on zns : mn nanocrystals can be produced as follows : zns : mn nanocrystal was put into deionized water and ultrasonically dispersed for 2 h. appropriate amount of zn(ch3coo)2 aqueous solution was slowly dropped into the zns : mn suspension under the conditions of vigorous stirring .
ten minutes later , appropriate amount of naoh aqueous solution was dropped into the suspension to form stoichiometric zn(oh)2 .
after 1 h of continuous stirring , the resulting precipitates were dealt in the same way as the zns : mn nanocrystals .
all the samples were characterized by powder x - ray diffraction ( xrd ) using a rigaku diffractometer with ni - filtered cuka radiation at room temperature .
the transmission electron microscopy ( tem ) images of the nanoparticles were obtained by using tem ( jem-2100f electron microscope / jeol co. 200 kv ) .
the optical properties of finely ground samples were collected by an uv vis spectrophotometer ( pe lambda950 ) using baso4 as a reference in the range of 200700 nm .
the photoluminescence ( pl ) spectra in the uv region were obtained using a fls-920 t fluorescence spectrophotometer equipped with xe 900 ( 450 w xenon arc lamp ) as the light source with spectral slits width of 1 nm . the quantum yield of the samples excited at 300 nm was recorded with horiba jobin - yvon fluorlog-3 spectrofluorometer system .
the xrd patterns of the zns : mn nanocrystals prepared in the condition of alcohol and water volume ratio of 1:1 are presented in fig
. 1 . the broadening of the diffraction peaks of all the mn : zns nanoparticles , a characteristic of nanocrystal , can be obviously observed .
for all the samples , the main three diffraction peak positions correspond to the lattice planes of ( 111 ) , ( 220 ) , and ( 311 ) , matching the cubic zinc blende zns structure ( jcpds no . 05 - 0566 ) .
scherrer formula , the mean crystalline sizes calculated from the full - width at half - maximum ( fwhm ) of these lines were about 3.02 nm . xrd patterns of zns : mn nanocrystals ( a / w = 1:1 ) this paper extends the method employing the a / w template to produce the zns : mn nanocrystals .
the nanocrystals distribute in a narrow size ; the shape of the particles is approximately spherical , and the mean diameter is about 35 nm . the nucleus formation mechanism in alcohol and water system is as follows : the reagents are over saturation in a / w template , and the relationship between super saturation and the radius of stable nuclei ( r ) can be expressed by kelvin equation : tem image of zns : mn nanocrystals ( a / w = 1:1 ) the degree of super saturation ( s ) is defined by the ratio of solute concentration ( c ) and saturation concentration ( c1 ) : when the s value is much larger than unity , a great number of primary nuclei formed . according to , the dependence of particle radius ( r ) on the dielectric constant ( ) of the solution can be further simplified as where 0 is the permittivity in vacuum and is the dielectric constant in a given solution .
the symbols r+ and r represent the radii of ions charged z+ and z , respectively , and e represents the elementary charge ( e = 1.602 10 c ) . for mixed solvents of homologous alcohol and water system , values of a and b
therefore , it is clear that the change in the dielectric constant of mixed solvent can remarkably affect the nucleation rate and the particle size .
in addition , for single water system , due to the small particle size there is a big specific surface area , so the products easily aggregate .
because alcohol and water can dissolve each other unlimitedly , most of water molecules adsorbing on the particle surface can be replaced by alcohol molecules , which diminish the particle surface tension and surface energy ; thus , the trend of reunion between the particles can be effectively declined . besides , the probability of collisions between particles can be reduced by the steric effect of organic system ( a / w ) , which contributes to the formation of nanoparticles with uniformity and good dispersion .
figure 3 illustrates photoluminescence ( pl ) spectra of zns : mn nanocrystals with different mn concentration excited at 300 nm in the room temperature .
the pl spectrum shows a weak emission at 430 nm and a broad strong emission band ranging from 520 to 700 nm .
the weak blue - light emission at 430 nm is attributed to a defect related emission of the zns host .
the orange broad emission centering at 605 nm ascribes to the transition of mn from the first excited state of t1 to the ground state of a1 .
the origin of typical emission of mn is as follows : when mn ions are incorporated into the zns lattice and substitute for host cation sites ; the hybrid between the sp electrons of the host zns and the d electrons of mn occurs and makes the forbidden transition of t1a1 partially allowed , as a result of the characteristic emission of mn .
it is observed that the strong pl position from zns : mn nanocrystals compared with other results redshifts to 605 nm ( fig .
a possible explanation is that the enhancement of quantum effect in nanocrystals increases the hybrid effect , according to tanabe - sugano cryatal field theory of transition metal , t1 energy in first excited state of mn decreases with the increase in crystal field intensity , but ground state a1 is not changed , so the shrunken gap between t1 and a1 causes a pl red shift .
the emission spectra of zns : mn nanocrystals for different mn concentrations ( inset is the intensity of emission as a function of mn concentration ) the doped manganese in the precursors may be introduced into inside or outside of zns nanocrystals .
they found that whether mn incorporates into the zns lattice will lead to the orange emission , while zns with mn on the surface - bound will yield the ultraviolet emission .
narayan pradhan studied the possible nucleation and growth process , they found that the successful doping and decoupling of doping must fulfill following conditions : with the increase in doped ions , the steady increase in the pl intensity from the doping centers at about 605 nm , the fixed pl positions of the host zns nanocrystals and the doping centers , and the gradual decrease in the pl intensity of the host zns nanocrystals .
3 demonstrates the functional relation between characteristic emission intensity of mn and doping amount . according to inset of fig .
3 , with the increase in mn , the emission intensity also increases and reaches its highest intensity at the mn content of 2% . at the same time ,
therefore , the mn precursor would be much less than zn if they both have the same carboxylate ligand . from above all
, it can be concluded that the mn ions in our samples are indeed introduced into the host zns nanocrystals . from the fig .
3 , the highest doping amount is 2% . at the same time , the amount of alcohol will affect the degree of agglomeration of nanoparticles . according to dlvo theory ,
the energy barrier between two particles that inhibits agglomeration can be expressed as : where a is the effective hamaker constant , k is the debye huckel parameter , r is the particle diameter , r is the relative dielectric constant of the liquid medium , 0 is the dielectric constant of vacuum , and is the surface potential . the dielectric constant of an a / w mixed solvent decreases with an increase in the volume ratio of a / w . in this study
, we kept the total a / w volume as a constant then adjusted the alcohol ratio in the template and investigated the effects of the alcohol volume on the pl properties .
mn concentration is fixed at 2% , the pl spectra of nanocrystals with various s are shown in fig .
the emission intensity of zns : mn enhances gradually with the increase in alcohol concentration . because of the integrity of a better particle namely passivated surface defect , the strongest emission could be obtained when the concentration of alcohol reached 33% ( alcohol and water volume ratio of 1:2 , inset of fig .
the emission spectra of zns : mn nanocrystals for different a / w volume ratio ( inset is the intensity of emission as a function of alcohol content in template ) according to the results , we draw a conclusion from the process of preparing nanocrystals that the function of a / w mixed system is as follows : at that moment , the hydrogen bonds , hydration , and ethanol water molecules are no longer separate water molecules or ethanol molecules but bridged molecular clusters formed by a number of ethanol molecules and water molecules .
it is deduced that there are three possible cluster structures : five ethanol molecules and six water molecules connect with each other to form a ring or chain structure through the hydrogen bond ; an ethanol molecule and two water molecules form the chain structure with hydrogen bond ; and an ethanol molecule and five water molecules link mutually to form a new molecular structure .
when the solution has a 60% ( vol ) of ethanol , the concentration of the first component cluster arrives at the maximum , the second and third types of the component cluster are , respectively , correspond to the alcohol concentration of 40 and 80% . from experiment results
, it could be inferred that in the course of obtaining integrated particle crystallization , the second cluster structure is the best template , because our sample with best emission intensity was produced in the condition of the closest approach to the second type molecular structure of alcohol ( 40%).the optimal volume of a / w is 1:2 .
optical absorption spectra of zns : mn nanocrystals for different a / w are shown in fig .
the excitonic absorption peaks for all these samples are blueshifted in the comparison with the bulk band gap ( 3.68 ev corresponding to the absorption edge at 336 nm ) .
it is clearly indicated the strong quantum size effects , and they have nearly the same particle size . according to the effective mass model , the radius of the particle is related to the absorption band .
the bandgap energy of nanocrystals as a function of their size is given by the brus equation , the absorption spectra of zns : mn nanocrystals for different a / w volume ratio in the case of zns , using a gap eg ( bulk ) = 3.68 ev , an effective mass for electrons me and holes mh = 0.61 m0(m0 is the electron mass ) and a dielectric constant r = 8.9 , the following relation between the bandgap eg ( r ) , in electron volt , and the particle radius r , in nanometers , is obtained : r was calculated ranges from 1.5 to 2.5 nm ( a diameter from 3 to 5 nm ) , in reasonable agreement with the xrd results . due to the sample synthesized in a / w volume ratio of 1:2
has the highest emission intensity and lowest absorption intensity ( figs . 4 , 5 )
, we can draw a conclusion that in our samples , this one has the best quantum efficiency , then we made surface treatment on this sample to improve its emission intensity and measure the quantum efficiency .
that is coated with a layer of zn(oh)2 on the surface of zns : mn .
figure 6 is the tem image of zns : mn coated with zn(oh)2 in a / w volume ratio of 1:2 and electron diffraction in the inset ( right ) , while inset ( left ) is the image of bare zns : mn nanoparticle .
the results indicate that zns : mn / zn(oh)2 nanoparticles with high distribution , and no aggregation was synthesized successfully .
tem shows the presence of zn(oh)2 coating layer on the surface of the zns : mn .
it is found that the emission intensity of coated sample enhanced by 30% . it is proven that the zns : mn surface had been modified by zn(oh)2 layer .
( a ) is the scatter spectra and ( b ) is the emission spectra of samples .
the quantum efficiency can be calculated by following formula : , where ec is the emission of sample , ea is the emission of integrating sphere , la is the scatter of integrating sphere , and lc is the scatter of sample , and after deducting the error of attenuating plate , the results are shown in table 1 . from table 1 , we find that the absolute quantum efficiency enhanced by 2.3% ( from 8.4% of uncoated sample to 10.7% of coated sample ) . based on the results , it is concluded that zn(oh)2 layer can passivate the surface of bare zns : mn to improve the emission intensity and quantum efficiency . tem image of zns : mn / zn(oh)2 nanocrystals ( inset of left ( a / w = 1:2 ) is bare zns : mn and right is electron diffraction pattern of zns : mn / zn(oh)2 ) the emission spectra of zns : mn and zns : mn / zn(oh)2 nanocrystals the schematic representation of quantum efficiency measurement quantum efficiency results luminescent decay curves and their fitting curves are shown in fig
curves a , b , and c , respectively , represent the samples synthesized under conditions of a / w volume ratio of 1:1 , 1:2 , and coated with a layer of zn(oh)2 with mn - doped concentration of 2% . because the mn ions in zns host are insular luminescent centers , the decay is evidently required a multiexponential function , which is shown through the solid smooth line from the data . where , 1 is the lifetime of luminescence ( at 1 , the intensity decays to e of its original intensity ) .
it can be found that shorter luminescent lifetime is fulfilled when a / w volume ratio is 1:2 with respect to 1:1 .
as nonradiative recombination centers , recombination rate of the surface mn ions is very fast .
once the number of the surface mn ions is reduced by the zn(oh)2 shell , the nonradiative transition paths will be blocked to some extent , leading to the lengthen of the luminescence decay .
the xrd patterns of the zns : mn nanocrystals prepared in the condition of alcohol and water volume ratio of 1:1 are presented in fig
. 1 . the broadening of the diffraction peaks of all the mn : zns nanoparticles , a characteristic of nanocrystal , can be obviously observed .
for all the samples , the main three diffraction peak positions correspond to the lattice planes of ( 111 ) , ( 220 ) , and ( 311 ) , matching the cubic zinc blende zns structure ( jcpds no . 05 - 0566 ) .
scherrer formula , the mean crystalline sizes calculated from the full - width at half - maximum ( fwhm ) of these lines were about 3.02 nm . xrd patterns of zns : mn nanocrystals ( a / w = 1:1 ) this paper extends the method employing the a / w template to produce the zns : mn nanocrystals .
the nanocrystals distribute in a narrow size ; the shape of the particles is approximately spherical , and the mean diameter is about 35 nm . the nucleus formation mechanism in alcohol and water system is as follows : the reagents are over saturation in a / w template , and the relationship between super saturation and the radius of stable nuclei ( r ) can be expressed by kelvin equation : tem image of zns : mn nanocrystals ( a / w = 1:1 ) the degree of super saturation ( s ) is defined by the ratio of solute concentration ( c ) and saturation concentration ( c1 ) : when the s value is much larger than unity , a great number of primary nuclei formed . according to , the dependence of particle radius ( r ) on the dielectric constant ( ) of the solution can be further simplified as where 0 is the permittivity in vacuum and is the dielectric constant in a given solution .
the symbols r+ and r represent the radii of ions charged z+ and z , respectively , and e represents the elementary charge ( e = 1.602 10 c ) . for mixed solvents of homologous alcohol and water system , values of a and b
therefore , it is clear that the change in the dielectric constant of mixed solvent can remarkably affect the nucleation rate and the particle size .
in addition , for single water system , due to the small particle size there is a big specific surface area , so the products easily aggregate .
because alcohol and water can dissolve each other unlimitedly , most of water molecules adsorbing on the particle surface can be replaced by alcohol molecules , which diminish the particle surface tension and surface energy ; thus , the trend of reunion between the particles can be effectively declined . besides , the probability of collisions between particles can be reduced by the steric effect of organic system ( a / w ) , which contributes to the formation of nanoparticles with uniformity and good dispersion .
figure 3 illustrates photoluminescence ( pl ) spectra of zns : mn nanocrystals with different mn concentration excited at 300 nm in the room temperature .
the pl spectrum shows a weak emission at 430 nm and a broad strong emission band ranging from 520 to 700 nm .
the weak blue - light emission at 430 nm is attributed to a defect related emission of the zns host .
the orange broad emission centering at 605 nm ascribes to the transition of mn from the first excited state of t1 to the ground state of a1 .
the origin of typical emission of mn is as follows : when mn ions are incorporated into the zns lattice and substitute for host cation sites ; the hybrid between the sp electrons of the host zns and the d electrons of mn occurs and makes the forbidden transition of t1a1 partially allowed , as a result of the characteristic emission of mn .
it is observed that the strong pl position from zns : mn nanocrystals compared with other results redshifts to 605 nm ( fig .
a possible explanation is that the enhancement of quantum effect in nanocrystals increases the hybrid effect , according to tanabe - sugano cryatal field theory of transition metal , t1 energy in first excited state of mn decreases with the increase in crystal field intensity , but ground state a1 is not changed , so the shrunken gap between t1 and a1 causes a pl red shift .
the emission spectra of zns : mn nanocrystals for different mn concentrations ( inset is the intensity of emission as a function of mn concentration ) the doped manganese in the precursors may be introduced into inside or outside of zns nanocrystals .
they found that whether mn incorporates into the zns lattice will lead to the orange emission , while zns with mn on the surface - bound will yield the ultraviolet emission .
narayan pradhan studied the possible nucleation and growth process , they found that the successful doping and decoupling of doping must fulfill following conditions : with the increase in doped ions , the steady increase in the pl intensity from the doping centers at about 605 nm , the fixed pl positions of the host zns nanocrystals and the doping centers , and the gradual decrease in the pl intensity of the host zns nanocrystals .
3 demonstrates the functional relation between characteristic emission intensity of mn and doping amount . according to inset of fig .
3 , with the increase in mn , the emission intensity also increases and reaches its highest intensity at the mn content of 2% . at the same time ,
therefore , the mn precursor would be much less than zn if they both have the same carboxylate ligand . from above all
, it can be concluded that the mn ions in our samples are indeed introduced into the host zns nanocrystals . from the fig .
at the same time , the amount of alcohol will affect the degree of agglomeration of nanoparticles . according to dlvo theory ,
the energy barrier between two particles that inhibits agglomeration can be expressed as : where a is the effective hamaker constant , k is the debye huckel parameter , r is the particle diameter , r is the relative dielectric constant of the liquid medium , 0 is the dielectric constant of vacuum , and is the surface potential . the dielectric constant of an a / w mixed solvent decreases with an increase in the volume ratio of a / w . in this study
, we kept the total a / w volume as a constant then adjusted the alcohol ratio in the template and investigated the effects of the alcohol volume on the pl properties .
mn concentration is fixed at 2% , the pl spectra of nanocrystals with various s are shown in fig .
the emission intensity of zns : mn enhances gradually with the increase in alcohol concentration . because of the integrity of a better particle namely passivated surface defect , the strongest emission could be obtained when the concentration of alcohol reached 33% ( alcohol and water volume ratio of 1:2 , inset of fig .
the emission spectra of zns : mn nanocrystals for different a / w volume ratio ( inset is the intensity of emission as a function of alcohol content in template ) according to the results , we draw a conclusion from the process of preparing nanocrystals that the function of a / w mixed system is as follows : at that moment , the hydrogen bonds , hydration , and ethanol water molecules are no longer separate water molecules or ethanol molecules but bridged molecular clusters formed by a number of ethanol molecules and water molecules .
it is deduced that there are three possible cluster structures : five ethanol molecules and six water molecules connect with each other to form a ring or chain structure through the hydrogen bond ; an ethanol molecule and two water molecules form the chain structure with hydrogen bond ; and an ethanol molecule and five water molecules link mutually to form a new molecular structure .
when the solution has a 60% ( vol ) of ethanol , the concentration of the first component cluster arrives at the maximum , the second and third types of the component cluster are , respectively , correspond to the alcohol concentration of 40 and 80% . from experiment results
, it could be inferred that in the course of obtaining integrated particle crystallization , the second cluster structure is the best template , because our sample with best emission intensity was produced in the condition of the closest approach to the second type molecular structure of alcohol ( 40%).the optimal volume of a / w is 1:2 .
optical absorption spectra of zns : mn nanocrystals for different a / w are shown in fig .
the excitonic absorption peaks for all these samples are blueshifted in the comparison with the bulk band gap ( 3.68 ev corresponding to the absorption edge at 336 nm ) .
it is clearly indicated the strong quantum size effects , and they have nearly the same particle size . according to the effective mass model , the radius of the particle is related to the absorption band .
the bandgap energy of nanocrystals as a function of their size is given by the brus equation , the absorption spectra of zns : mn nanocrystals for different a / w volume ratio in the case of zns , using a gap eg ( bulk ) = 3.68 ev , an effective mass for electrons me and holes mh = 0.61 m0(m0 is the electron mass ) and a dielectric constant r = 8.9 , the following relation between the bandgap eg ( r ) , in electron volt , and the particle radius r , in nanometers , is obtained : r was calculated ranges from 1.5 to 2.5 nm ( a diameter from 3 to 5 nm ) , in reasonable agreement with the xrd results .
due to the sample synthesized in a / w volume ratio of 1:2 has the highest emission intensity and lowest absorption intensity ( figs . 4 , 5 )
, we can draw a conclusion that in our samples , this one has the best quantum efficiency , then we made surface treatment on this sample to improve its emission intensity and measure the quantum efficiency .
that is coated with a layer of zn(oh)2 on the surface of zns : mn .
figure 6 is the tem image of zns : mn coated with zn(oh)2 in a / w volume ratio of 1:2 and electron diffraction in the inset ( right ) , while inset ( left ) is the image of bare zns : mn nanoparticle .
the results indicate that zns : mn / zn(oh)2 nanoparticles with high distribution , and no aggregation was synthesized successfully .
tem shows the presence of zn(oh)2 coating layer on the surface of the zns : mn .
it is found that the emission intensity of coated sample enhanced by 30% . it is proven that the zns : mn surface had been modified by zn(oh)2 layer .
( a ) is the scatter spectra and ( b ) is the emission spectra of samples .
the quantum efficiency can be calculated by following formula : , where ec is the emission of sample , ea is the emission of integrating sphere , la is the scatter of integrating sphere , and lc is the scatter of sample , and after deducting the error of attenuating plate , the results are shown in table 1 . from table 1 , we find that the absolute quantum efficiency enhanced by 2.3% ( from 8.4% of uncoated sample to 10.7% of coated sample ) . based on the results , it is concluded that zn(oh)2 layer can passivate the surface of bare zns : mn to improve the emission intensity and quantum efficiency . tem image of zns : mn / zn(oh)2 nanocrystals ( inset of left ( a / w = 1:2 ) is bare zns : mn and right is electron diffraction pattern of zns : mn / zn(oh)2 ) the emission spectra of zns : mn and zns : mn / zn(oh)2 nanocrystals the schematic representation of quantum efficiency measurement quantum efficiency results luminescent decay curves and their fitting curves are shown in fig .
9 . curves a , b , and c , respectively , represent the samples synthesized under conditions of a / w volume ratio of 1:1 , 1:2 , and coated with a layer of zn(oh)2 with mn - doped concentration of 2% .
because the mn ions in zns host are insular luminescent centers , the decay is evidently required a multiexponential function , which is shown through the solid smooth line from the data .
where , 1 is the lifetime of luminescence ( at 1 , the intensity decays to e of its original intensity ) .
it can be found that shorter luminescent lifetime is fulfilled when a / w volume ratio is 1:2 with respect to 1:1 .
sample coated with zn(oh)2 shell leads to the increased lifetime . as nonradiative recombination centers ,
once the number of the surface mn ions is reduced by the zn(oh)2 shell , the nonradiative transition paths will be blocked to some extent , leading to the lengthen of the luminescence decay .
in summary , zns : mn and zns : mn / zn(oh)2 nanocrystals were synthesized by soft template and precipitation reaction method .
as the mn concentration increased , the emission intensity increases and reaches highest with the mn content at 2% .
meanwhile , the emission intensity varies with the ratio of alcohol . when alcohol and water volume ratio is 1:2 , the strongest emission intensity and shortest lifetime decay is achieved , which is attributed to the integrity of better particle getting from structure of alcohol / water template .
when zns : mn is coated with zn(oh)2 , the emission intensity of mn enhances by 30% , and its absolute quantum efficiency increases by 2.3% .
this work was supported by the national natural science foundation of china ( 10874061 ) and research fund for the doctoral program of higher education ( 200807300010 ) .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . | mn - doped zns nanocrystals based on low dopant concentrations ( 02% ) and coated with a shell of zn(oh)2 have been prepared via soft template and precipitation reaction .
the results indicate that the zns : mn nanocrystal is cubic zinc blende structure and its diameter is 3.02 nm as demonstrated by xrd . measured by tem , the morphology of nanocrystals is a spherical shape , and their particle size ( 35 nm ) is similar to that of xrd results .
photoluminescence spectra under ultraviolet region shows that the volume ratio of alcohol to water in the template has a great effect on the luminescence properties of zns : mn particles .
compared with unpassivated zns : mn nanocrystals , zns : mn / zn(oh)2 core / shell nanocrystal exhibits much improved luminescence and higher absolute quantum efficiency .
meanwhile , we simply explore the formation mechanism of zns : mn nanocrystals in alcohol and water system and analyze the reason why alcohol and water cluster structures can affect the luminescent properties of nanoparticle . | Introduction
Experimental Procedure
Result and Discussion
The Volume Ratio of A/W is 1:1
Effect of Volume Ratio of A/W on Luminescence Behavior
Conclusions
Acknowledgments
Open Access | initiatively proposed luminescence properties of mn - doped zns nanocrystals that were prepared by a room temperature chemical process . in the bulk form
vi semiconductor with a large band gap 3.68 ev ( 340 nm ) . the mn ion , used as a dopant in many luminescent materials , has a d configuration that exhibits a broad emission peak , and its position strongly depends on the host lattice , which lies on the change in strength of crystal field with host . to obtain nanometer - sized particles ,
a variety of methods have been proposed , including microemulsion method , sol gel processing , competitive reaction chemistry method , and aqueous chemical method . in this paper , we focus on preparation of stabilized mn - doped zns nanocrystals by soft template method and coated zn(oh)2 shells through precipitation reaction . photoenhanced luminescence and higher quantum efficiency have been observed in zns : mn / zn(oh)2 nanoparticle . then , zn(ch3coo)2 was added and ground in soft template for the regrowth of zns on the surface of mns layer . finally , the products were ultrasonically dispersed in alcohol and dried at 40c in vacuum . zn(oh)2 shell , coating on zns : mn nanocrystals can be produced as follows : zns : mn nanocrystal was put into deionized water and ultrasonically dispersed for 2 h. appropriate amount of zn(ch3coo)2 aqueous solution was slowly dropped into the zns : mn suspension under the conditions of vigorous stirring . after 1 h of continuous stirring , the resulting precipitates were dealt in the same way as the zns : mn nanocrystals . the xrd patterns of the zns : mn nanocrystals prepared in the condition of alcohol and water volume ratio of 1:1 are presented in fig
. for all the samples , the main three diffraction peak positions correspond to the lattice planes of ( 111 ) , ( 220 ) , and ( 311 ) , matching the cubic zinc blende zns structure ( jcpds no . scherrer formula , the mean crystalline sizes calculated from the full - width at half - maximum ( fwhm ) of these lines were about 3.02 nm . xrd patterns of zns : mn nanocrystals ( a / w = 1:1 ) this paper extends the method employing the a / w template to produce the zns : mn nanocrystals . the nanocrystals distribute in a narrow size ; the shape of the particles is approximately spherical , and the mean diameter is about 35 nm . the nucleus formation mechanism in alcohol and water system is as follows : the reagents are over saturation in a / w template , and the relationship between super saturation and the radius of stable nuclei ( r ) can be expressed by kelvin equation : tem image of zns : mn nanocrystals ( a / w = 1:1 ) the degree of super saturation ( s ) is defined by the ratio of solute concentration ( c ) and saturation concentration ( c1 ) : when the s value is much larger than unity , a great number of primary nuclei formed . according to , the dependence of particle radius ( r ) on the dielectric constant ( ) of the solution can be further simplified as where 0 is the permittivity in vacuum and is the dielectric constant in a given solution . for mixed solvents of homologous alcohol and water system , values of a and b
therefore , it is clear that the change in the dielectric constant of mixed solvent can remarkably affect the nucleation rate and the particle size . in addition , for single water system , due to the small particle size there is a big specific surface area , so the products easily aggregate . because alcohol and water can dissolve each other unlimitedly , most of water molecules adsorbing on the particle surface can be replaced by alcohol molecules , which diminish the particle surface tension and surface energy ; thus , the trend of reunion between the particles can be effectively declined . figure 3 illustrates photoluminescence ( pl ) spectra of zns : mn nanocrystals with different mn concentration excited at 300 nm in the room temperature . it is observed that the strong pl position from zns : mn nanocrystals compared with other results redshifts to 605 nm ( fig . the emission spectra of zns : mn nanocrystals for different mn concentrations ( inset is the intensity of emission as a function of mn concentration ) the doped manganese in the precursors may be introduced into inside or outside of zns nanocrystals . narayan pradhan studied the possible nucleation and growth process , they found that the successful doping and decoupling of doping must fulfill following conditions : with the increase in doped ions , the steady increase in the pl intensity from the doping centers at about 605 nm , the fixed pl positions of the host zns nanocrystals and the doping centers , and the gradual decrease in the pl intensity of the host zns nanocrystals . from above all
, it can be concluded that the mn ions in our samples are indeed introduced into the host zns nanocrystals . at the same time , the amount of alcohol will affect the degree of agglomeration of nanoparticles . according to dlvo theory ,
the energy barrier between two particles that inhibits agglomeration can be expressed as : where a is the effective hamaker constant , k is the debye huckel parameter , r is the particle diameter , r is the relative dielectric constant of the liquid medium , 0 is the dielectric constant of vacuum , and is the surface potential . the dielectric constant of an a / w mixed solvent decreases with an increase in the volume ratio of a / w . in this study
, we kept the total a / w volume as a constant then adjusted the alcohol ratio in the template and investigated the effects of the alcohol volume on the pl properties . mn concentration is fixed at 2% , the pl spectra of nanocrystals with various s are shown in fig . the emission intensity of zns : mn enhances gradually with the increase in alcohol concentration . because of the integrity of a better particle namely passivated surface defect , the strongest emission could be obtained when the concentration of alcohol reached 33% ( alcohol and water volume ratio of 1:2 , inset of fig . the emission spectra of zns : mn nanocrystals for different a / w volume ratio ( inset is the intensity of emission as a function of alcohol content in template ) according to the results , we draw a conclusion from the process of preparing nanocrystals that the function of a / w mixed system is as follows : at that moment , the hydrogen bonds , hydration , and ethanol water molecules are no longer separate water molecules or ethanol molecules but bridged molecular clusters formed by a number of ethanol molecules and water molecules . from experiment results
, it could be inferred that in the course of obtaining integrated particle crystallization , the second cluster structure is the best template , because our sample with best emission intensity was produced in the condition of the closest approach to the second type molecular structure of alcohol ( 40%).the optimal volume of a / w is 1:2 . optical absorption spectra of zns : mn nanocrystals for different a / w are shown in fig . the excitonic absorption peaks for all these samples are blueshifted in the comparison with the bulk band gap ( 3.68 ev corresponding to the absorption edge at 336 nm ) . the bandgap energy of nanocrystals as a function of their size is given by the brus equation , the absorption spectra of zns : mn nanocrystals for different a / w volume ratio in the case of zns , using a gap eg ( bulk ) = 3.68 ev , an effective mass for electrons me and holes mh = 0.61 m0(m0 is the electron mass ) and a dielectric constant r = 8.9 , the following relation between the bandgap eg ( r ) , in electron volt , and the particle radius r , in nanometers , is obtained : r was calculated ranges from 1.5 to 2.5 nm ( a diameter from 3 to 5 nm ) , in reasonable agreement with the xrd results . due to the sample synthesized in a / w volume ratio of 1:2
has the highest emission intensity and lowest absorption intensity ( figs . 4 , 5 )
, we can draw a conclusion that in our samples , this one has the best quantum efficiency , then we made surface treatment on this sample to improve its emission intensity and measure the quantum efficiency . that is coated with a layer of zn(oh)2 on the surface of zns : mn . figure 6 is the tem image of zns : mn coated with zn(oh)2 in a / w volume ratio of 1:2 and electron diffraction in the inset ( right ) , while inset ( left ) is the image of bare zns : mn nanoparticle . the results indicate that zns : mn / zn(oh)2 nanoparticles with high distribution , and no aggregation was synthesized successfully . tem shows the presence of zn(oh)2 coating layer on the surface of the zns : mn . it is proven that the zns : mn surface had been modified by zn(oh)2 layer . the quantum efficiency can be calculated by following formula : , where ec is the emission of sample , ea is the emission of integrating sphere , la is the scatter of integrating sphere , and lc is the scatter of sample , and after deducting the error of attenuating plate , the results are shown in table 1 . from table 1 , we find that the absolute quantum efficiency enhanced by 2.3% ( from 8.4% of uncoated sample to 10.7% of coated sample ) . based on the results , it is concluded that zn(oh)2 layer can passivate the surface of bare zns : mn to improve the emission intensity and quantum efficiency . tem image of zns : mn / zn(oh)2 nanocrystals ( inset of left ( a / w = 1:2 ) is bare zns : mn and right is electron diffraction pattern of zns : mn / zn(oh)2 ) the emission spectra of zns : mn and zns : mn / zn(oh)2 nanocrystals the schematic representation of quantum efficiency measurement quantum efficiency results luminescent decay curves and their fitting curves are shown in fig
curves a , b , and c , respectively , represent the samples synthesized under conditions of a / w volume ratio of 1:1 , 1:2 , and coated with a layer of zn(oh)2 with mn - doped concentration of 2% . the xrd patterns of the zns : mn nanocrystals prepared in the condition of alcohol and water volume ratio of 1:1 are presented in fig
. for all the samples , the main three diffraction peak positions correspond to the lattice planes of ( 111 ) , ( 220 ) , and ( 311 ) , matching the cubic zinc blende zns structure ( jcpds no . scherrer formula , the mean crystalline sizes calculated from the full - width at half - maximum ( fwhm ) of these lines were about 3.02 nm . xrd patterns of zns : mn nanocrystals ( a / w = 1:1 ) this paper extends the method employing the a / w template to produce the zns : mn nanocrystals . the nanocrystals distribute in a narrow size ; the shape of the particles is approximately spherical , and the mean diameter is about 35 nm . the nucleus formation mechanism in alcohol and water system is as follows : the reagents are over saturation in a / w template , and the relationship between super saturation and the radius of stable nuclei ( r ) can be expressed by kelvin equation : tem image of zns : mn nanocrystals ( a / w = 1:1 ) the degree of super saturation ( s ) is defined by the ratio of solute concentration ( c ) and saturation concentration ( c1 ) : when the s value is much larger than unity , a great number of primary nuclei formed . for mixed solvents of homologous alcohol and water system , values of a and b
therefore , it is clear that the change in the dielectric constant of mixed solvent can remarkably affect the nucleation rate and the particle size . in addition , for single water system , due to the small particle size there is a big specific surface area , so the products easily aggregate . because alcohol and water can dissolve each other unlimitedly , most of water molecules adsorbing on the particle surface can be replaced by alcohol molecules , which diminish the particle surface tension and surface energy ; thus , the trend of reunion between the particles can be effectively declined . besides , the probability of collisions between particles can be reduced by the steric effect of organic system ( a / w ) , which contributes to the formation of nanoparticles with uniformity and good dispersion . figure 3 illustrates photoluminescence ( pl ) spectra of zns : mn nanocrystals with different mn concentration excited at 300 nm in the room temperature . it is observed that the strong pl position from zns : mn nanocrystals compared with other results redshifts to 605 nm ( fig . the emission spectra of zns : mn nanocrystals for different mn concentrations ( inset is the intensity of emission as a function of mn concentration ) the doped manganese in the precursors may be introduced into inside or outside of zns nanocrystals . they found that whether mn incorporates into the zns lattice will lead to the orange emission , while zns with mn on the surface - bound will yield the ultraviolet emission . narayan pradhan studied the possible nucleation and growth process , they found that the successful doping and decoupling of doping must fulfill following conditions : with the increase in doped ions , the steady increase in the pl intensity from the doping centers at about 605 nm , the fixed pl positions of the host zns nanocrystals and the doping centers , and the gradual decrease in the pl intensity of the host zns nanocrystals . at the same time , the amount of alcohol will affect the degree of agglomeration of nanoparticles . the dielectric constant of an a / w mixed solvent decreases with an increase in the volume ratio of a / w . in this study
, we kept the total a / w volume as a constant then adjusted the alcohol ratio in the template and investigated the effects of the alcohol volume on the pl properties . the emission intensity of zns : mn enhances gradually with the increase in alcohol concentration . because of the integrity of a better particle namely passivated surface defect , the strongest emission could be obtained when the concentration of alcohol reached 33% ( alcohol and water volume ratio of 1:2 , inset of fig . the emission spectra of zns : mn nanocrystals for different a / w volume ratio ( inset is the intensity of emission as a function of alcohol content in template ) according to the results , we draw a conclusion from the process of preparing nanocrystals that the function of a / w mixed system is as follows : at that moment , the hydrogen bonds , hydration , and ethanol water molecules are no longer separate water molecules or ethanol molecules but bridged molecular clusters formed by a number of ethanol molecules and water molecules . from experiment results
, it could be inferred that in the course of obtaining integrated particle crystallization , the second cluster structure is the best template , because our sample with best emission intensity was produced in the condition of the closest approach to the second type molecular structure of alcohol ( 40%).the optimal volume of a / w is 1:2 . optical absorption spectra of zns : mn nanocrystals for different a / w are shown in fig . the bandgap energy of nanocrystals as a function of their size is given by the brus equation , the absorption spectra of zns : mn nanocrystals for different a / w volume ratio in the case of zns , using a gap eg ( bulk ) = 3.68 ev , an effective mass for electrons me and holes mh = 0.61 m0(m0 is the electron mass ) and a dielectric constant r = 8.9 , the following relation between the bandgap eg ( r ) , in electron volt , and the particle radius r , in nanometers , is obtained : r was calculated ranges from 1.5 to 2.5 nm ( a diameter from 3 to 5 nm ) , in reasonable agreement with the xrd results . that is coated with a layer of zn(oh)2 on the surface of zns : mn . figure 6 is the tem image of zns : mn coated with zn(oh)2 in a / w volume ratio of 1:2 and electron diffraction in the inset ( right ) , while inset ( left ) is the image of bare zns : mn nanoparticle . the results indicate that zns : mn / zn(oh)2 nanoparticles with high distribution , and no aggregation was synthesized successfully . tem shows the presence of zn(oh)2 coating layer on the surface of the zns : mn . it is proven that the zns : mn surface had been modified by zn(oh)2 layer . the quantum efficiency can be calculated by following formula : , where ec is the emission of sample , ea is the emission of integrating sphere , la is the scatter of integrating sphere , and lc is the scatter of sample , and after deducting the error of attenuating plate , the results are shown in table 1 . from table 1 , we find that the absolute quantum efficiency enhanced by 2.3% ( from 8.4% of uncoated sample to 10.7% of coated sample ) . based on the results , it is concluded that zn(oh)2 layer can passivate the surface of bare zns : mn to improve the emission intensity and quantum efficiency . tem image of zns : mn / zn(oh)2 nanocrystals ( inset of left ( a / w = 1:2 ) is bare zns : mn and right is electron diffraction pattern of zns : mn / zn(oh)2 ) the emission spectra of zns : mn and zns : mn / zn(oh)2 nanocrystals the schematic representation of quantum efficiency measurement quantum efficiency results luminescent decay curves and their fitting curves are shown in fig . curves a , b , and c , respectively , represent the samples synthesized under conditions of a / w volume ratio of 1:1 , 1:2 , and coated with a layer of zn(oh)2 with mn - doped concentration of 2% . in summary , zns : mn and zns : mn / zn(oh)2 nanocrystals were synthesized by soft template and precipitation reaction method . meanwhile , the emission intensity varies with the ratio of alcohol . when alcohol and water volume ratio is 1:2 , the strongest emission intensity and shortest lifetime decay is achieved , which is attributed to the integrity of better particle getting from structure of alcohol / water template . when zns : mn is coated with zn(oh)2 , the emission intensity of mn enhances by 30% , and its absolute quantum efficiency increases by 2.3% . | [
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maintaining the health of australia 's ageing population is a priority for governments and healthcare providers , not only to improve quality of life but also to minimise the burden of ill health on existing resources . ensuring that older people get the recommended amount of daily physical activity is a key way to improve and maintain health in this important group .
just 40% of australian adults who aged 5574 years achieve the recommended amount of physical activity each week , and after 75 years , the proportion falls to 24% ; however identifying physical activities that are enjoyable and accessible for older people can be challenging .
many older adults prefer to exercise alone or with others of their own age , and more than half of older australians undertake their physical activity in unstructured forms such as walking to local shops .
barriers to exercise for older people can include cost , time , and difficulty , travelling to group programs .
it is nonweight bearing and therefore has less impact on the joints than jogging or other running sports , and several longitudinal epidemiological studies have shown significant risk reduction for all - cause and cancer mortality , cardiovascular disease , colon and breast cancer , and obesity morbidity in middle - aged and elderly cyclists [ 79 ] .
cycling may also contribute to improved quality of life for older people , by enhancing social networks and building empowerment , and can be incorporated easily into a daily routine . despite these benefits , cycling participation by older adults in australia is low .
in contrast to many european cities where older people continue to cycle , in australia just 18% of those who aged over 50 cycled in the past year , compared with 50% of those aged 1029 and 39% of those aged 3049 years . a small number of published studies have investigated the barriers and facilitators of adult cycling in australia , but none have looked specifically at the important and growing population of older people .
these studies found that poor confidence and cycling ability are concerns for inexperienced riders [ 14 , 15 ] .
lack of cycle paths is often cited as a barrier to regular cycling , as is fear of cars and negative attitudes from motorists [ 14 , 16 , 17 ] .
this study investigated the motivators , barriers and facilitators of cycling in older people who participated in a pilot cycling program .
this qualitative study was part of a cycling promotion study among older people , looking at improvements in leg strength and balance as risk factors for falls .
adults who aged 5075 years , who were willing to cycle for two or more hours per week over a 12-week period , were recruited through local advertising and word of mouth in a discrete geographic area of sydney ( canada bay ) , australia .
canada bay is an innerwest suburb of sydney , located around 8 km west of the city .
the area scores 1,076.5 on the seifa index of disadvantage , indicating that it is less disadvantaged that the national average , and the median / average age of the population is 39 years of age , 2 years above the australian average .
the site was chosen for this study to build on existing relationships between one of the researchers ( cm ) and the community centre and bicycle user group in that area .
we aimed to recruit around 20 participants for the pilot study because it was feasible to support this quantity with the resources available , and the researchers felt that around 20 participants would be sufficient for the purposes of this trial .
semistructured interviews were conducted one - on - one before participants attended a cycling skills course and 12-weeks later .
the study 's chief investigator conducted baseline interviews , and follow - up interviews were conducted by another author .
the chief investigator was experienced in qualitative interviewing techniques , is personally within the age bracket for participation in the study , and is an experienced cyclist who rides to work daily .
the second interviewer has less experience in qualitative interviewing techniques , is younger than the study participants , and cycles to work often .
five themes were investigated ; motivators and barriers were the focus of the baseline interview , while the follow - up interview focused on benefits / outcomes , enablers , and cycling promotion .
interviews were audio - recorded but not transcribed , and analysed by an individual researcher using thematic analysis .
interviews were coded and then data linked by codes were collated into potential themes . in this way , the researcher generated a thematic map of the entire analysis .
the person conducting the analysis was younger than the study participants , is female , and cycles to work occasionally .
participants were supported to complete their 2 hr / week of cycling by providing cycling skills training , mentors , and a resource pack .
the program commenced with a cycling skills course to develop participants ' cycling ability , safety knowledge , and confidence , as poor skills and confidence are known barriers to taking up cycling across all ages .
the course was delivered by accredited trainers bikewise , and funded by the city of sydney council ( see http://www.bikewise.com.au/ ) .
the course lasted 4.5 hours and covered basic bicycle control skills ( starting , braking , and turning ) , route planning , and on - road riding skills .
participants were matched with mentors from their local area , who were experienced cyclists and could be contacted either directly or via a google group .
the research team also made brief informal phone calls to participants at one and six weeks to maintain participant engagement and identify any additional support required .
strong social support and encouragement have been shown to enable and motivate people to take up and sustain physical activity , and these interventions targeted these constructs .
participants received a resource pack which included local cycling maps , information about local bicycle user groups , and where and when local group rides were held .
these interventions targeted the potential barrier of participants not knowing where to ride and also encouraged participants and supported social connectedness .
this study received ethical approval on september 7 , 2011 from the university of sydney research integrity human research ethics committee ( protocol no . : 09 - 2011/14065 ) .
seventeen participants were recruited , ranging from 49 to 72 years of age ( mean 61 years ) . the majority ( twelve ) were female .
most ( twelve ) had not ridden in the past year , but fifteen were undertaking some form of physical activity ; however the amount varied widely , ranging from a little bit of walking to one woman running 50 km per week .
two participants withdrew from the program for personal reasons after the baseline interview , and researchers were unable to schedule a suitable time for a followup interview with four participants .
the four participants who were unable to attend a follow - up interview were at the younger end of the age bracket ( more likely to be in their 50s ) and more likely to be working .
as the sample was self - selected , all enrolled participants were highly motivated to start cycling .
the main reason for wanting to cycle was for exercise , and the biggest perceived barrier was fear of cars and traffic .
this study was advertised as a way to potentially improve balance and leg strength , and this should be considered a motivator for people to take part .
all participants cited exercise as a major reason to take up cycling , as many did not like gyms and thought that cycling would be a more fun way to exercise , and would fit more easily into their lifestyle .
if it fits into my lifestyle , then i 'll do it and love it ... but if i have to ... go to the gym ... then i 'm not very good at following through
if it fits into my lifestyle , then i 'll do it and love it ... but if i have to ... go to the gym ... then i 'm not very good at following through ( 59yo female ) .
some participants added that cycling is a form of exercise that they can continue to do now that they are older.we [ my wife and i ] used to run ... but i ca n't run anymore because i damaged one of my knees so i 'm past running , so that 's why we 're thinking well bikes will be good
we [ my wife and i ] used to run ... but i ca n't run anymore because i damaged one of my knees so i 'm past running , so that 's why we 're thinking well bikes will be good
all participants also mentioned enjoyment as a reason to cycle , but this was not as strong a motivator as exercise .
cycling was perceived as a fun way to spend time with family or make new friends .
participants expected to enjoy being outdoors and anticipated the feeling of freedom that cycling brings.i think it 's quite nice because i see my husband going off and it 's like he has a social group and ... they always seem to stop somewhere nice for coffee ... i think that would be quite nice to be in a similar sort of social group ( 67yo female ) .
i think it 's quite nice because i see my husband going off and it 's like he has a social group and ... they always seem to stop somewhere nice for coffee ... i think that would be quite nice to be in a similar sort of social group ( 67yo female ) . joy , exercise , enjoyment , getting out , you know ? getting out and getting about
( 59yo female ) . around a third intended to use the bicycle for local utility trips .
motivators included convenience , exercise , enjoyment , and the environment.i drive to work , i drive to dad , i drive back from dad , i drive to church , i drive ... even to the shops now ... i 'm just driving all the time and i 'm hating it ... i 'd love to ... just jump on my bike to go and get the milk ( 59yo female ) .
i drive to work , i drive to dad , i drive back from dad , i drive to church , i drive ... even to the shops now ... i 'm just driving all the time and i 'm hating it ... i 'd love to ... just jump on my bike to go and get the milk ( 59yo female ) .
several participants had plans to go on a cycling holiday with family members and were building their confidence and fitness.i said to my son ... next year , why do n't you meet me in copenhagen and we can cycle up the east coast ?
i said to my son ... next year , why do n't you meet me in copenhagen and we can cycle up the east coast ?
the primary barrier to cycling for all participants was fear of cars and riding on streets .
most would only consider riding on cycle paths and very quiet roads , and many mentioned friends who had had a
close call while cycling on roads.i've got a lot of friends ... and i ca n't find a single friend who thinks that it 's safe enough for them to be on the road
i 've got a lot of friends ... and i ca n't find a single friend who thinks that it 's safe enough for them to be on the road
many were not confident in their riding ability , mentioning they might be a bit wobbly on the bike to begin with because they had not ridden in years and had never ridden a bicycle with gears .
many were relying on the cycling skills course to practice and to build confidence.i feel very nervous about riding a bike because i do n't feel i can have adequate control over it ... in terms of wobbling where there 's traffic
i feel very nervous about riding a bike because i do n't feel i can have adequate control over it ... in terms of wobbling where there 's traffic
interestingly , participants did not feel particularly concerned about falling off their bikes as older people .
participants were certainly aware that they may fall , but they reported that their fear was no worse than when they were younger . even when asked about being frail or having brittle bones , this group were not particularly concerned with this aspect of cycling .
of these , one had been unable to cycle due to an unrelated injury , and one found she lacked the skill to cycle so she gave up on her 2 hours / week target .
all of the remaining nine participants had met their 2 hr per week target and many had done much more .
most were cycling for recreation and fitness , and just over half had also used the bicycle to travel locally to work , the gym , or local shops and events .
all who had met their target reported a very positive experience from cycling.you enjoy it , you 're getting to work , you 're missing the traffic , it makes you feel more healthy in your mind ... and you end up stronger !
you enjoy it , you 're getting to work , you 're missing the traffic , it makes you feel more healthy in your mind ... and you end up stronger !
overwhelmingly , taking up cycling was a liberating , fun experience , and many participants reported that it felt lovely to be outdoors , exercising in nature .
many recalled cycling in childhood and recaptured a sense of joy and freedom.i loved the wind blowing through your hair , that was lovely ! ... that was the exhilarating part of it ( 71-year - old female ) .
i loved the wind blowing through your hair , that was lovely ! ... that was the exhilarating part of it
this was a distressing realisation for this participant and she expressed disappointment in herself . at baseline , this participant explained that she had fallen from her bike recently and had lost her riding confidence .
she hoped that participating in this program would give her the resources and motivation that she needed to get back on the bicycle .
many participants also enjoyed a benefit to their social life as a result of cycling .
they had a new experience to talk about with friends and inspired others to take up cycling themselves .
in addition , cycling plugged participants into a new community , allowing them to interact with people outside their usual social group .
the best example of this was one local cycling group , where two participants rode on weekends with a father and his two daughters .
participants loved interacting with the two young girls , as well as strengthening their own friendship .
finally , most of the women expressed a strong sense of pride at their cycling achievements and felt empowered by overcoming their fear of cycling and improving their skills , especially as an older person .
friends told them they were an inspiration , which heightened the positive association.some of my friends , they 're amazed that i 've gone out ( well , i am too ! ) ... she said
( 71yo female ) . some of my friends , they 're amazed that i 've gone out ( well , i am too ! ) ... she said
participants were not asked about this feeling specifically ; it came up spontaneously in their interviews .
feeling proud and/or empowerment was not mentioned by any of the male participants in their interviews .
participants reported feeling more energetic , having a better mood , and being able to relax and sleep better after a bike ride .
this improvement in mental health was the most commonly reported health - related benefit of cycling ; however they also reported improved cardiovascular fitness and feeling stronger , especially in the legs .
one individual 's diabetic control had improved so much that he was using less insulin .
the strongest determinants of confidence were bike - handling skills and knowing their intended route .
the majority began cycling in parks , before moving to cycle paths , and eventually back - roads as confidence increased . while finding the confidence to cycle was a struggle for some , the cycling skills course was a hugely positive experience for all participants , and for many it was the main factor in getting them
back on a bike.fantastic ! if it was n't for that , i would n't be cycling now
participants were the happiest when they were grouped with other older riders or riders with a similar level of skill , as they did not feel they were holding the group back and did not feel embarrassed.i did not want to be the duffer in the class
three participants attended the course a second time , and two more asked whether they were allowed to attend again , emphasising the importance of repetition and reinforcement for older riders .
they valued the safety knowledge they gained through the course , such as road positioning and how to approach intersections , and practicing basic skills , which improved their confidence .
many had recommended the course to friends and colleagues and could not speak about it more highly.it's probably the best course i 've ever done in my life ( 66yo male ) .
( 66yo male ) . due to low confidence , participants needed to feel secure about where they were riding . mentors played a big role by either providing cycle maps or showing the way . for example , one participant 's mentor rode with her to work one day , and after that she was able to ride to work a few times a week , always along that same route .
local cycle maps were also highly valued , but one participant could not use the map given because she could not locate her house on it .
participants were impressed with the well - maintained cycling facilities available in local parks , and some had not known such facilities existed until they saw them on the map.well , i was amazed [ when i saw the map ] at the extent of bike paths and cycle ways in sydney ( 71yo female ) .
well , i was amazed [ when i saw the map ] at the extent of bike paths and cycle ways in sydney
i 'm more concerned about traffic than anything , so if anyone in sydney wants to build more cycleways , i think that 's wonderful !
i 'm more concerned about traffic than anything , so if anyone in sydney wants to build more cycleways , i think that 's wonderful !
many participants felt that a media campaign about the benefits of cycling would encourage more older people to cycle , but that campaign would need to be backed - up with more cycle ways .
other suggestions included advertising in seniors magazines and at seniors groups and word of mouth or presentations from other older cyclists .
they felt the messages needed to be that cycling is achievable , fun , and beneficial for health and that it could be done safely . despite this
, participants noted that many older people would never be responsive to this type of encouragement as cycling is perceived as too dangerous and some older people simply will not undertake physical activity .
the strategies used to promote cycling in this study were simple and inexpensive , mainly linking motivated people to existing resources , but had a high success rate for increasing cycling participation .
this work explored motivators , barriers , and outcomes to taking up or increasing cycling and highlighted successful strategies for promoting cycling in older people .
key findings included that cycling promotion for this age group must focus on improving confidence and must consider the need for reinforcement and repetition .
encouragement for bicycle user groups to reach out to older people , widespread availability of cycling maps , and advertising the multiple benefits of cycling are also helpful . continued improvement to cycle paths was strongly supported by participants .
fear of cars and traffic is a strong barrier to cycling across all age groups so investment in infrastructure should also have benefits across the population .
the participant who found she was unable to cycle adds weight to the theory that confidence is extremely important in cycling promotion .
this participant was given additional time , resources , and encouragement ( she was lent a bike to use and strongly encouraged by her mentor ) ; however even with these additional resources she was not able to overcome her fear of falling off her bike again although she persisted in trying .
none of the other participants mentioned previous bad experiences with cycling , indicating that a previous bad experience may well cause a significant loss of confidence which would need additional , tailored strategies to overcome .
another interesting finding is that the sense of empowerment and pride was felt very strongly by female participants but not even mentioned by men .
it is possible that men may be inherently more physically confident and assume that they will be able to ride without problems while for women this is a realisation rather than an assumption .
it could also be that both sexes felt empowered and proud of cycling but that women were more likely to talk about it at interview , especially as the topic was not specifically discussed or explored .
contrary to our own preconceived ideas and that of colleagues who we spoke to informally , the participants themselves did not express concerns about being frail and bicycle riding .
there may be a feeling among the general population that older people need to be physically cautious that is not held by older people themselves , or worse that older people participating in physical activity are being reckless .
if this view is held widely , it may partly explain why older people are not routinely targeted and encouraged in physical activity campaigns , despite the many benefits that physical activity provides in older age .
our study showed that exercise is a strong initial motivator when encouraging older people to cycle , as are sociability and fun , but the best outcomes are empowerment and improved quality of life .
participants preferred to be in a group with others like themselves and some needed the reinforcement and repetition of attending the cycling skills course a second time . following our program ,
the city of sydney has set up a cycling skills course specifically for older people , called rusty riders . having a mentor for support and encouragement and to ride with a participant along a new route was another effective method to support new riders , and for some new riders , group rides were a fun way to experience the social aspect of riding , gain confidence , and learn about where to ride while getting their physical activity . a public health campaign focusing on the health benefits of cycling and showing personal stories of other older cyclists might be effective and is worthy of further research .
local cycle maps were highly valued and might be improved if someone ( perhaps a mentor ) could interpret the map and highlight a route for an older person to take . as with all qualitative work ,
our findings are specific to the older people in our study group and not necessarily representative of the broad older population in sydney .
however , the broad themes identified in this group of older adults appear to be readily applicable to older adults generally .
similarly as with all qualitative work , differences between interviewers may have influenced the way the interviews were conducted and the way participants related to the interviewers .
interview data were analysed by one researcher , which may be seen as a weakness to the study ; however analysis of qualitative data involves interpretation of study findings , and this process is inherently subjective .
it is well accepted in qualitative research that a definitive , objective view of social reality does not exist and different researchers may well interpret the same data differently .
therefore validation by an additional researcher would not necessarily give a correct interpretation , simply another , equally valid one .
the participants who were lost to followup were all contacted multiple times , but the researchers were unable to schedule a time which was convenient for them for their followup interview . on reflection , these participants were more likely to be younger and working , meaning they were probably busy and had less time to devote to the study .
unfortunately , we are unable to say whether these participants had actually increased their cycling behaviour but an area for further research may be investigating the differences for cycling promotion in working , compared with nonworking older adults .
other areas for further research could expand on our finding that empowerment , pride , and external feedback ( admiration from peers ) were very important positive outcomes for female participants but not mentioned by men .
another relevant topic would be looking at the use of electric bikes ( ebikes ) as enablers ( or perhaps deterrents ) to cycling in this age group .
this study suggests that cycling can have a positive influence on the quality of life of older adults , especially through a sense of empowerment and pride , broadening and invigoration of social networks , and simple pleasure .
older people could successfully be encouraged to cycle relatively cheaply and easily by linking them with existing resources in an age - targeted way . while additional cycling infrastructure remains an important goal , in the meantime ,
programs such as the one used here are simple and low - cost to implement .
cycling promotion should centre around improving older people 's cycling confidence through improving skills and practice and repetition , promoting existing cycle paths , increasing safe cycle paths , and promoting cycling to older adults for its health , social , and well - being benefits . |
introduction . cycling can be an enjoyable way to meet physical activity recommendations and is suitable for older people ; however cycling participation by older australians is low .
this qualitative study explored motivators , enablers , and barriers to cycling among older people through an age - targeted cycling promotion program
.
methods .
seventeen adults who aged 5075 years participated in a 12-week cycling promotion program which included a cycling skills course , mentor , and resource pack .
semistructured interviews at the beginning and end of the program explored motivators , enablers , and barriers to cycling .
results . fitness and recreation
were the primary motivators for cycling .
the biggest barrier was fear of cars and traffic , and the cycling skills course was the most important enabler for improving participants ' confidence
. reported outcomes from cycling included improved quality of life ( better mental health , social benefit , and empowerment ) and improved physical health .
conclusions .
a simple cycling program increased cycling participation among older people .
this work confirms the importance of improving confidence in this age group through a skills course , mentors , and maps and highlights additional strategies for promoting cycling , such as ongoing improvement to infrastructure and advertising . | 1. Introduction
2. Methods
3. Ethical Approval
4. Results
5. Discussion
6. Conclusions | maintaining the health of australia 's ageing population is a priority for governments and healthcare providers , not only to improve quality of life but also to minimise the burden of ill health on existing resources . ensuring that older people get the recommended amount of daily physical activity is a key way to improve and maintain health in this important group . just 40% of australian adults who aged 5574 years achieve the recommended amount of physical activity each week , and after 75 years , the proportion falls to 24% ; however identifying physical activities that are enjoyable and accessible for older people can be challenging . many older adults prefer to exercise alone or with others of their own age , and more than half of older australians undertake their physical activity in unstructured forms such as walking to local shops . barriers to exercise for older people can include cost , time , and difficulty , travelling to group programs . it is nonweight bearing and therefore has less impact on the joints than jogging or other running sports , and several longitudinal epidemiological studies have shown significant risk reduction for all - cause and cancer mortality , cardiovascular disease , colon and breast cancer , and obesity morbidity in middle - aged and elderly cyclists [ 79 ] . cycling may also contribute to improved quality of life for older people , by enhancing social networks and building empowerment , and can be incorporated easily into a daily routine . despite these benefits , cycling participation by older adults in australia is low . in contrast to many european cities where older people continue to cycle , in australia just 18% of those who aged over 50 cycled in the past year , compared with 50% of those aged 1029 and 39% of those aged 3049 years . a small number of published studies have investigated the barriers and facilitators of adult cycling in australia , but none have looked specifically at the important and growing population of older people . these studies found that poor confidence and cycling ability are concerns for inexperienced riders [ 14 , 15 ] . lack of cycle paths is often cited as a barrier to regular cycling , as is fear of cars and negative attitudes from motorists [ 14 , 16 , 17 ] . this study investigated the motivators , barriers and facilitators of cycling in older people who participated in a pilot cycling program . this qualitative study was part of a cycling promotion study among older people , looking at improvements in leg strength and balance as risk factors for falls . adults who aged 5075 years , who were willing to cycle for two or more hours per week over a 12-week period , were recruited through local advertising and word of mouth in a discrete geographic area of sydney ( canada bay ) , australia . canada bay is an innerwest suburb of sydney , located around 8 km west of the city . the area scores 1,076.5 on the seifa index of disadvantage , indicating that it is less disadvantaged that the national average , and the median / average age of the population is 39 years of age , 2 years above the australian average . the site was chosen for this study to build on existing relationships between one of the researchers ( cm ) and the community centre and bicycle user group in that area . we aimed to recruit around 20 participants for the pilot study because it was feasible to support this quantity with the resources available , and the researchers felt that around 20 participants would be sufficient for the purposes of this trial . semistructured interviews were conducted one - on - one before participants attended a cycling skills course and 12-weeks later . the study 's chief investigator conducted baseline interviews , and follow - up interviews were conducted by another author . the chief investigator was experienced in qualitative interviewing techniques , is personally within the age bracket for participation in the study , and is an experienced cyclist who rides to work daily . the second interviewer has less experience in qualitative interviewing techniques , is younger than the study participants , and cycles to work often . five themes were investigated ; motivators and barriers were the focus of the baseline interview , while the follow - up interview focused on benefits / outcomes , enablers , and cycling promotion . interviews were audio - recorded but not transcribed , and analysed by an individual researcher using thematic analysis . interviews were coded and then data linked by codes were collated into potential themes . in this way , the researcher generated a thematic map of the entire analysis . the person conducting the analysis was younger than the study participants , is female , and cycles to work occasionally . participants were supported to complete their 2 hr / week of cycling by providing cycling skills training , mentors , and a resource pack . the program commenced with a cycling skills course to develop participants ' cycling ability , safety knowledge , and confidence , as poor skills and confidence are known barriers to taking up cycling across all ages . the course was delivered by accredited trainers bikewise , and funded by the city of sydney council ( see http://www.bikewise.com.au/ ) . the course lasted 4.5 hours and covered basic bicycle control skills ( starting , braking , and turning ) , route planning , and on - road riding skills . strong social support and encouragement have been shown to enable and motivate people to take up and sustain physical activity , and these interventions targeted these constructs . participants received a resource pack which included local cycling maps , information about local bicycle user groups , and where and when local group rides were held . this study received ethical approval on september 7 , 2011 from the university of sydney research integrity human research ethics committee ( protocol no . : 09 - 2011/14065 ) . the majority ( twelve ) were female . most ( twelve ) had not ridden in the past year , but fifteen were undertaking some form of physical activity ; however the amount varied widely , ranging from a little bit of walking to one woman running 50 km per week . two participants withdrew from the program for personal reasons after the baseline interview , and researchers were unable to schedule a suitable time for a followup interview with four participants . the four participants who were unable to attend a follow - up interview were at the younger end of the age bracket ( more likely to be in their 50s ) and more likely to be working . the main reason for wanting to cycle was for exercise , and the biggest perceived barrier was fear of cars and traffic . this study was advertised as a way to potentially improve balance and leg strength , and this should be considered a motivator for people to take part . all participants cited exercise as a major reason to take up cycling , as many did not like gyms and thought that cycling would be a more fun way to exercise , and would fit more easily into their lifestyle . some participants added that cycling is a form of exercise that they can continue to do now that they are older.we [ my wife and i ] used to run ... but i ca n't run anymore because i damaged one of my knees so i 'm past running , so that 's why we 're thinking well bikes will be good
we [ my wife and i ] used to run ... but i ca n't run anymore because i damaged one of my knees so i 'm past running , so that 's why we 're thinking well bikes will be good
all participants also mentioned enjoyment as a reason to cycle , but this was not as strong a motivator as exercise . cycling was perceived as a fun way to spend time with family or make new friends . participants expected to enjoy being outdoors and anticipated the feeling of freedom that cycling brings.i think it 's quite nice because i see my husband going off and it 's like he has a social group and ... they always seem to stop somewhere nice for coffee ... i think that would be quite nice to be in a similar sort of social group ( 67yo female ) . i think it 's quite nice because i see my husband going off and it 's like he has a social group and ... they always seem to stop somewhere nice for coffee ... i think that would be quite nice to be in a similar sort of social group ( 67yo female ) . motivators included convenience , exercise , enjoyment , and the environment.i drive to work , i drive to dad , i drive back from dad , i drive to church , i drive ... even to the shops now ... i 'm just driving all the time and i 'm hating it ... i 'd love to ... just jump on my bike to go and get the milk ( 59yo female ) . several participants had plans to go on a cycling holiday with family members and were building their confidence and fitness.i said to my son ... next year , why do n't you meet me in copenhagen and we can cycle up the east coast ? the primary barrier to cycling for all participants was fear of cars and riding on streets . most would only consider riding on cycle paths and very quiet roads , and many mentioned friends who had had a
close call while cycling on roads.i've got a lot of friends ... and i ca n't find a single friend who thinks that it 's safe enough for them to be on the road
i 've got a lot of friends ... and i ca n't find a single friend who thinks that it 's safe enough for them to be on the road
many were not confident in their riding ability , mentioning they might be a bit wobbly on the bike to begin with because they had not ridden in years and had never ridden a bicycle with gears . many were relying on the cycling skills course to practice and to build confidence.i feel very nervous about riding a bike because i do n't feel i can have adequate control over it ... in terms of wobbling where there 's traffic
i feel very nervous about riding a bike because i do n't feel i can have adequate control over it ... in terms of wobbling where there 's traffic
interestingly , participants did not feel particularly concerned about falling off their bikes as older people . even when asked about being frail or having brittle bones , this group were not particularly concerned with this aspect of cycling . of these , one had been unable to cycle due to an unrelated injury , and one found she lacked the skill to cycle so she gave up on her 2 hours / week target . all of the remaining nine participants had met their 2 hr per week target and many had done much more . most were cycling for recreation and fitness , and just over half had also used the bicycle to travel locally to work , the gym , or local shops and events . all who had met their target reported a very positive experience from cycling.you enjoy it , you 're getting to work , you 're missing the traffic , it makes you feel more healthy in your mind ... and you end up stronger ! you enjoy it , you 're getting to work , you 're missing the traffic , it makes you feel more healthy in your mind ... and you end up stronger ! overwhelmingly , taking up cycling was a liberating , fun experience , and many participants reported that it felt lovely to be outdoors , exercising in nature . many recalled cycling in childhood and recaptured a sense of joy and freedom.i loved the wind blowing through your hair , that was lovely ! ... that was the exhilarating part of it ( 71-year - old female ) . i loved the wind blowing through your hair , that was lovely ! ... that was the exhilarating part of it
this was a distressing realisation for this participant and she expressed disappointment in herself . she hoped that participating in this program would give her the resources and motivation that she needed to get back on the bicycle . finally , most of the women expressed a strong sense of pride at their cycling achievements and felt empowered by overcoming their fear of cycling and improving their skills , especially as an older person . friends told them they were an inspiration , which heightened the positive association.some of my friends , they 're amazed that i 've gone out ( well , i am too ! ) feeling proud and/or empowerment was not mentioned by any of the male participants in their interviews . participants reported feeling more energetic , having a better mood , and being able to relax and sleep better after a bike ride . this improvement in mental health was the most commonly reported health - related benefit of cycling ; however they also reported improved cardiovascular fitness and feeling stronger , especially in the legs . one individual 's diabetic control had improved so much that he was using less insulin . the majority began cycling in parks , before moving to cycle paths , and eventually back - roads as confidence increased . while finding the confidence to cycle was a struggle for some , the cycling skills course was a hugely positive experience for all participants , and for many it was the main factor in getting them
back on a bike.fantastic ! if it was n't for that , i would n't be cycling now
participants were the happiest when they were grouped with other older riders or riders with a similar level of skill , as they did not feel they were holding the group back and did not feel embarrassed.i did not want to be the duffer in the class
three participants attended the course a second time , and two more asked whether they were allowed to attend again , emphasising the importance of repetition and reinforcement for older riders . they valued the safety knowledge they gained through the course , such as road positioning and how to approach intersections , and practicing basic skills , which improved their confidence . many had recommended the course to friends and colleagues and could not speak about it more highly.it's probably the best course i 've ever done in my life ( 66yo male ) . for example , one participant 's mentor rode with her to work one day , and after that she was able to ride to work a few times a week , always along that same route . local cycle maps were also highly valued , but one participant could not use the map given because she could not locate her house on it . participants were impressed with the well - maintained cycling facilities available in local parks , and some had not known such facilities existed until they saw them on the map.well , i was amazed [ when i saw the map ] at the extent of bike paths and cycle ways in sydney ( 71yo female ) . well , i was amazed [ when i saw the map ] at the extent of bike paths and cycle ways in sydney
i 'm more concerned about traffic than anything , so if anyone in sydney wants to build more cycleways , i think that 's wonderful ! i 'm more concerned about traffic than anything , so if anyone in sydney wants to build more cycleways , i think that 's wonderful ! many participants felt that a media campaign about the benefits of cycling would encourage more older people to cycle , but that campaign would need to be backed - up with more cycle ways . they felt the messages needed to be that cycling is achievable , fun , and beneficial for health and that it could be done safely . despite this
, participants noted that many older people would never be responsive to this type of encouragement as cycling is perceived as too dangerous and some older people simply will not undertake physical activity . the strategies used to promote cycling in this study were simple and inexpensive , mainly linking motivated people to existing resources , but had a high success rate for increasing cycling participation . this work explored motivators , barriers , and outcomes to taking up or increasing cycling and highlighted successful strategies for promoting cycling in older people . key findings included that cycling promotion for this age group must focus on improving confidence and must consider the need for reinforcement and repetition . encouragement for bicycle user groups to reach out to older people , widespread availability of cycling maps , and advertising the multiple benefits of cycling are also helpful . continued improvement to cycle paths was strongly supported by participants . fear of cars and traffic is a strong barrier to cycling across all age groups so investment in infrastructure should also have benefits across the population . the participant who found she was unable to cycle adds weight to the theory that confidence is extremely important in cycling promotion . this participant was given additional time , resources , and encouragement ( she was lent a bike to use and strongly encouraged by her mentor ) ; however even with these additional resources she was not able to overcome her fear of falling off her bike again although she persisted in trying . none of the other participants mentioned previous bad experiences with cycling , indicating that a previous bad experience may well cause a significant loss of confidence which would need additional , tailored strategies to overcome . another interesting finding is that the sense of empowerment and pride was felt very strongly by female participants but not even mentioned by men . contrary to our own preconceived ideas and that of colleagues who we spoke to informally , the participants themselves did not express concerns about being frail and bicycle riding . there may be a feeling among the general population that older people need to be physically cautious that is not held by older people themselves , or worse that older people participating in physical activity are being reckless . if this view is held widely , it may partly explain why older people are not routinely targeted and encouraged in physical activity campaigns , despite the many benefits that physical activity provides in older age . our study showed that exercise is a strong initial motivator when encouraging older people to cycle , as are sociability and fun , but the best outcomes are empowerment and improved quality of life . participants preferred to be in a group with others like themselves and some needed the reinforcement and repetition of attending the cycling skills course a second time . following our program ,
the city of sydney has set up a cycling skills course specifically for older people , called rusty riders . having a mentor for support and encouragement and to ride with a participant along a new route was another effective method to support new riders , and for some new riders , group rides were a fun way to experience the social aspect of riding , gain confidence , and learn about where to ride while getting their physical activity . a public health campaign focusing on the health benefits of cycling and showing personal stories of other older cyclists might be effective and is worthy of further research . as with all qualitative work ,
our findings are specific to the older people in our study group and not necessarily representative of the broad older population in sydney . however , the broad themes identified in this group of older adults appear to be readily applicable to older adults generally . similarly as with all qualitative work , differences between interviewers may have influenced the way the interviews were conducted and the way participants related to the interviewers . interview data were analysed by one researcher , which may be seen as a weakness to the study ; however analysis of qualitative data involves interpretation of study findings , and this process is inherently subjective . therefore validation by an additional researcher would not necessarily give a correct interpretation , simply another , equally valid one . unfortunately , we are unable to say whether these participants had actually increased their cycling behaviour but an area for further research may be investigating the differences for cycling promotion in working , compared with nonworking older adults . other areas for further research could expand on our finding that empowerment , pride , and external feedback ( admiration from peers ) were very important positive outcomes for female participants but not mentioned by men . another relevant topic would be looking at the use of electric bikes ( ebikes ) as enablers ( or perhaps deterrents ) to cycling in this age group . this study suggests that cycling can have a positive influence on the quality of life of older adults , especially through a sense of empowerment and pride , broadening and invigoration of social networks , and simple pleasure . older people could successfully be encouraged to cycle relatively cheaply and easily by linking them with existing resources in an age - targeted way . while additional cycling infrastructure remains an important goal , in the meantime ,
programs such as the one used here are simple and low - cost to implement . cycling promotion should centre around improving older people 's cycling confidence through improving skills and practice and repetition , promoting existing cycle paths , increasing safe cycle paths , and promoting cycling to older adults for its health , social , and well - being benefits . | [
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sleep deprivation is defined as a state in which adequate sleep has not been attained ( 19 ) .
acute sleep deprivation has well known effects on mood , cognitive function and rate of perceived exertion . in order to help combat the effects of sleep deprivation , athlete and non - athletes
the international olympic committee recently lifted the partial ban on caffeine , hence making the supplement more attractable to athletes for performance enhancement ( 3 ) .
cook et al . reported increased voluntary workload in professional athletes following caffeine supplementation in a sleep - restricted state ( 7 ) .
reported greater improvement in endurance performance following supplementation with 6 compared to 3 and 9 mg.kg of caffeine ( 9 , 15 ) .
cortisol is considered a catabolic hormone responsible for muscle proteolysis , stimulation of gluconeogenesis , and mobilization of ffas .
it is the major glucocorticoid released from the adrenal cortex and rises in times of stress .
researchers have reported increased cortisol concentrations in a sleep deprived state when compared to baseline ( 18 , 20 ) . regarding testosterone concentrations , the majority of its daily release occurs when an individual is asleep ( 14 ) . a lack of sleep will therefore result in a decrease in testosterone concentration compared to baseline ( 14 ) .
testosterone is considered an anabolic hormone , responsible for several functions including development of secondary sexual characteristics in the young male , development of lean muscle mass , bone density and sex - drive ( 4 ) .
studies ( 2 , 12 ) investigating testosterone concentration in the sleep deprived participant have documented a significant decrease in testosterone concentration when compared to the non - sleep restricted participant . by assessing testosterone and cortisol , the ratio of testosterone to cortisol ( t / c ) can be calculated .
the t / c ratio was originally examined as a means of diagnosing overtraining syndrome ( ots ) in athletes ( 10 ) .
a high ratio reflects an anabolic state , whereas a low ratio reflects a catabolic state .
however , the t / c ratio was found to more accurately reflect a means of assessing current training load rather than as a means of diagnosing ots ( 10 ) .
the current study aims to investigate changes in the t / c ratio induced by sleep deprivation .
documented an increase in testosterone concentration as a result of caffeine supplementation ( 3 ) .
interestingly , the same study also revealed a marked rise in cortisol concentration , thus maintaining a high t / c ratio .
therefore , several factors are at play in determining the effects of sleep deprivation on testosterone and cortisol concentrations .
our hypothesis is that there will be an alteration in the t / c ratio as a result of sleep deprivation and that caffeine supplementation may alter these effects .
inclusion criteria were , healthy male amateur games players as assessed by subjective questionnaire and medical examination , actively participating in a competitive sport on a seasonal basis and aged 1835 yr .
athletes were excluded from the study if they did not participate in competitive games , were female , or if they were deemed unfit to participate medically . in total , 11 athletes were enrolled into the current study .
data in table 1 presents participants age , height , mass , percent body fat and body mass index data .
the trinity college dublin research ethics committee granted ethical approval for the study in may 2015 .
each participant required 4 test sessions ; namely , non - sleep deprived familiarization session , a non - sleep deprived night ( nsd ) with no supplementation , a sleep - deprived night ( sdc ) with caffeine supplementation ( 6mg.kg ) and a sleep deprived night ( sdp ) with placebo supplementation .
each test day was separated by at least one week to facilitate adequate recovery between sessions .
all testing was performed indoors in a gymnasium to minimize environmental factors influencing test data .
the modified hoff test was based on the originally devised hoff test ( 11 ) .
the modified hoff test involves each participant dribbling a size 5 oneills football ( oneills , dublin , ireland ) around a series of cones and hurdles , the total distance covered in 10-min was recorded .
the original test was modified to fit the exercise hall available for the current study .
each participant was followed by a tester to ensure all cones and hurdles were adequately cleared .
testers also replaced any cones or hurdles which were moved while the participant was completing the course .
the second part involves dribbling backwards with the ball until a pair of cones 10-m away was reached .
the third section involves dribbling the ball through three by 1-m wide , 70-cm high hurdles and hopping over them , hurdles was spaced 4-m apart .
the fourth section involves a 2.5-m sprint to a cone and then a 9-m sprint to the next cone .
the fifth section involves dribbling from side to side at an angle around a series of 12 cones .
each circuit equated to 150-m and participants completed as many circuits as possible in the allotted time .
the yo - yo intermittent recovery test level 1 ( yo - yo ir1 ) was used to assess the aerobic fitness of participants ( 13 ) this involved using a cd player to play a copy of the testing procedure .
participants lined up on the first set of cones and were required to complete shuttles from the first to the second set of cones and back in times instructed by the cd .
the area between the first and third set of cones formed a jog / walk area .
participants received a verbal warning when they did not complete a successful shuttle in the allocated time .
a maximum of 4 participants attended on any test day , and to ensure a competitive environment competed in pairs based on performance data collected during their familiarization session .
consequently , each group of 4 participants was sub - divided into 2 sub - groups : sub - group a and sub - group b. for the purpose of this study , acute sleep deprivation was defined as remaining awake , with no sleep , for a period of 24-h prior to commencing testing .
participants arrived in dublin on the night prior to testing and remained at an independent accommodation for the night prior .
a standardized timetable of events to entertain athletes occurred each night ; including video gaming and board games . on each test day , a uniform breakfast of 5 kcal.kg body mass , consisting of cornflakes and low - fat milk was served to participants at 07:00 .
the caffeine ( anhydrous caffeine , sigma aldrich , mo , usa ) or placebo ( lactose bdh , poole , uk ) supplements were pre - measured by the project supervisor , and amounted to 6 mg per kg body mass for each individual .
neither tester nor athlete ( double blinded study design ) knew the content of the randomly assigned capsule .
each individual s mass , denoted by their initials to maintain anonymity , assessed during familiarization , was provided to the project supervisor for capsule preparation .
the placebo ( lactose ) or caffeine supplement was then accurately weighed using a laboratory grade scales and placed into either brown or white capsules at random .
consequently , the project supervisor , an individual not involved in data collection , was the only member of the team with knowledge of the contents of each athlete s assigned capsules .
samples were collected via passive drooling through a 7.4 by 20 mm straw into a clean dry pre - labeled 1.5ml eppendorf tube . the first saliva sample ( t1 ) was collected before ingestion of the caffeine or placebo supplement on sleep deprived nights ( sdc and sdp ) . on the non - sleep
the second saliva sample ( t2 ) was collected just before the modified hoff aerobic test , following a low - intensity 10 min warm - up . following termination of the hoff test another saliva sample ( t3 )
the 4 and final sample ( t4 ) was collected immediately following the yo - yo aerobic test , see table 2 . all samples ( t1 to t4 )
were immediately placed in an on - site pre - cooled icebox maintained at 23c during testing .
once testing was completed the collected samples were immediately transported to an off - site freezer , and stored frozen ( 20c ) for batch analysis .
samples were assayed for cortisol and testosterone concentrations using highly sensitive enzyme linked immunoassay ( elisa ) kits ( salimetrics , pa , usa ) .
the cortisol assay had a lower limit sensitivity of 0.007 g.dl and mean intra- and inter - cv of 6.8 and 15% , respectively .
the testosterone assay had a lower limit sensitivity of 3.7 pg.ml , range of 3.7 to 360 pg.ml and mean intra- and inter - cv of 6.5 and less than 15% , respectively .
for each elisa plate , the mean optical density ( mod ) was computed from duplicate wells .
in line with manufacturer guidelines nsb data were subtracted from the mod of , standards , controls and unknowns .
the percent bound ( b1/b0 ) for each standard , control and unknown , was computed by multiplying mod by the inverse optical density for zero ( 1/b0 ) .
the concentration of controls and their equivalent b1/b0 data were entered into a graphical interpolation package . for each elisa plate , a graph of log control concentration versus b / b0 was constructed .
a 3 order polynomial was fitted to the data and the line of best fit calculated for each elisa plate .
the resultant cubic equations were used to assess concentrations of cortisol and testosterone of the unknown samples on each plate .
initially an a priori power analysis was conducted for expected outcomes with a type 1 error probability of 0.05 and a power of 0.8 , this analysis indicated that n=8 would provide a statistical power of 82% ( g*power v3.0.10 ) . to avoid discrepancies between plate readings , each participant
s saliva from their three test sessions were analyzed in duplicate on an elisa plate .
each plate , for testosterone and cortisol , could accommodate three test sessions for two participants in duplicate with 4 wells remaining for additional test standards .
any unexpectedly high or low assays were subsequently re - assayed on an additional plate .
a two - way ( time by test ) repeated measures anova was utilized for statistical comparison of hormonal data .
a single factor repeated measures anova was utilized to compare performance data ( distance completed in hoff and yo - yo tests ) and area under the curve ( auc ) data for cortisol and testosterone across tests .
post - hoc bonferroni test quantified detected significant differences and p < 0.05 inferred statistical significance .
inclusion criteria were , healthy male amateur games players as assessed by subjective questionnaire and medical examination , actively participating in a competitive sport on a seasonal basis and aged 1835 yr .
athletes were excluded from the study if they did not participate in competitive games , were female , or if they were deemed unfit to participate medically . in total , 11 athletes were enrolled into the current study .
data in table 1 presents participants age , height , mass , percent body fat and body mass index data .
the trinity college dublin research ethics committee granted ethical approval for the study in may 2015 .
each participant required 4 test sessions ; namely , non - sleep deprived familiarization session , a non - sleep deprived night ( nsd ) with no supplementation , a sleep - deprived night ( sdc ) with caffeine supplementation ( 6mg.kg ) and a sleep deprived night ( sdp ) with placebo supplementation .
each test day was separated by at least one week to facilitate adequate recovery between sessions .
all testing was performed indoors in a gymnasium to minimize environmental factors influencing test data .
the modified hoff test was based on the originally devised hoff test ( 11 ) .
the modified hoff test involves each participant dribbling a size 5 oneills football ( oneills , dublin , ireland ) around a series of cones and hurdles , the total distance covered in 10-min was recorded .
the original test was modified to fit the exercise hall available for the current study .
each participant was followed by a tester to ensure all cones and hurdles were adequately cleared .
testers also replaced any cones or hurdles which were moved while the participant was completing the course .
the second part involves dribbling backwards with the ball until a pair of cones 10-m away was reached .
the third section involves dribbling the ball through three by 1-m wide , 70-cm high hurdles and hopping over them , hurdles was spaced 4-m apart .
the fourth section involves a 2.5-m sprint to a cone and then a 9-m sprint to the next cone .
the fifth section involves dribbling from side to side at an angle around a series of 12 cones .
each circuit equated to 150-m and participants completed as many circuits as possible in the allotted time .
the yo - yo intermittent recovery test level 1 ( yo - yo ir1 ) was used to assess the aerobic fitness of participants ( 13 ) this involved using a cd player to play a copy of the testing procedure .
participants lined up on the first set of cones and were required to complete shuttles from the first to the second set of cones and back in times instructed by the cd .
the area between the first and third set of cones formed a jog / walk area .
participants received a verbal warning when they did not complete a successful shuttle in the allocated time .
a maximum of 4 participants attended on any test day , and to ensure a competitive environment competed in pairs based on performance data collected during their familiarization session .
consequently , each group of 4 participants was sub - divided into 2 sub - groups : sub - group a and sub - group b. for the purpose of this study , acute sleep deprivation was defined as remaining awake , with no sleep , for a period of 24-h prior to commencing testing .
participants arrived in dublin on the night prior to testing and remained at an independent accommodation for the night prior .
a standardized timetable of events to entertain athletes occurred each night ; including video gaming and board games . on each test day , a uniform breakfast of 5 kcal.kg body mass , consisting of cornflakes and low - fat milk was served to participants at 07:00 .
the caffeine ( anhydrous caffeine , sigma aldrich , mo , usa ) or placebo ( lactose bdh , poole , uk ) supplements were pre - measured by the project supervisor , and amounted to 6 mg per kg body mass for each individual .
neither tester nor athlete ( double blinded study design ) knew the content of the randomly assigned capsule .
each individual s mass , denoted by their initials to maintain anonymity , assessed during familiarization , was provided to the project supervisor for capsule preparation .
the placebo ( lactose ) or caffeine supplement was then accurately weighed using a laboratory grade scales and placed into either brown or white capsules at random .
consequently , the project supervisor , an individual not involved in data collection , was the only member of the team with knowledge of the contents of each athlete s assigned capsules .
samples were collected via passive drooling through a 7.4 by 20 mm straw into a clean dry pre - labeled 1.5ml eppendorf tube . the first saliva sample ( t1 )
was collected before ingestion of the caffeine or placebo supplement on sleep deprived nights ( sdc and sdp ) . on the non - sleep
the second saliva sample ( t2 ) was collected just before the modified hoff aerobic test , following a low - intensity 10 min warm - up . following termination of the hoff test another saliva sample ( t3 )
the 4 and final sample ( t4 ) was collected immediately following the yo - yo aerobic test , see table 2 . all samples ( t1 to t4 )
were immediately placed in an on - site pre - cooled icebox maintained at 23c during testing .
once testing was completed the collected samples were immediately transported to an off - site freezer , and stored frozen ( 20c ) for batch analysis .
samples were assayed for cortisol and testosterone concentrations using highly sensitive enzyme linked immunoassay ( elisa ) kits ( salimetrics , pa , usa ) .
the cortisol assay had a lower limit sensitivity of 0.007 g.dl and mean intra- and inter - cv of 6.8 and 15% , respectively .
the testosterone assay had a lower limit sensitivity of 3.7 pg.ml , range of 3.7 to 360 pg.ml and mean intra- and inter - cv of 6.5 and less than 15% , respectively .
for each elisa plate , the mean optical density ( mod ) was computed from duplicate wells .
in line with manufacturer guidelines nsb data were subtracted from the mod of , standards , controls and unknowns .
the percent bound ( b1/b0 ) for each standard , control and unknown , was computed by multiplying mod by the inverse optical density for zero ( 1/b0 ) .
the concentration of controls and their equivalent b1/b0 data were entered into a graphical interpolation package . for each elisa plate , a graph of log control concentration versus b / b0 was constructed .
a 3 order polynomial was fitted to the data and the line of best fit calculated for each elisa plate .
the resultant cubic equations were used to assess concentrations of cortisol and testosterone of the unknown samples on each plate .
initially an a priori power analysis was conducted for expected outcomes with a type 1 error probability of 0.05 and a power of 0.8 , this analysis indicated that n=8 would provide a statistical power of 82% ( g*power v3.0.10 ) . to avoid discrepancies between plate readings , each participant
s saliva from their three test sessions were analyzed in duplicate on an elisa plate .
each plate , for testosterone and cortisol , could accommodate three test sessions for two participants in duplicate with 4 wells remaining for additional test standards .
any unexpectedly high or low assays were subsequently re - assayed on an additional plate .
a two - way ( time by test ) repeated measures anova was utilized for statistical comparison of hormonal data .
a single factor repeated measures anova was utilized to compare performance data ( distance completed in hoff and yo - yo tests ) and area under the curve ( auc ) data for cortisol and testosterone across tests .
post - hoc bonferroni test quantified detected significant differences and p < 0.05 inferred statistical significance .
of the 11 participants involved , 2 were excluded due to inadequate quantities of saliva collected at designated time - points .
t1 infers pre - supplementation , t2 infers pre - hoff , t3 infers post - hoff and t4 infers post - yo - yo .
globally , no significant changes were detected comparing non - sleep restricted , with sleep restricted following caffeine or placebo supplementation .
however , a significant time effect was detected using post - hoc testing in sdp , see figure 1 .
salivary cortisol concentrations were significantly lower comparing pre- and post - hoff with post - yo - yo data ( t2 vs. t4 ; p < 0.05 and t3 vs. t4 ; p < 0.01 ) .
there were no significant differences detected in testosterone concentration recorded at any time - point comparing the investigated sleep states .
in addition , unlike data recorded for cortisol concentration within a test session , no significant differences were detected across time within any investigated session .
there was an overall significant time effect detected at t3 ( p < 0.05 ) .
post - hoc analysis revealed significant time effects comparing t1 and t3 and comparing t3 and t4 in the nsd state ( p < 0.05 ) . within discrete time - points post - hoc analysis revealed a significant intervention effect comparing nsd and sdc at time - point t3 ( p < 0.05 ) .
the nsd state exhibited the highest testosterone to cortisol ( t / c ) ratio across the assessed conditions .
the t / c ratio rose in the nsd state from t1 to t3 , respectively , and fell to its lowest at t4 .
the sdp state exhibited a similar t / c response , showing a rise from t2 to t3 and a drop from t3 to t4 .
in contrast to the nsd state , there was a decrease in t / c ratio from t1 to t2 recorded in sdp .
the sdc state exhibited a near linear decrease in t / c ratio from t1 to t4 .
comparison of computed auc data for assayed hormones failed to detect significant differences across states , ( cortisol , f=1.21 , p > 0.05 ; testosterone , f=0.70 , p > 0.05 ) .
in addition , analysis of distance data completed in the hoff and yo - yo aerobic tests failed to detect significant differences ( p > 0.05 ) across assessed states .
changes in mean cortisol concentration between the 3 different assessed states did not attain statistical significance for the population involved in the current study .
however , there was a significant time difference detected in the sdp state ( t3 vs. t4 ) , see figure 1 .
maximal mean cortisol concentrations were recorded in all groups at t4 , following the yo - yo aerobic test .
the highest cortisol concentrations were recorded in sdp with the sdc state demonstrating intermediate data , see figure 1 .
the modified hoff test is a 10-min aerobic test involving careful and skillful control of a football around a fixed circuit .
as skills execution was a limiting factor participants did not attain a state of true volitional exhaustion .
this was highlighted by mean post - test lactate data ( 9.3 0.6 , 8.1 0.8 and 8.9 0.8 mmol .
no significant differences were recorded in hoff distance completed across assessed states ( 1138 18 , 1143 14 and 1115 14 m for nsd , sdp and sdc , respectively ) .
although participants did not attain volitional exhaustion during this test , there were noticeable increases in cortisol data recorded at t3 .
cortisol concentration at t3 was lowest in nsd , followed by sdc , with the highest concentration recorded for sdp , see figure 1 .
the final time - point , t4 , exhibited the highest cortisol concentrations in all participants , regardless of assessed state .
participants at this time - point had just performed a maximal volitional aerobic exercise test ( yo - yo test ) .
the increase in cortisol would have been expected in all assessed states resultant from maximal exercise . in similarity with the hoff test
, no significant differences were detected in yo - yo distance completed across assessed states ( 1295 6 , 1325 7 and 1315 8 m for nsd , sdp and sdc , respectively ) . nevertheless , the increases in cortisol concentration from baseline were most pronounced in sdp and sdc when compared to nsd .
the addition of sleep deprivation resulted in a higher cortisol concentration at all time - points . from the results attained
if the athlete was sleep deprived and exercised , they would exhibit a higher cortisol response .
furthermore , in the sleep - deprived states , participants who ingested caffeine ( sdc ) did not exhibit as extensive a cortisol increase as placebo ( sdp ) .
therefore one could postulate that caffeine had a mild blunting effect on the endogenous cortisol response . in the current study
, caffeine appeared to blunt the aerobic - exercise - induced increase in cortisol concentration following sleep deprivation .
as depicted in figure 2 , no statistically significant differences were detected across sleep states for testosterone .
furthermore , unlike data recorded for cortisol within a test session , no significant differences were detected across time within any investigated session .
maximal testosterone concentrations were recorded in the nsd state , this was evident across time - points t1t4 .
this is in keeping with the expected results where sleep restriction or sleep fragmentation have been reported to negatively affect testosterone concentration ( 2 , 12 ) .
testosterone is thought to peak prior to rem sleep ; therefore , lack of sleep will result in a decrease in total testosterone .
mean testosterone concentrations across the sleep - deprived states were equivocal for the first three time - points : t1 , t2 and t3 , see figure 2 .
maximal data were recorded at t1 for the sleep deprived stated and were highest at t3 for the nsd state .
a high testosterone concentration for both sleep deprived states relative to the other 3 time - points may be expected due to the diurnal variation of the hormone in question ( peaking in the am ) , as there was a dip in testosterone concentration across time from t1 ( pre - supplementation ) to t2 ( the pre - exercise testing ) .
depicted an increase in testosterone concentration following intermittent sprinting in sleep restricted athletes ( 1 ) .
this is also seen in the sleep - deprived states transitioning from t2 to t3 . at this stage
, there was a measurable increase in mean testosterone in the sleep - restricted states , also evident in the non - sleep restricted state .
this increase in testosterone concentration is potentially due to the non - exhausting exercise evoked in the hoff test .
it can be postulated therefore that when athletes undergo intermittent high - intensity ( non - exhausting ) aerobic exercise that an increase in testosterone concentration was expected .
reported overall increased testosterone concentrations in young male athletes when performing intermittent high - intensity exercise bouts involving indoor cycling ( 16 ) .
testosterone has also been shown to increase linearly in response to exercise , reaching a peak once activity has ceased ( 21 ) .
cadouxhudson et al . believed that the increase was secondary to decreased testosterone clearance ( 6 ) ; others ( 11 ) have postulated increased testosterone secretion as a direct result of catecholamine stimulation .
the testosterone concentration at t4 did not reflect the same rise in testosterone concentration portrayed at t3 . at t4
there was a decrease in testosterone concentration evident for sdp and also nsd states , see figure 2 .
the yo - yo test induced a greater decline in testosterone concentration for sdp when compared to sdc at t4 .
it would seem from these results that caffeine ingestion helped to offset the decrease in testosterone expected post - exhausting aerobic exercise .
the smaller decrease recorded in testosterone concentration in sdc was in concordance with the literature .
wu ( 22 ) documented that , following caffeine supplementation ( up to 6 mg.kg ) , testosterone and cortisol concentrations increased significantly .
this direct increase in testosterone as a result of caffeine ingestion also seems to have an effect in the sleep - deprived athlete . in this sleep - deprived state ( sdc ) , individuals following caffeine ingestion exhibited an intermediate concentration when compared to nsd ( highest concentration ) and sdp ( lowest concentration ) , see figure 2 .
interestingly , sdc was the only sleep state that recorded an increase in mean testosterone concentration from t3 to t4 . from this data
one can infer that the caffeine supplementation provided a more pronounced effect on testosterone metabolism than the exhausting yo - yo aerobic test .
illustrating an increase in testosterone concentration after high dose caffeine ingestion ( 800 mg ) in relation to anaerobic exercise ( 3 ) .
as testosterone is a potent stimulator for lean muscle formation ( 4 ) , supplementation with caffeine to promote testosterone secretion would be of benefit for athletes .
improved muscle growth and recovery would enable athletes to recover quicker and ensure a more prompt return to training in a repaired / recovered state .
overall there was a significant time difference detected when comparing nsd and sdc states ( p < 0.05 ) . from figure 3
, one can see that the t / c ratio was highest at all time points in the nsd state and lowest in the sdc state .
this is accordance with figures 1 and 2 , respectively , which depict the nsd state as having the lowest cortisol concentrations across all 4 time points and also depict the nsd state as having double the testosterone concentrations at t3 and t4 when compared to sdc state . as described previously a heightened testosterone concentration in the nsd state would be expected .
although it appears that caffeine may play a role in offsetting central stress responses , the overall picture points to a decrease in the t / c ratio .
this can be accounted for by a more exaggerated decrease in testosterone in sdc compared to nsd .
furthermore , increased cortisol concentrations which were not evident at the t3 in the nsd state were evident in the sdc state .
depicting an overall decrease in t / c in caffeinated rugby players ( supplemented with 800 mg.kg ) due to an overall exaggerated increase in cortisol , despite having an initial increase in testosterone ( 3 ) .
this exaggerated increase in one hormone with respect to the other can also explain the intervention - difference noted at t3 between nsd and sdc states .
the significant time effect recorded in nsd athletes between t1 and t3 and between t3 and t4 can be explained by examining figures 1 and 2 .
overall there were no significant differences detected comparing the sleep - state investigated for performance data , testosterone or cortisol concentrations or t / c ratio .
potential reasons for not attaining statistical significance include a small end population size and possible skewing of data by individual participants .
a trend did exist whereby the sdc state mirrored the nsd state for mean cortisol concentration .
an increase in overall mean testosterone concentration post - aerobic exercise was only observed in the sdc state , a potentially beneficial effect of caffeine supplementation .
overall further studies are required to establish whether caffeine has a significant effect on both testosterone and cortisol in the sleep deprived athlete .
this may be achieved with a larger population size , venous sampling and comparison with baseline circadian variation . | caffeine has become a popular ergogenic aid amongst athletes and usage to improve athletic performance has been well documented .
the effect of caffeine on anabolic and catabolic hormones in a sleep - deprived s tate has had little investigation to date .
the purpose of the current study was to investigate the potential of caffeine to offset the effects , if any , of short - term sleep deprivation and exercise on an athlete s testosterone and cortisol concentrations via salivary technique .
eleven competitive male athletes volunteered to be part of this prospective double - blinded study .
three test days were scheduled for each athlete ; one non - sleep deprived , one sleep - deprived with caffeine supplementation ( 6 mg.kg1 ) and one sleep - deprived with placebo ingestion .
sleep deprivation was defined as 24-h without sleep .
each test day was composed of 2 aerobic components : a modified hoff test and a yo - yo test . testosterone and cortisol concentrations were measured via salivary analysis at 4 different time - points ; t1 to t4 , representing baseline , and pre- and post - aerobic components , respectively .
overall no significant differences were detected comparing the different sleep states for testosterone or cortisol concentrations .
a trend existed whereby the sleep - deprived with caffeine ingestion state mirrored the non - sleep deprived state for cortisol concentration .
therefore , caffeine supplementation may have potential benefits for athletes during short - term aerobic exercise when sleep - deprived .
an increase in mean testosterone concentration post - aerobic exercise was only observed in the sleep - deprived with caffeine ingestion state . | INTRODUCTION
METHODS
Participants
Protocol
Statistical Analysis
RESULTS
DISCUSSION | sleep deprivation is defined as a state in which adequate sleep has not been attained ( 19 ) . acute sleep deprivation has well known effects on mood , cognitive function and rate of perceived exertion . in order to help combat the effects of sleep deprivation , athlete and non - athletes
the international olympic committee recently lifted the partial ban on caffeine , hence making the supplement more attractable to athletes for performance enhancement ( 3 ) . reported increased voluntary workload in professional athletes following caffeine supplementation in a sleep - restricted state ( 7 ) . reported greater improvement in endurance performance following supplementation with 6 compared to 3 and 9 mg.kg of caffeine ( 9 , 15 ) . cortisol is considered a catabolic hormone responsible for muscle proteolysis , stimulation of gluconeogenesis , and mobilization of ffas . researchers have reported increased cortisol concentrations in a sleep deprived state when compared to baseline ( 18 , 20 ) . a lack of sleep will therefore result in a decrease in testosterone concentration compared to baseline ( 14 ) . studies ( 2 , 12 ) investigating testosterone concentration in the sleep deprived participant have documented a significant decrease in testosterone concentration when compared to the non - sleep restricted participant . by assessing testosterone and cortisol , the ratio of testosterone to cortisol ( t / c ) can be calculated . the current study aims to investigate changes in the t / c ratio induced by sleep deprivation . documented an increase in testosterone concentration as a result of caffeine supplementation ( 3 ) . interestingly , the same study also revealed a marked rise in cortisol concentration , thus maintaining a high t / c ratio . therefore , several factors are at play in determining the effects of sleep deprivation on testosterone and cortisol concentrations . our hypothesis is that there will be an alteration in the t / c ratio as a result of sleep deprivation and that caffeine supplementation may alter these effects . inclusion criteria were , healthy male amateur games players as assessed by subjective questionnaire and medical examination , actively participating in a competitive sport on a seasonal basis and aged 1835 yr . in total , 11 athletes were enrolled into the current study . each participant required 4 test sessions ; namely , non - sleep deprived familiarization session , a non - sleep deprived night ( nsd ) with no supplementation , a sleep - deprived night ( sdc ) with caffeine supplementation ( 6mg.kg ) and a sleep deprived night ( sdp ) with placebo supplementation . each test day was separated by at least one week to facilitate adequate recovery between sessions . all testing was performed indoors in a gymnasium to minimize environmental factors influencing test data . the modified hoff test was based on the originally devised hoff test ( 11 ) . the modified hoff test involves each participant dribbling a size 5 oneills football ( oneills , dublin , ireland ) around a series of cones and hurdles , the total distance covered in 10-min was recorded . the original test was modified to fit the exercise hall available for the current study . each circuit equated to 150-m and participants completed as many circuits as possible in the allotted time . the yo - yo intermittent recovery test level 1 ( yo - yo ir1 ) was used to assess the aerobic fitness of participants ( 13 ) this involved using a cd player to play a copy of the testing procedure . participants received a verbal warning when they did not complete a successful shuttle in the allocated time . a maximum of 4 participants attended on any test day , and to ensure a competitive environment competed in pairs based on performance data collected during their familiarization session . consequently , each group of 4 participants was sub - divided into 2 sub - groups : sub - group a and sub - group b. for the purpose of this study , acute sleep deprivation was defined as remaining awake , with no sleep , for a period of 24-h prior to commencing testing . on each test day , a uniform breakfast of 5 kcal.kg body mass , consisting of cornflakes and low - fat milk was served to participants at 07:00 . the caffeine ( anhydrous caffeine , sigma aldrich , mo , usa ) or placebo ( lactose bdh , poole , uk ) supplements were pre - measured by the project supervisor , and amounted to 6 mg per kg body mass for each individual . neither tester nor athlete ( double blinded study design ) knew the content of the randomly assigned capsule . consequently , the project supervisor , an individual not involved in data collection , was the only member of the team with knowledge of the contents of each athlete s assigned capsules . the first saliva sample ( t1 ) was collected before ingestion of the caffeine or placebo supplement on sleep deprived nights ( sdc and sdp ) . on the non - sleep
the second saliva sample ( t2 ) was collected just before the modified hoff aerobic test , following a low - intensity 10 min warm - up . following termination of the hoff test another saliva sample ( t3 )
the 4 and final sample ( t4 ) was collected immediately following the yo - yo aerobic test , see table 2 . all samples ( t1 to t4 )
were immediately placed in an on - site pre - cooled icebox maintained at 23c during testing . once testing was completed the collected samples were immediately transported to an off - site freezer , and stored frozen ( 20c ) for batch analysis . samples were assayed for cortisol and testosterone concentrations using highly sensitive enzyme linked immunoassay ( elisa ) kits ( salimetrics , pa , usa ) . the cortisol assay had a lower limit sensitivity of 0.007 g.dl and mean intra- and inter - cv of 6.8 and 15% , respectively . the testosterone assay had a lower limit sensitivity of 3.7 pg.ml , range of 3.7 to 360 pg.ml and mean intra- and inter - cv of 6.5 and less than 15% , respectively . for each elisa plate , the mean optical density ( mod ) was computed from duplicate wells . the percent bound ( b1/b0 ) for each standard , control and unknown , was computed by multiplying mod by the inverse optical density for zero ( 1/b0 ) . for each elisa plate , a graph of log control concentration versus b / b0 was constructed . to avoid discrepancies between plate readings , each participant
s saliva from their three test sessions were analyzed in duplicate on an elisa plate . each plate , for testosterone and cortisol , could accommodate three test sessions for two participants in duplicate with 4 wells remaining for additional test standards . a single factor repeated measures anova was utilized to compare performance data ( distance completed in hoff and yo - yo tests ) and area under the curve ( auc ) data for cortisol and testosterone across tests . post - hoc bonferroni test quantified detected significant differences and p < 0.05 inferred statistical significance . in total , 11 athletes were enrolled into the current study . each participant required 4 test sessions ; namely , non - sleep deprived familiarization session , a non - sleep deprived night ( nsd ) with no supplementation , a sleep - deprived night ( sdc ) with caffeine supplementation ( 6mg.kg ) and a sleep deprived night ( sdp ) with placebo supplementation . each test day was separated by at least one week to facilitate adequate recovery between sessions . all testing was performed indoors in a gymnasium to minimize environmental factors influencing test data . the modified hoff test was based on the originally devised hoff test ( 11 ) . the modified hoff test involves each participant dribbling a size 5 oneills football ( oneills , dublin , ireland ) around a series of cones and hurdles , the total distance covered in 10-min was recorded . the original test was modified to fit the exercise hall available for the current study . the yo - yo intermittent recovery test level 1 ( yo - yo ir1 ) was used to assess the aerobic fitness of participants ( 13 ) this involved using a cd player to play a copy of the testing procedure . a maximum of 4 participants attended on any test day , and to ensure a competitive environment competed in pairs based on performance data collected during their familiarization session . consequently , each group of 4 participants was sub - divided into 2 sub - groups : sub - group a and sub - group b. for the purpose of this study , acute sleep deprivation was defined as remaining awake , with no sleep , for a period of 24-h prior to commencing testing . on each test day , a uniform breakfast of 5 kcal.kg body mass , consisting of cornflakes and low - fat milk was served to participants at 07:00 . the caffeine ( anhydrous caffeine , sigma aldrich , mo , usa ) or placebo ( lactose bdh , poole , uk ) supplements were pre - measured by the project supervisor , and amounted to 6 mg per kg body mass for each individual . neither tester nor athlete ( double blinded study design ) knew the content of the randomly assigned capsule . consequently , the project supervisor , an individual not involved in data collection , was the only member of the team with knowledge of the contents of each athlete s assigned capsules . the first saliva sample ( t1 )
was collected before ingestion of the caffeine or placebo supplement on sleep deprived nights ( sdc and sdp ) . on the non - sleep
the second saliva sample ( t2 ) was collected just before the modified hoff aerobic test , following a low - intensity 10 min warm - up . following termination of the hoff test another saliva sample ( t3 )
the 4 and final sample ( t4 ) was collected immediately following the yo - yo aerobic test , see table 2 . all samples ( t1 to t4 )
were immediately placed in an on - site pre - cooled icebox maintained at 23c during testing . the cortisol assay had a lower limit sensitivity of 0.007 g.dl and mean intra- and inter - cv of 6.8 and 15% , respectively . the testosterone assay had a lower limit sensitivity of 3.7 pg.ml , range of 3.7 to 360 pg.ml and mean intra- and inter - cv of 6.5 and less than 15% , respectively . for each elisa plate , the mean optical density ( mod ) was computed from duplicate wells . the percent bound ( b1/b0 ) for each standard , control and unknown , was computed by multiplying mod by the inverse optical density for zero ( 1/b0 ) . for each elisa plate , a graph of log control concentration versus b / b0 was constructed . initially an a priori power analysis was conducted for expected outcomes with a type 1 error probability of 0.05 and a power of 0.8 , this analysis indicated that n=8 would provide a statistical power of 82% ( g*power v3.0.10 ) . to avoid discrepancies between plate readings , each participant
s saliva from their three test sessions were analyzed in duplicate on an elisa plate . each plate , for testosterone and cortisol , could accommodate three test sessions for two participants in duplicate with 4 wells remaining for additional test standards . a single factor repeated measures anova was utilized to compare performance data ( distance completed in hoff and yo - yo tests ) and area under the curve ( auc ) data for cortisol and testosterone across tests . post - hoc bonferroni test quantified detected significant differences and p < 0.05 inferred statistical significance . of the 11 participants involved , 2 were excluded due to inadequate quantities of saliva collected at designated time - points . t1 infers pre - supplementation , t2 infers pre - hoff , t3 infers post - hoff and t4 infers post - yo - yo . globally , no significant changes were detected comparing non - sleep restricted , with sleep restricted following caffeine or placebo supplementation . however , a significant time effect was detected using post - hoc testing in sdp , see figure 1 . salivary cortisol concentrations were significantly lower comparing pre- and post - hoff with post - yo - yo data ( t2 vs. t4 ; p < 0.05 and t3 vs. t4 ; p < 0.01 ) . there were no significant differences detected in testosterone concentration recorded at any time - point comparing the investigated sleep states . in addition , unlike data recorded for cortisol concentration within a test session , no significant differences were detected across time within any investigated session . post - hoc analysis revealed significant time effects comparing t1 and t3 and comparing t3 and t4 in the nsd state ( p < 0.05 ) . within discrete time - points post - hoc analysis revealed a significant intervention effect comparing nsd and sdc at time - point t3 ( p < 0.05 ) . the t / c ratio rose in the nsd state from t1 to t3 , respectively , and fell to its lowest at t4 . the sdp state exhibited a similar t / c response , showing a rise from t2 to t3 and a drop from t3 to t4 . in contrast to the nsd state , there was a decrease in t / c ratio from t1 to t2 recorded in sdp . the sdc state exhibited a near linear decrease in t / c ratio from t1 to t4 . in addition , analysis of distance data completed in the hoff and yo - yo aerobic tests failed to detect significant differences ( p > 0.05 ) across assessed states . changes in mean cortisol concentration between the 3 different assessed states did not attain statistical significance for the population involved in the current study . however , there was a significant time difference detected in the sdp state ( t3 vs. t4 ) , see figure 1 . maximal mean cortisol concentrations were recorded in all groups at t4 , following the yo - yo aerobic test . the highest cortisol concentrations were recorded in sdp with the sdc state demonstrating intermediate data , see figure 1 . the modified hoff test is a 10-min aerobic test involving careful and skillful control of a football around a fixed circuit . this was highlighted by mean post - test lactate data ( 9.3 0.6 , 8.1 0.8 and 8.9 0.8 mmol . no significant differences were recorded in hoff distance completed across assessed states ( 1138 18 , 1143 14 and 1115 14 m for nsd , sdp and sdc , respectively ) . the final time - point , t4 , exhibited the highest cortisol concentrations in all participants , regardless of assessed state . participants at this time - point had just performed a maximal volitional aerobic exercise test ( yo - yo test ) . in similarity with the hoff test
, no significant differences were detected in yo - yo distance completed across assessed states ( 1295 6 , 1325 7 and 1315 8 m for nsd , sdp and sdc , respectively ) . nevertheless , the increases in cortisol concentration from baseline were most pronounced in sdp and sdc when compared to nsd . the addition of sleep deprivation resulted in a higher cortisol concentration at all time - points . furthermore , in the sleep - deprived states , participants who ingested caffeine ( sdc ) did not exhibit as extensive a cortisol increase as placebo ( sdp ) . in the current study
, caffeine appeared to blunt the aerobic - exercise - induced increase in cortisol concentration following sleep deprivation . as depicted in figure 2 , no statistically significant differences were detected across sleep states for testosterone . furthermore , unlike data recorded for cortisol within a test session , no significant differences were detected across time within any investigated session . maximal testosterone concentrations were recorded in the nsd state , this was evident across time - points t1t4 . this is in keeping with the expected results where sleep restriction or sleep fragmentation have been reported to negatively affect testosterone concentration ( 2 , 12 ) . testosterone is thought to peak prior to rem sleep ; therefore , lack of sleep will result in a decrease in total testosterone . mean testosterone concentrations across the sleep - deprived states were equivocal for the first three time - points : t1 , t2 and t3 , see figure 2 . maximal data were recorded at t1 for the sleep deprived stated and were highest at t3 for the nsd state . a high testosterone concentration for both sleep deprived states relative to the other 3 time - points may be expected due to the diurnal variation of the hormone in question ( peaking in the am ) , as there was a dip in testosterone concentration across time from t1 ( pre - supplementation ) to t2 ( the pre - exercise testing ) . depicted an increase in testosterone concentration following intermittent sprinting in sleep restricted athletes ( 1 ) . this is also seen in the sleep - deprived states transitioning from t2 to t3 . at this stage
, there was a measurable increase in mean testosterone in the sleep - restricted states , also evident in the non - sleep restricted state . this increase in testosterone concentration is potentially due to the non - exhausting exercise evoked in the hoff test . it can be postulated therefore that when athletes undergo intermittent high - intensity ( non - exhausting ) aerobic exercise that an increase in testosterone concentration was expected . the testosterone concentration at t4 did not reflect the same rise in testosterone concentration portrayed at t3 . the yo - yo test induced a greater decline in testosterone concentration for sdp when compared to sdc at t4 . it would seem from these results that caffeine ingestion helped to offset the decrease in testosterone expected post - exhausting aerobic exercise . wu ( 22 ) documented that , following caffeine supplementation ( up to 6 mg.kg ) , testosterone and cortisol concentrations increased significantly . this direct increase in testosterone as a result of caffeine ingestion also seems to have an effect in the sleep - deprived athlete . in this sleep - deprived state ( sdc ) , individuals following caffeine ingestion exhibited an intermediate concentration when compared to nsd ( highest concentration ) and sdp ( lowest concentration ) , see figure 2 . interestingly , sdc was the only sleep state that recorded an increase in mean testosterone concentration from t3 to t4 . from this data
one can infer that the caffeine supplementation provided a more pronounced effect on testosterone metabolism than the exhausting yo - yo aerobic test . illustrating an increase in testosterone concentration after high dose caffeine ingestion ( 800 mg ) in relation to anaerobic exercise ( 3 ) . as testosterone is a potent stimulator for lean muscle formation ( 4 ) , supplementation with caffeine to promote testosterone secretion would be of benefit for athletes . from figure 3
, one can see that the t / c ratio was highest at all time points in the nsd state and lowest in the sdc state . this is accordance with figures 1 and 2 , respectively , which depict the nsd state as having the lowest cortisol concentrations across all 4 time points and also depict the nsd state as having double the testosterone concentrations at t3 and t4 when compared to sdc state . as described previously a heightened testosterone concentration in the nsd state would be expected . although it appears that caffeine may play a role in offsetting central stress responses , the overall picture points to a decrease in the t / c ratio . furthermore , increased cortisol concentrations which were not evident at the t3 in the nsd state were evident in the sdc state . depicting an overall decrease in t / c in caffeinated rugby players ( supplemented with 800 mg.kg ) due to an overall exaggerated increase in cortisol , despite having an initial increase in testosterone ( 3 ) . this exaggerated increase in one hormone with respect to the other can also explain the intervention - difference noted at t3 between nsd and sdc states . overall there were no significant differences detected comparing the sleep - state investigated for performance data , testosterone or cortisol concentrations or t / c ratio . a trend did exist whereby the sdc state mirrored the nsd state for mean cortisol concentration . an increase in overall mean testosterone concentration post - aerobic exercise was only observed in the sdc state , a potentially beneficial effect of caffeine supplementation . overall further studies are required to establish whether caffeine has a significant effect on both testosterone and cortisol in the sleep deprived athlete . | [
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fifty years ago , experiments have demonstrated that perinatal asphyxia could induce brain injury in primates . since then
, different patterns of brain injury have been established , which were dependent on the severity and duration of the hypoxic
these findings in animal studies were comparable to postmortem findings in asphyxiated human neonates . during the 1960s to 1980s ,
imaging of brain injury was performed in human neonates using positron emission tomography scans and ultrasound . since the introduction of magnetic resonance imaging ( mri )
, the knowledge of localisation and severity of brain injury following perinatal asphyxia ( hypoxic ischaemic brain lesions ) in surviving neonates has expanded tremendously [ 35 ] .
diffusion - weighted mri has enabled us to diagnose lesions much earlier than conventional mri .
in addition , phosphorus and proton mr spectroscopy ( mrs ) have enabled us to detect metabolic changes in the neonatal brain following hypoxia ischaemia [ 6 , 7 ] . in this review
, we will describe the use of mri and proton mrs in detecting different patterns of brain injury in ( full - term ) human neonates following hypoxic
although cranial ultrasound can still play a role in the full - term infant with hypoxic
ischaemic brain injury , mri is the method of choice to obtain more detailed and accurate information .
the american academy of neurology recommended in 2002 that a computed tomography ( ct ) should be performed to detect haemorrhagic lesions in encephalopathic term infants and mri only if findings are inconclusive .
we do , however , recommend the use of mri in all full - term infants who present with neonatal encephalopathy and/or seizures . the use of ct is restricted to infants who present with a large intracranial haemorrhage with a shift of the midline seen on cranial ultrasound .
these infants may need neurosurgical intervention and access to a ct may be easier , and associated skull fractures may also be better visualised than with mri [ 10 , 11 ] .
the increased use of neuroimaging techniques and mri , in particular , has been a tremendous help in timing of brain injury and recognising patterns of injury [ 4 , 12 , 13 ] .
performing mri within the first 2 weeks after birth , cowan et al .
were able to show that more than 90% of affected newborns had evidence of perinatally acquired lesions on their mri , with a very low rate of established antenatal brain injury .
the presence of ventricular dilatation , widening of the subarachnoid space and interhemispheric fissure , presence of germinolytic cysts or cystic lesions in the white matter , seen at birth or during the first week , are all suggestive of an antenatal insult or an underlying problem , for instance a metabolic disorder . performing cranial ultrasound on admission
is also of value , as most of these lesions suggestive of an antenatal insult or an underlying problem will be recognised with ultrasound .
the presence of increased echogenicity in the white matter on a day 1 ultrasound examination is also strongly suggestive of an antenatal insult as this echogenicity takes time to develop .
these findings may be important for genetic counselling as well as for medicolegal issues ( fig . 1 ) .
1cranial ultrasound , coronal view , day 1 , showing severe echogenicity in the white matter .
mri ( t2se ( tr 6284/te 120 ) and adc ) performed on day 3 showing increased signal intensity in the white matter on t2se and low signal intensity in the deep white matter on the adc map with sparing of the anterior periventricular white matter and asymmetrical distribution in the parieto - occipital white matter .
the child died and was subsequently diagnosed to have molybdenum cofactor deficiency cranial ultrasound , coronal view , day 1 , showing severe echogenicity in the white matter .
mri ( t2se ( tr 6284/te 120 ) and adc ) performed on day 3 showing increased signal intensity in the white matter on t2se and low signal intensity in the deep white matter on the adc map with sparing of the anterior periventricular white matter and asymmetrical distribution in the parieto - occipital white matter .
the child died and was subsequently diagnosed to have molybdenum cofactor deficiency the use of diffusion - weighted imaging ( dwi ) has also greatly improved our ability to time the onset of brain lesions .
a reduced apparent diffusion coefficient can be calculated , showing reduced values ( restricted diffusion ) during the first few days after the insult , with pseudonormalisation by the end of the first week [ 1517 ] .
sequential imaging has shown that lesions in the basal ganglia may increase in size and site during the first week after birth [ 18 , 19 ] .
performed sequential imaging in ten newborn infants and noted that patterns of injury varied considerably during the first 2 weeks after injury .
the appearance of new areas of reduced diffusion simultaneous with the pseudonormalisation of areas that had reduced diffusion at earlier times could result in an entirely different pattern of injury on adc maps acquired at different time points .
comparable to studies in a primate model , two main patterns of injury can be distinguished with mri in the full - term neonate :
basal ganglia
thalamus pattern ( bgt ) predominantly affecting bilaterally the central grey nuclei ( ventrolateral thalami and posterior putamina ) and perirolandic cortex .
associated involvement of the hippocampus and brain stem is not uncommon ( fig . 2 ) .
this pattern of injury is most often seen following an acute sentinel event , for instance a ruptured uterus , placental abruption or a prolapsed cord , and is also referred to as a pattern following acute near total asphyxia [ 12 , 20 ] . using conventional mri , it was first shown by rutherford et al .
that absence of a normal high - signal intensity of the posterior limb of the internal capsule ( plic ) is highly predictive of severe adverse sequelae . using conventional mri , an inversion of the signal within the plic
is only seen from 48 to 72 h onwards . when mri is performed early
more accurate information about timing of injury can sometimes be obtained when measuring the apparent diffusion coefficients , but due to evolution over time , this is mainly helpful in the most severely affected infants , who have an mri performed within the first few days after birth [ 16 , 22 ] .
measured adc values within the plic in 28 term infants with a clinical diagnosis of hypoxic ischaemic encephalopathy ( hie ) at a mean age of 5.6 days .
measuring fractional anisotropy ( fa ) was noted to be superior to measuring adc values in predicting outcome .
while reduced adc values were only found in infants with severe encephalopathy , reduced fa values were found in infants with severe and moderate encephalopathy .
children with the bgt pattern of injury tend to be severely disabled due to dyskinetic cerebral palsy ( cp ) .
studied 48 children at a mean age of 9 years ( range 413 years ) with dyskinetic cp mostly due to bgt injury and found that most children had gross motor function classification system levels of level iv , n = 10 , and level v , n = 28 .
the rate of learning disability ( n = 35 ) and epilepsy ( n = 30 ) increased with the severity of the motor disability .
fig . 2full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . a inversion recovery sequence ( tr 5038/te 30/ti 600 )
does not show a normal signal within the posterior limb of the internal capsule , but areas of increased signal intensity within thalami and basal ganglia .
dwi ( b d ) shows restricted diffusion in the ventrolateral thalami , lentiform nuclei , cerebral peduncles , and in the perirolandic cortex .
also note involvement of the hippocampiwatershed predominant pattern of injury ( ws ) is the other pattern of injury which is also referred to as a pattern seen following prolonged partial asphyxia. the vascular watershed zones ( anterior middle cerebral artery and posterior middle cerebral artery ) are involved , affecting white matter and in more severely affected infants also the overlying cortex ( fig .
although loss of the cortical ribbon and therefore the grey white matter differentiation can be seen on conventional mri , dwi highlights the abnormalities and is especially helpful in making an early diagnosis [ 26 , 27 ] .
a repeat mri may show cystic evolution , but more often atrophy and gliotic changes will be recognised .
it is also more common after hypotension , infection and hypoglycaemia , all of which may be associated with a more protracted course .
neurological manifestations at birth may be mild and do not always meet the perinatal asphyxia criteria and onset of neurological signs can be delayed .
severe motor impairment is uncommon in this group of infants , and they are often considered to have an early normal outcome , when seen at 1218 months . when seen up till early childhood suboptimal head growth , behavioural problems and delay in language are , however , common [ 31 , 32 ] .
miller et al . were first able to recognise cognitive deficits associated with the watershed pattern of injury at 30 months , while the problems were largely overlooked , when seen at 12 months .
more recently , they also showed a correlation with verbal iq at 4 years of age .
symptomatic parieto - occipital epilepsy may occur later in childhood , often associated with reduced intelligence quotients and visuospatial cognitive functions .
born with severe anaemia ( hb 2.2 mmol / l ) following fetomaternal transfusion and a short period of hypoglycaemia ( < 1.1
loss of cortical ribbon is noted on the t2se ( tr 6284/te 120 ; a ) , and the corpus callosum appears to be swollen with increased signal intensity .
b ) the adc map shows low - signal intensity in the posterior watershed areas , as well as the splenium of the corpus callosum and the optic radiationalthough not very common , severe involvement of the subcortical white matter and cortex can be seen with relative sparing of the immediate periventricular white matter and central grey matter , referred to as the white cerebrum , as dwi shows an almost completely white cerebrum , contrasted to a normal looking cerebellum ( fig . 4 ) .
this condition tends to be fatal , but in case of survival , muticystic encephalomalacia will develop .
the prevalence of the 677c > t allele was studied in 11 children with hie , their respective mothers and 85 healthy individuals .
the variant allele frequency was higher in the group of mothers with affected children than in the controls and was associated with an increase in plasma homocysteine after methionine loading .
the 677c > t mutation in mothers , either in a homozygous or heterozygous state , together with poor nutritional status ( probable folate deficiency ) may represent a risk factor for irreversible brain injury in the offspring .
fig . 4full - term infant with white brain pattern of injury . a , c t2se ( tr 6284/te 120 ) shows increased signal intensity in the white matter with loss of cortical ribbon .
dwi ( b , d ) confirms the abnormalities and shows a striking discrepancy in signal intensity with the cerebellum .
note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d)another pattern of brain injury consists of lesions restricted to the periventricular white matter
, not dissimilar from the so - called punctate white matter lesions in the preterm infant ( fig .
diagnosed this pattern of injury in 23% of their infants and pointed out that infants with this type of injury are significantly less mature with a milder degree of encephalopathy and fewer clinical seizures relative to other newborns in their cohort , who were diagnosed to have the two more common patterns of injury .
this pattern of brain injury is also seen in newborn infants with congenital heart defects .
5full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi .
there is also mild involvement of the corpus callosum and plic , seen on dwiperinatal arterial ischaemic stroke ( pais ) , perinatal haemorrhagic stroke ( phs ) and sinovenous thrombosis can also be seen in newborn infants presenting with neonatal encephalopathy and/or seizures .
pais was found to be related to perinatal asphyxia in only six out of 124 infants . our own experience and that of others ,
however , do suggest that the obstetric history is more complicated in infants presenting with pais , and there is often a history of an abnormal cardiotocograph , an instrumental delivery or an emergency caesarean section .
cranial ultrasound may be normal if the stroke is superficial and ischaemic , or it may reveal a wedge - shaped area of increased echogenicity with a linear demarcation line , usually within the territory of the middle cerebral artery .
mri , including dwi , and mr angiography are by far superior to head ultrasound and should be performed in any newborn presenting with neonatal seizures and especially hemiconvulsions .
the role of dwi in the prediction of motor outcome was recently shown [ 42 , 43 ] with restricted diffusion within the descending corticospinal tracts .
restricted diffusion at the level of the internal capsule and especially the middle part of the cerebral peduncle is now referred to as pre - wallerian degeneration as it is followed by wallerian degeneration at 612 weeks and beyond .
presence of wallerian degeneration at birth suggests an antenatal onset of the insult ( fig .
mri , t2se ( tr 6284/te 120 ) performed on day 3 , shows a large left - sided middle cerebral artery infarct of antenatal onset , with evidence of wallerian degeneration and presence of cysts within the area of infarction .
diffusion tensor tractography shows loss of fibres with the corticospinal tract of the affected hemisphere basal ganglia
thalamus pattern ( bgt ) predominantly affecting bilaterally the central grey nuclei ( ventrolateral thalami and posterior putamina ) and perirolandic cortex .
associated involvement of the hippocampus and brain stem is not uncommon ( fig . 2 ) .
this pattern of injury is most often seen following an acute sentinel event , for instance a ruptured uterus , placental abruption or a prolapsed cord , and is also referred to as a pattern following acute near total asphyxia [ 12 , 20 ] . using conventional mri , it was first shown by rutherford et al .
that absence of a normal high - signal intensity of the posterior limb of the internal capsule ( plic ) is highly predictive of severe adverse sequelae . using conventional mri , an inversion of the signal within the plic
is only seen from 48 to 72 h onwards . when mri is performed early
more accurate information about timing of injury can sometimes be obtained when measuring the apparent diffusion coefficients , but due to evolution over time , this is mainly helpful in the most severely affected infants , who have an mri performed within the first few days after birth [ 16 , 22 ] .
measured adc values within the plic in 28 term infants with a clinical diagnosis of hypoxic ischaemic encephalopathy ( hie ) at a mean age of 5.6 days .
measuring fractional anisotropy ( fa ) was noted to be superior to measuring adc values in predicting outcome .
while reduced adc values were only found in infants with severe encephalopathy , reduced fa values were found in infants with severe and moderate encephalopathy .
children with the bgt pattern of injury tend to be severely disabled due to dyskinetic cerebral palsy ( cp ) .
studied 48 children at a mean age of 9 years ( range 413 years ) with dyskinetic cp mostly due to bgt injury and found that most children had gross motor function classification system levels of level iv , n = 10 , and level v , n = 28 .
the rate of learning disability ( n = 35 ) and epilepsy ( n = 30 ) increased with the severity of the motor disability .
fig . 2full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . a inversion recovery sequence ( tr 5038/te 30/ti 600 )
does not show a normal signal within the posterior limb of the internal capsule , but areas of increased signal intensity within thalami and basal ganglia .
dwi ( b d ) shows restricted diffusion in the ventrolateral thalami , lentiform nuclei , cerebral peduncles , and in the perirolandic cortex .
also note involvement of the hippocampi full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia .
a inversion recovery sequence ( tr 5038/te 30/ti 600 ) does not show a normal signal within the posterior limb of the internal capsule , but areas of increased signal intensity within thalami and basal ganglia .
dwi ( b d ) shows restricted diffusion in the ventrolateral thalami , lentiform nuclei , cerebral peduncles , and in the perirolandic cortex .
also note involvement of the hippocampi watershed predominant pattern of injury ( ws ) is the other pattern of injury which is also referred to as a pattern seen following prolonged partial asphyxia. the vascular watershed zones ( anterior middle cerebral artery and posterior middle cerebral artery ) are involved , affecting white matter and in more severely affected infants also the overlying cortex ( fig . 3 ) .
although loss of the cortical ribbon and therefore the grey white matter differentiation can be seen on conventional mri , dwi highlights the abnormalities and is especially helpful in making an early diagnosis [ 26 , 27 ] .
a repeat mri may show cystic evolution , but more often atrophy and gliotic changes will be recognised .
it is also more common after hypotension , infection and hypoglycaemia , all of which may be associated with a more protracted course .
neurological manifestations at birth may be mild and do not always meet the perinatal asphyxia criteria and onset of neurological signs can be delayed .
severe motor impairment is uncommon in this group of infants , and they are often considered to have an early normal outcome , when seen at 1218 months . when seen up till early childhood suboptimal head growth , behavioural problems and delay in language are , however , common [ 31 , 32 ] .
miller et al . were first able to recognise cognitive deficits associated with the watershed pattern of injury at 30 months , while the problems were largely overlooked , when seen at 12 months .
more recently , they also showed a correlation with verbal iq at 4 years of age .
symptomatic parieto - occipital epilepsy may occur later in childhood , often associated with reduced intelligence quotients and visuospatial cognitive functions .
born with severe anaemia ( hb 2.2 mmol / l ) following fetomaternal transfusion and a short period of hypoglycaemia ( < 1.1
loss of cortical ribbon is noted on the t2se ( tr 6284/te 120 ; a ) , and the corpus callosum appears to be swollen with increased signal intensity .
b ) the adc map shows low - signal intensity in the posterior watershed areas , as well as the splenium of the corpus callosum and the optic radiation full - term infant with watershed pattern of injury .
born with severe anaemia ( hb 2.2 mmol / l ) following fetomaternal transfusion and a short period of hypoglycaemia ( < 1.1
loss of cortical ribbon is noted on the t2se ( tr 6284/te 120 ; a ) , and the corpus callosum appears to be swollen with increased signal intensity .
b ) the adc map shows low - signal intensity in the posterior watershed areas , as well as the splenium of the corpus callosum and the optic radiation although not very common , severe involvement of the subcortical white matter and cortex can be seen with relative sparing of the immediate periventricular white matter and central grey matter , referred to as the white cerebrum , as dwi shows an almost completely white cerebrum , contrasted to a normal looking cerebellum ( fig . 4 ) .
this condition tends to be fatal , but in case of survival , muticystic encephalomalacia will develop .
the prevalence of the 677c > t allele was studied in 11 children with hie , their respective mothers and 85 healthy individuals .
the variant allele frequency was higher in the group of mothers with affected children than in the controls and was associated with an increase in plasma homocysteine after methionine loading .
the 677c > t mutation in mothers , either in a homozygous or heterozygous state , together with poor nutritional status ( probable folate deficiency ) may represent a risk factor for irreversible brain injury in the offspring .
fig . 4full - term infant with white brain pattern of injury . a , c t2se ( tr 6284/te 120 ) shows increased signal intensity in the white matter with loss of cortical ribbon .
dwi ( b , d ) confirms the abnormalities and shows a striking discrepancy in signal intensity with the cerebellum .
note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d ) full - term infant with white brain pattern of injury .
a , c t2se ( tr 6284/te 120 ) shows increased signal intensity in the white matter with loss of cortical ribbon .
dwi ( b , d ) confirms the abnormalities and shows a striking discrepancy in signal intensity with the cerebellum .
note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d ) another pattern of brain injury consists of lesions restricted to the periventricular white matter , not dissimilar from the so - called punctate white matter lesions in the preterm infant ( fig .
diagnosed this pattern of injury in 23% of their infants and pointed out that infants with this type of injury are significantly less mature with a milder degree of encephalopathy and fewer clinical seizures relative to other newborns in their cohort , who were diagnosed to have the two more common patterns of injury .
this pattern of brain injury is also seen in newborn infants with congenital heart defects .
5full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi .
there is also mild involvement of the corpus callosum and plic , seen on dwi full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi .
there is also mild involvement of the corpus callosum and plic , seen on dwi perinatal arterial ischaemic stroke ( pais ) , perinatal haemorrhagic stroke ( phs ) and sinovenous thrombosis can also be seen in newborn infants presenting with neonatal encephalopathy and/or seizures .
pais was found to be related to perinatal asphyxia in only six out of 124 infants . our own experience and that of others , however ,
do suggest that the obstetric history is more complicated in infants presenting with pais , and there is often a history of an abnormal cardiotocograph , an instrumental delivery or an emergency caesarean section .
cranial ultrasound may be normal if the stroke is superficial and ischaemic , or it may reveal a wedge - shaped area of increased echogenicity with a linear demarcation line , usually within the territory of the middle cerebral artery .
mri , including dwi , and mr angiography are by far superior to head ultrasound and should be performed in any newborn presenting with neonatal seizures and especially hemiconvulsions .
the role of dwi in the prediction of motor outcome was recently shown [ 42 , 43 ] with restricted diffusion within the descending corticospinal tracts .
restricted diffusion at the level of the internal capsule and especially the middle part of the cerebral peduncle is now referred to as pre - wallerian degeneration as it is followed by wallerian degeneration at 612 weeks and beyond .
presence of wallerian degeneration at birth suggests an antenatal onset of the insult ( fig .
mri , t2se ( tr 6284/te 120 ) performed on day 3 , shows a large left - sided middle cerebral artery infarct of antenatal onset , with evidence of wallerian degeneration and presence of cysts within the area of infarction .
diffusion tensor tractography shows loss of fibres with the corticospinal tract of the affected hemisphere born at 37 weeks , following antenatal diagnosis of foetal supraventricular tachycardia .
mri , t2se ( tr 6284/te 120 ) performed on day 3 , shows a large left - sided middle cerebral artery infarct of antenatal onset , with evidence of wallerian degeneration and presence of cysts within the area of infarction .
diffusion tensor tractography shows loss of fibres with the corticospinal tract of the affected hemisphere recently developed techniques , such as diffusion tensor imaging ( dti ) , will allow quantification and visualisation of white matter pathways in vivo .
water diffuses preferentially along the direction of the axons and is restricted perpendicular to axons by myelin . this directional dependency is referred to as anisotropy .
directionality encoded colour maps ( red green blue ) or fibre trackings are commonly used .
a fa map can show asymmetry of the plic as early as the neonatal period . in a study of 15 patients with congenital hemiparesis due to different causes , studied at a median age of 2 years and compared with 17 age - matched controls , clinical severity of hemiparesis
was noted to correlate with asymmetry in fractional anisotropy ( p < 0.0001 ) , transverse diffusivity ( p < 0.0001 ) and mean diffusivity ( p < 0.03 ) .
another promising technique is volumetric determination of stroke volumes , which was noted to predict motor outcome in animal studies .
functional mri tends to be used in childhood or adolescence to study reorganisation of the sensorimotor cortex [ 47 , 48 ] , but it was recently shown that passive unilateral sensorimotor stimulation is feasible even in the preterm infant resulting in bilateral activation of the sensorimotor cortex [ 49 , 50 ] .
the term phs was recently coined by armstrong - wells et al . and appeared to include term infants with parenchymal haemorrhage due to different underlying problems .
all infants presented with encephalopathy and more than half ( 65% ) with seizures . perinatal haemorrhagic stroke was typically unifocal ( 74% ) and unilateral ( 83% ) .
etiologies included thrombocytopenia ( n = 4 ) and cavernous malformation ( n = 1 ) ; 15 ( 75% ) were idiopathic .
while some of the larger lesions will be recognised with ultrasound , mri will provide more detailed information , and early dwi will be able to show associated areas with restricted diffusion .
this diagnosis should especially be considered in infants who present without a history of perinatal asphyxia but with seizures and/or lethargy , sometimes in the context of infection or dehydration .
wu et al . first pointed out that csvt should always be considered in the presence of an intraventricular haemorrhage ( ivh ) , especially when associated with a unilateral thalamic haemorrhage .
thirty - one percent of 29 infants born > 36 weeks gestation , who were diagnosed with csvt , presented with an ivh .
thalamic haemorrhage was diagnosed in 16% of infants with csvt [ 53 , 54 ] .
cranial ultrasound may detect csvt , particularly in the presence of a midline thrombus in the superior sagittal sinus , or a unilateral thalamic haemorrhage . power doppler may be superior to colour doppler when available [ 55 , 56 ] . additional imaging is required to exclude csvt in more peripheral locations and to confirm the extent of the thrombus .
empty delta sign which is a filling defect in the posterior portion of the superior sagittal sinus due to thrombus .
there are false positives and missed diagnoses in up to 40% of children with csvt .
susceptibility weighted imaging has recently been reported as another useful sequence in confirming the presence of csvt and following for progression or resolution [ 59 , 60 ] .
the increased use of neuroimaging techniques and mri , in particular , has been a tremendous help in timing of brain injury and recognising patterns of injury [ 4 , 12 , 13 ] .
performing mri within the first 2 weeks after birth , cowan et al .
were able to show that more than 90% of affected newborns had evidence of perinatally acquired lesions on their mri , with a very low rate of established antenatal brain injury .
the presence of ventricular dilatation , widening of the subarachnoid space and interhemispheric fissure , presence of germinolytic cysts or cystic lesions in the white matter , seen at birth or during the first week , are all suggestive of an antenatal insult or an underlying problem , for instance a metabolic disorder . performing cranial ultrasound on admission is also of value , as most of these lesions suggestive of an antenatal insult or an underlying problem will be recognised with ultrasound .
the presence of increased echogenicity in the white matter on a day 1 ultrasound examination is also strongly suggestive of an antenatal insult as this echogenicity takes time to develop .
these findings may be important for genetic counselling as well as for medicolegal issues ( fig . 1 ) .
1cranial ultrasound , coronal view , day 1 , showing severe echogenicity in the white matter .
mri ( t2se ( tr 6284/te 120 ) and adc ) performed on day 3 showing increased signal intensity in the white matter on t2se and low signal intensity in the deep white matter on the adc map with sparing of the anterior periventricular white matter and asymmetrical distribution in the parieto - occipital white matter .
the child died and was subsequently diagnosed to have molybdenum cofactor deficiency cranial ultrasound , coronal view , day 1 , showing severe echogenicity in the white matter .
mri ( t2se ( tr 6284/te 120 ) and adc ) performed on day 3 showing increased signal intensity in the white matter on t2se and low signal intensity in the deep white matter on the adc map with sparing of the anterior periventricular white matter and asymmetrical distribution in the parieto - occipital white matter .
the child died and was subsequently diagnosed to have molybdenum cofactor deficiency the use of diffusion - weighted imaging ( dwi ) has also greatly improved our ability to time the onset of brain lesions .
a reduced apparent diffusion coefficient can be calculated , showing reduced values ( restricted diffusion ) during the first few days after the insult , with pseudonormalisation by the end of the first week [ 1517 ] .
sequential imaging has shown that lesions in the basal ganglia may increase in size and site during the first week after birth [ 18 , 19 ] .
performed sequential imaging in ten newborn infants and noted that patterns of injury varied considerably during the first 2 weeks after injury .
the appearance of new areas of reduced diffusion simultaneous with the pseudonormalisation of areas that had reduced diffusion at earlier times could result in an entirely different pattern of injury on adc maps acquired at different time points .
comparable to studies in a primate model , two main patterns of injury can be distinguished with mri in the full - term neonate :
basal ganglia
thalamus pattern ( bgt ) predominantly affecting bilaterally the central grey nuclei ( ventrolateral thalami and posterior putamina ) and perirolandic cortex .
associated involvement of the hippocampus and brain stem is not uncommon ( fig . 2 ) .
this pattern of injury is most often seen following an acute sentinel event , for instance a ruptured uterus , placental abruption or a prolapsed cord , and is also referred to as a pattern following acute near total asphyxia [ 12 , 20 ] . using conventional mri , it was first shown by rutherford et al .
that absence of a normal high - signal intensity of the posterior limb of the internal capsule ( plic ) is highly predictive of severe adverse sequelae . using conventional mri , an inversion of the signal within the plic
is only seen from 48 to 72 h onwards . when mri is performed early
more accurate information about timing of injury can sometimes be obtained when measuring the apparent diffusion coefficients , but due to evolution over time , this is mainly helpful in the most severely affected infants , who have an mri performed within the first few days after birth [ 16 , 22 ] .
measured adc values within the plic in 28 term infants with a clinical diagnosis of hypoxic ischaemic encephalopathy ( hie ) at a mean age of 5.6 days .
measuring fractional anisotropy ( fa ) was noted to be superior to measuring adc values in predicting outcome .
while reduced adc values were only found in infants with severe encephalopathy , reduced fa values were found in infants with severe and moderate encephalopathy .
children with the bgt pattern of injury tend to be severely disabled due to dyskinetic cerebral palsy ( cp ) .
studied 48 children at a mean age of 9 years ( range 413 years ) with dyskinetic cp mostly due to bgt injury and found that most children had gross motor function classification system levels of level iv , n = 10 , and level v , n = 28 .
the rate of learning disability ( n = 35 ) and epilepsy ( n = 30 ) increased with the severity of the motor disability .
fig . 2full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . a inversion recovery sequence ( tr 5038/te 30/ti 600 )
does not show a normal signal within the posterior limb of the internal capsule , but areas of increased signal intensity within thalami and basal ganglia .
dwi ( b d ) shows restricted diffusion in the ventrolateral thalami , lentiform nuclei , cerebral peduncles , and in the perirolandic cortex .
also note involvement of the hippocampiwatershed predominant pattern of injury ( ws ) is the other pattern of injury which is also referred to as a pattern seen following prolonged partial asphyxia. the vascular watershed zones ( anterior middle cerebral artery and posterior middle cerebral artery ) are involved , affecting white matter and in more severely affected infants also the overlying cortex ( fig . 3 ) .
although loss of the cortical ribbon and therefore the grey white matter differentiation can be seen on conventional mri , dwi highlights the abnormalities and is especially helpful in making an early diagnosis [ 26 , 27 ] .
a repeat mri may show cystic evolution , but more often atrophy and gliotic changes will be recognised .
it is also more common after hypotension , infection and hypoglycaemia , all of which may be associated with a more protracted course .
neurological manifestations at birth may be mild and do not always meet the perinatal asphyxia criteria and onset of neurological signs can be delayed .
severe motor impairment is uncommon in this group of infants , and they are often considered to have an early normal outcome , when seen at 1218 months . when seen up till early childhood suboptimal head growth , behavioural problems and delay in language are , however , common [ 31 , 32 ] .
miller et al . were first able to recognise cognitive deficits associated with the watershed pattern of injury at 30 months , while the problems were largely overlooked , when seen at 12 months .
more recently , they also showed a correlation with verbal iq at 4 years of age .
symptomatic parieto - occipital epilepsy may occur later in childhood , often associated with reduced intelligence quotients and visuospatial cognitive functions .
born with severe anaemia ( hb 2.2 mmol / l ) following fetomaternal transfusion and a short period of hypoglycaemia ( < 1.1
loss of cortical ribbon is noted on the t2se ( tr 6284/te 120 ; a ) , and the corpus callosum appears to be swollen with increased signal intensity .
b ) the adc map shows low - signal intensity in the posterior watershed areas , as well as the splenium of the corpus callosum and the optic radiationalthough not very common , severe involvement of the subcortical white matter and cortex can be seen with relative sparing of the immediate periventricular white matter and central grey matter , referred to as the white cerebrum , as dwi shows an almost completely white cerebrum , contrasted to a normal looking cerebellum ( fig . 4 ) .
this condition tends to be fatal , but in case of survival , muticystic encephalomalacia will develop .
the prevalence of the 677c > t allele was studied in 11 children with hie , their respective mothers and 85 healthy individuals .
the variant allele frequency was higher in the group of mothers with affected children than in the controls and was associated with an increase in plasma homocysteine after methionine loading .
the 677c > t mutation in mothers , either in a homozygous or heterozygous state , together with poor nutritional status ( probable folate deficiency ) may represent a risk factor for irreversible brain injury in the offspring .
white brain pattern of injury . a , c t2se ( tr 6284/te 120 ) shows increased signal intensity in the white matter with loss of cortical ribbon .
dwi ( b , d ) confirms the abnormalities and shows a striking discrepancy in signal intensity with the cerebellum .
note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d)another pattern of brain injury consists of lesions restricted to the periventricular white matter
, not dissimilar from the so - called punctate white matter lesions in the preterm infant ( fig . 5 ) .
diagnosed this pattern of injury in 23% of their infants and pointed out that infants with this type of injury are significantly less mature with a milder degree of encephalopathy and fewer clinical seizures relative to other newborns in their cohort , who were diagnosed to have the two more common patterns of injury .
this pattern of brain injury is also seen in newborn infants with congenital heart defects .
5full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi .
there is also mild involvement of the corpus callosum and plic , seen on dwiperinatal arterial ischaemic stroke ( pais ) , perinatal haemorrhagic stroke ( phs ) and sinovenous thrombosis can also be seen in newborn infants presenting with neonatal encephalopathy and/or seizures .
pais was found to be related to perinatal asphyxia in only six out of 124 infants . our own experience and that of others , however
, do suggest that the obstetric history is more complicated in infants presenting with pais , and there is often a history of an abnormal cardiotocograph , an instrumental delivery or an emergency caesarean section .
cranial ultrasound may be normal if the stroke is superficial and ischaemic , or it may reveal a wedge - shaped area of increased echogenicity with a linear demarcation line , usually within the territory of the middle cerebral artery .
mri , including dwi , and mr angiography are by far superior to head ultrasound and should be performed in any newborn presenting with neonatal seizures and especially hemiconvulsions .
the role of dwi in the prediction of motor outcome was recently shown [ 42 , 43 ] with restricted diffusion within the descending corticospinal tracts .
restricted diffusion at the level of the internal capsule and especially the middle part of the cerebral peduncle is now referred to as pre - wallerian degeneration as it is followed by wallerian degeneration at 612 weeks and beyond .
presence of wallerian degeneration at birth suggests an antenatal onset of the insult ( fig .
mri , t2se ( tr 6284/te 120 ) performed on day 3 , shows a large left - sided middle cerebral artery infarct of antenatal onset , with evidence of wallerian degeneration and presence of cysts within the area of infarction .
diffusion tensor tractography shows loss of fibres with the corticospinal tract of the affected hemisphere basal ganglia
thalamus pattern ( bgt ) predominantly affecting bilaterally the central grey nuclei ( ventrolateral thalami and posterior putamina ) and perirolandic cortex .
associated involvement of the hippocampus and brain stem is not uncommon ( fig . 2 ) .
this pattern of injury is most often seen following an acute sentinel event , for instance a ruptured uterus , placental abruption or a prolapsed cord , and is also referred to as a pattern following acute near total asphyxia [ 12 , 20 ] . using conventional mri , it was first shown by rutherford et al .
that absence of a normal high - signal intensity of the posterior limb of the internal capsule ( plic ) is highly predictive of severe adverse sequelae . using conventional mri , an inversion of the signal within the plic
is only seen from 48 to 72 h onwards . when mri is performed early
more accurate information about timing of injury can sometimes be obtained when measuring the apparent diffusion coefficients , but due to evolution over time , this is mainly helpful in the most severely affected infants , who have an mri performed within the first few days after birth [ 16 , 22 ] .
measured adc values within the plic in 28 term infants with a clinical diagnosis of hypoxic ischaemic encephalopathy ( hie ) at a mean age of 5.6 days .
measuring fractional anisotropy ( fa ) was noted to be superior to measuring adc values in predicting outcome .
while reduced adc values were only found in infants with severe encephalopathy , reduced fa values were found in infants with severe and moderate encephalopathy .
children with the bgt pattern of injury tend to be severely disabled due to dyskinetic cerebral palsy ( cp ) .
studied 48 children at a mean age of 9 years ( range 413 years ) with dyskinetic cp mostly due to bgt injury and found that most children had gross motor function classification system levels of level iv , n = 10 , and level v , n = 28 .
the rate of learning disability ( n = 35 ) and epilepsy ( n = 30 ) increased with the severity of the motor disability .
fig . 2full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia .
a inversion recovery sequence ( tr 5038/te 30/ti 600 ) does not show a normal signal within the posterior limb of the internal capsule , but areas of increased signal intensity within thalami and basal ganglia .
dwi ( b d ) shows restricted diffusion in the ventrolateral thalami , lentiform nuclei , cerebral peduncles , and in the perirolandic cortex .
also note involvement of the hippocampi full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia .
a inversion recovery sequence ( tr 5038/te 30/ti 600 ) does not show a normal signal within the posterior limb of the internal capsule , but areas of increased signal intensity within thalami and basal ganglia .
dwi ( b d ) shows restricted diffusion in the ventrolateral thalami , lentiform nuclei , cerebral peduncles , and in the perirolandic cortex .
also note involvement of the hippocampi watershed predominant pattern of injury ( ws ) is the other pattern of injury which is also referred to as a pattern seen following prolonged partial asphyxia. the vascular watershed zones ( anterior middle cerebral artery and posterior middle cerebral artery ) are involved , affecting white matter and in more severely affected infants also the overlying cortex ( fig . 3 ) .
although loss of the cortical ribbon and therefore the grey white matter differentiation can be seen on conventional mri , dwi highlights the abnormalities and is especially helpful in making an early diagnosis [ 26 , 27 ] .
a repeat mri may show cystic evolution , but more often atrophy and gliotic changes will be recognised .
it is also more common after hypotension , infection and hypoglycaemia , all of which may be associated with a more protracted course .
neurological manifestations at birth may be mild and do not always meet the perinatal asphyxia criteria and onset of neurological signs can be delayed .
severe motor impairment is uncommon in this group of infants , and they are often considered to have an early normal outcome , when seen at 1218 months . when seen up till early childhood suboptimal head growth , behavioural problems and delay in language are , however , common [ 31 , 32 ] .
miller et al . were first able to recognise cognitive deficits associated with the watershed pattern of injury at 30 months , while the problems were largely overlooked , when seen at 12 months .
more recently , they also showed a correlation with verbal iq at 4 years of age .
symptomatic parieto - occipital epilepsy may occur later in childhood , often associated with reduced intelligence quotients and visuospatial cognitive functions .
born with severe anaemia ( hb 2.2 mmol / l ) following fetomaternal transfusion and a short period of hypoglycaemia ( < 1.1
loss of cortical ribbon is noted on the t2se ( tr 6284/te 120 ; a ) , and the corpus callosum appears to be swollen with increased signal intensity .
b ) the adc map shows low - signal intensity in the posterior watershed areas , as well as the splenium of the corpus callosum and the optic radiation full - term infant with watershed pattern of injury .
born with severe anaemia ( hb 2.2 mmol / l ) following fetomaternal transfusion and a short period of hypoglycaemia ( < 1.1
loss of cortical ribbon is noted on the t2se ( tr 6284/te 120 ; a ) , and the corpus callosum appears to be swollen with increased signal intensity .
b ) the adc map shows low - signal intensity in the posterior watershed areas , as well as the splenium of the corpus callosum and the optic radiation although not very common , severe involvement of the subcortical white matter and cortex can be seen with relative sparing of the immediate periventricular white matter and central grey matter , referred to as the white cerebrum , as dwi shows an almost completely white cerebrum , contrasted to a normal looking cerebellum ( fig . 4 ) .
this condition tends to be fatal , but in case of survival , muticystic encephalomalacia will develop .
the prevalence of the 677c > t allele was studied in 11 children with hie , their respective mothers and 85 healthy individuals .
the variant allele frequency was higher in the group of mothers with affected children than in the controls and was associated with an increase in plasma homocysteine after methionine loading .
the 677c > t mutation in mothers , either in a homozygous or heterozygous state , together with poor nutritional status ( probable folate deficiency ) may represent a risk factor for irreversible brain injury in the offspring .
fig . 4full - term infant with white brain pattern of injury . a , c t2se ( tr 6284/te 120 ) shows increased signal intensity in the white matter with loss of cortical ribbon .
dwi ( b , d ) confirms the abnormalities and shows a striking discrepancy in signal intensity with the cerebellum .
note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d ) full - term infant with white brain pattern of injury .
a , c t2se ( tr 6284/te 120 ) shows increased signal intensity in the white matter with loss of cortical ribbon .
dwi ( b , d ) confirms the abnormalities and shows a striking discrepancy in signal intensity with the cerebellum .
note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d ) another pattern of brain injury consists of lesions restricted to the periventricular white matter
, not dissimilar from the so - called punctate white matter lesions in the preterm infant ( fig . 5 ) .
diagnosed this pattern of injury in 23% of their infants and pointed out that infants with this type of injury are significantly less mature with a milder degree of encephalopathy and fewer clinical seizures relative to other newborns in their cohort , who were diagnosed to have the two more common patterns of injury .
this pattern of brain injury is also seen in newborn infants with congenital heart defects .
5full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi .
there is also mild involvement of the corpus callosum and plic , seen on dwi full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi .
there is also mild involvement of the corpus callosum and plic , seen on dwi perinatal arterial ischaemic stroke ( pais ) , perinatal haemorrhagic stroke ( phs ) and sinovenous thrombosis can also be seen in newborn infants presenting with neonatal encephalopathy and/or seizures .
pais was found to be related to perinatal asphyxia in only six out of 124 infants . our own experience and that of others , however , do suggest that the obstetric history is more complicated in infants presenting with pais , and there is often a history of an abnormal cardiotocograph , an instrumental delivery or an emergency caesarean section .
cranial ultrasound may be normal if the stroke is superficial and ischaemic , or it may reveal a wedge - shaped area of increased echogenicity with a linear demarcation line , usually within the territory of the middle cerebral artery .
mri , including dwi , and mr angiography are by far superior to head ultrasound and should be performed in any newborn presenting with neonatal seizures and especially hemiconvulsions .
the role of dwi in the prediction of motor outcome was recently shown [ 42 , 43 ] with restricted diffusion within the descending corticospinal tracts . restricted diffusion at the level of the internal capsule and especially the middle part of the cerebral peduncle
is now referred to as pre - wallerian degeneration as it is followed by wallerian degeneration at 612 weeks and beyond .
presence of wallerian degeneration at birth suggests an antenatal onset of the insult ( fig .
mri , t2se ( tr 6284/te 120 ) performed on day 3 , shows a large left - sided middle cerebral artery infarct of antenatal onset , with evidence of wallerian degeneration and presence of cysts within the area of infarction .
diffusion tensor tractography shows loss of fibres with the corticospinal tract of the affected hemisphere born at 37 weeks , following antenatal diagnosis of foetal supraventricular tachycardia .
mri , t2se ( tr 6284/te 120 ) performed on day 3 , shows a large left - sided middle cerebral artery infarct of antenatal onset , with evidence of wallerian degeneration and presence of cysts within the area of infarction .
diffusion tensor tractography shows loss of fibres with the corticospinal tract of the affected hemisphere recently developed techniques , such as diffusion tensor imaging ( dti ) , will allow quantification and visualisation of white matter pathways in vivo .
water diffuses preferentially along the direction of the axons and is restricted perpendicular to axons by myelin . this directional dependency is referred to as anisotropy .
directionality encoded colour maps ( red green blue ) or fibre trackings are commonly used .
a fa map can show asymmetry of the plic as early as the neonatal period . in a study of 15 patients with congenital hemiparesis due to different causes , studied at a median age of 2 years and compared with 17 age - matched controls , clinical severity of hemiparesis was noted to correlate with asymmetry in fractional anisotropy ( p < 0.0001 ) , transverse diffusivity ( p < 0.0001 ) and mean diffusivity ( p < 0.03 ) .
another promising technique is volumetric determination of stroke volumes , which was noted to predict motor outcome in animal studies .
functional mri tends to be used in childhood or adolescence to study reorganisation of the sensorimotor cortex [ 47 , 48 ] , but it was recently shown that passive unilateral sensorimotor stimulation is feasible even in the preterm infant resulting in bilateral activation of the sensorimotor cortex [ 49 , 50 ] .
the term phs was recently coined by armstrong - wells et al . and appeared to include term infants with parenchymal haemorrhage due to different underlying problems .
all infants presented with encephalopathy and more than half ( 65% ) with seizures . perinatal haemorrhagic stroke was typically unifocal ( 74% ) and unilateral ( 83% ) .
etiologies included thrombocytopenia ( n = 4 ) and cavernous malformation ( n = 1 ) ; 15 ( 75% ) were idiopathic .
while some of the larger lesions will be recognised with ultrasound , mri will provide more detailed information , and early dwi will be able to show associated areas with restricted diffusion .
this diagnosis should especially be considered in infants who present without a history of perinatal asphyxia but with seizures and/or lethargy , sometimes in the context of infection or dehydration .
wu et al . first pointed out that csvt should always be considered in the presence of an intraventricular haemorrhage ( ivh ) , especially when associated with a unilateral thalamic haemorrhage .
thirty - one percent of 29 infants born > 36 weeks gestation , who were diagnosed with csvt , presented with an ivh .
thalamic haemorrhage was diagnosed in 16% of infants with csvt [ 53 , 54 ] .
cranial ultrasound may detect csvt , particularly in the presence of a midline thrombus in the superior sagittal sinus , or a unilateral thalamic haemorrhage . power doppler may be superior to colour doppler when available [ 55 , 56 ] . additional imaging is required to exclude csvt in more peripheral locations and to confirm the extent of the thrombus .
empty delta sign which is a filling defect in the posterior portion of the superior sagittal sinus due to thrombus .
there are false positives and missed diagnoses in up to 40% of children with csvt .
susceptibility weighted imaging has recently been reported as another useful sequence in confirming the presence of csvt and following for progression or resolution [ 59 , 60 ] .
nuclei that have been used clinically for mrs are p and h. thereby , in vivo brain metabolism can be assessed , and changes can be documented .
mrs has been used to study changes in cerebral metabolism of neonates following ( perinatal ) hypoxia ischaemia .
p - mrs was one of the first mrs techniques to be used in neonates more than two decades ago [ 6 , 7 ] . with this technique , metabolites such as high - energy phosphates ( phosphocreatine ( pcr ) , nucleotide triphosphates ( ntp ) ) and inorganic phosphate ( pi ) , phosphomonoesters and phosphodiesters can be detected .
it is time - consuming to measure absolute concentrations , and therefore , metabolite ratios have been calculated used instead to demonstrate changes in the brain . in addition , intracellular ph can be calculated using the formula published by petroff et al . .
it has been shown that these metabolite ratios change during development , especially during the first years of life . in animal experiments
full - term neonates with perinatal asphyxia have also been studied [ 64 , 65 ] . after a successful resuscitation ,
brain energy metabolism returned to normal to become abnormal after 612 h to decrease even further after 2448 h [ 6567 ] .
the concept of secondary energy failure has been elaborated in animal models , in particular in newborn piglets [ 6870 ] . using p - mrs in these animal models ,
an example of the changes in p - mrs during cerebral hypoxia ischaemia in the newborn piglet is presented in ( fig .
p - mrs was found to correlate with long - term neurodevelopmental outcome , especially when performed during the first few weeks after the insult [ 66 , 67 ] . in neonates with a very poor outcome , elevated intracellular ph , the so - called ph paradox , existed even for weeks after the insult , possibly due to changes in the na / h transporter .
7p - mrs of a newborn piglet at baseline ( top ) and after 1 h of cerebral hypoxia
the pcr / pi ratio and atp peaks decreased , whereas the distance between pcr and pi decreased , indicating a decrease in the intracellular ph p - mrs of a newborn piglet at baseline ( top ) and after 1 h of cerebral hypoxia
the pcr / pi ratio and atp peaks decreased , whereas the distance between pcr and pi decreased , indicating a decrease in the intracellular ph unfortunately , with the magnetic field strength of the current clinical systems , it is impossible to perform localised p - mrs , so only relatively large brain areas can be examined . due to these limitations , p - mrs has not become a routine clinical tool to assess asphyxiated full - term neonates .
proton mrs has also been used in neonates since the early 1990s [ 62 , 73 ] .
metabolites that can be demonstrated with h - mrs and echo times of 136 or more milliseconds are choline ( cho ) , creatine and phosphocreatine as a single peak ( cr ) , n - acetylaspartate ( naa ) and when present lactate ( lac ) . with shorter echo times , myo - inositol and the combined glutamate
given the large amounts of water in the neonatal brain , high - quality water suppression is essential for h - mrs .
the detection level of the aforementioned metabolites is in the millimolar range . as with p - mrs ,
although some have used water as an internal standard , this may not be appropriate in asphyxiated full - term neonates with changing amounts of intracellular and extracellular brain water .
therefore , metabolite ratios such as naa / cho , naa / cr or lac / naa are used instead .
it has been used as a marker of neuronal integrity . with selective loss of neurons ,
8h - mrs of a full - term neonate , 4 days after perinatal asphyxia . naa / cho is still within the normal range for full - term neonates , but a very large lactate resonance can be identified at1.33 ppm
. a original spectrum of te 35 ms , b original spectrum of te 144 ms , c fitted spectrum of te 35 ms , and d fitted spectrum of te 144 ms h - mrs of a full - term neonate , 4 days after perinatal asphyxia .
naa / cho is still within the normal range for full - term neonates , but a very large lactate resonance can be identified at1.33 ppm
. a original spectrum of te 35 ms , b original spectrum of te 144 ms , c fitted spectrum of te 35 ms , and d fitted spectrum of te 144 ms previously , the changes in h - mrs during normal development have been demonstrated . in a previous study , we have shown an increase in the naa / cho ratio between preterm at a gestational age of 32 weeks and term equivalent age .
we have demonstrated lactate in the brain of the asphyxiated full - term neonate days after the insult .
experimental work in animals showed accumulation of lactate in the brain during secondary energy failure which could be decreased with appropriate neuroprotective strategies .
an advantage of h - mrs over p - mrs is the possibility to perform localised examinations .
volumes of brain tissue as small as 1 cc can be assessed in 1.5-t mr systems .
thereby , changes in brain areas that are particularly vulnerable , such as the basal ganglia and thalamus , can be demonstrated .
in addition , chemical shift imaging enables us to measure metabolites in a matrix of voxels overlying the brain .
9 , where h - mrs chemical shift imaging is presented of a neonate with a large infarct in the territory of the right middle cerebral artery .
9chemical shift imaging a neonate with a large stroke in the territory of the right middle cerebral artery ( a ) . in the infarcted area , naa concentration
is decreased ( b , red ) , whereas lac / naa ratio is increased ( c , yellow ) chemical shift imaging a neonate with a large stroke in the territory of the right middle cerebral artery ( a ) . in the infarcted area , naa concentration is decreased ( b , red ) , whereas lac / naa ratio is increased ( c , yellow ) one of the limitations of h - mrs is the loss of quality by susceptibility .
we and others have shown decreases in naa / cho or naa / cr and elevations of lac / naa to predict a poor neurodevelopmental outcome [ 75 , 80 ] .
measurements performed after the second week of life may show normalised pcr / pi ratios .
mr imaging performed before the fourth day after the insult may not yet demonstrate changes on t1- and t2-weighted images .
diffusion - weighted images may show pseudo - normalisation , which complicates assessment of the severity of the insult .
abnormalities using h - mrs may only be missed , when this technique is performed very early , i.e. during the first day of life , before the development of secondary energy failure .
based on these aspects , it has been suggested that h - mrs is the best mr biomarker to predict neurodevelopmental outcome in asphyxiated full - term neonates .
however , since brain metabolite ratios may vary between mr systems and coils , development of normal values in one s own setting is required , and support of physicists is mandatory .
the limiting factor in the development of these normal values may be the lack of
p - mrs was one of the first mrs techniques to be used in neonates more than two decades ago [ 6 , 7 ] . with this technique , metabolites such as high - energy phosphates ( phosphocreatine ( pcr ) , nucleotide triphosphates ( ntp ) ) and inorganic phosphate ( pi ) , phosphomonoesters and phosphodiesters can be detected .
it is time - consuming to measure absolute concentrations , and therefore , metabolite ratios have been calculated used instead to demonstrate changes in the brain . in addition , intracellular ph can be calculated using the formula published by petroff et al . .
it has been shown that these metabolite ratios change during development , especially during the first years of life . in animal experiments hypoxia decreased pcr / pi and ntp / total phosphate ratios .
full - term neonates with perinatal asphyxia have also been studied [ 64 , 65 ] . after a successful resuscitation , brain energy metabolism returned to normal to become abnormal after 612 h to decrease even further after 2448 h [ 6567 ] .
the concept of secondary energy failure has been elaborated in animal models , in particular in newborn piglets [ 6870 ] . using p - mrs in these animal models , neuroprotective strategies like hypothermia or 2-iminobiotin
an example of the changes in p - mrs during cerebral hypoxia ischaemia in the newborn piglet is presented in ( fig .
p - mrs was found to correlate with long - term neurodevelopmental outcome , especially when performed during the first few weeks after the insult [ 66 , 67 ] . in neonates with a very poor outcome , elevated intracellular ph , the so - called ph paradox , existed even for weeks after the insult , possibly due to changes in the na / h transporter .
7p - mrs of a newborn piglet at baseline ( top ) and after 1 h of cerebral hypoxia
the pcr / pi ratio and atp peaks decreased , whereas the distance between pcr and pi decreased , indicating a decrease in the intracellular ph p - mrs of a newborn piglet at baseline ( top ) and after 1 h of cerebral hypoxia
the pcr / pi ratio and atp peaks decreased , whereas the distance between pcr and pi decreased , indicating a decrease in the intracellular ph unfortunately , with the magnetic field strength of the current clinical systems , it is impossible to perform localised p - mrs , so only relatively large brain areas can be examined . due to these limitations , p - mrs has not become a routine clinical tool to assess asphyxiated full - term neonates .
proton mrs has also been used in neonates since the early 1990s [ 62 , 73 ] .
metabolites that can be demonstrated with h - mrs and echo times of 136 or more milliseconds are choline ( cho ) , creatine and phosphocreatine as a single peak ( cr ) , n - acetylaspartate ( naa ) and when present lactate ( lac ) . with shorter echo times , myo - inositol and the combined glutamate glutamine
given the large amounts of water in the neonatal brain , high - quality water suppression is essential for h - mrs .
the detection level of the aforementioned metabolites is in the millimolar range . as with p - mrs ,
although some have used water as an internal standard , this may not be appropriate in asphyxiated full - term neonates with changing amounts of intracellular and extracellular brain water .
therefore , metabolite ratios such as naa / cho , naa / cr or lac / naa are used instead .
it has been used as a marker of neuronal integrity . with selective loss of neurons ,
8h - mrs of a full - term neonate , 4 days after perinatal asphyxia .
naa / cho is still within the normal range for full - term neonates , but a very large lactate resonance can be identified at1.33 ppm
. a original spectrum of te 35 ms , b original spectrum of te 144 ms , c fitted spectrum of te 35 ms , and d fitted spectrum of te 144 ms h - mrs of a full - term neonate , 4 days after perinatal asphyxia .
naa / cho is still within the normal range for full - term neonates , but a very large lactate resonance can be identified at1.33 ppm
. a original spectrum of te 35 ms , b original spectrum of te 144 ms , c fitted spectrum of te 35 ms , and d fitted spectrum of te 144 ms previously , the changes in h - mrs during normal development have been demonstrated . in a previous study , we have shown an increase in the naa / cho ratio between preterm at a gestational age of 32 weeks and term equivalent age .
others have shown that lactate is a normal component in the preterm brain . we have demonstrated lactate in the brain of the asphyxiated full - term neonate days after the insult .
experimental work in animals showed accumulation of lactate in the brain during secondary energy failure which could be decreased with appropriate neuroprotective strategies .
an advantage of h - mrs over p - mrs is the possibility to perform localised examinations .
volumes of brain tissue as small as 1 cc can be assessed in 1.5-t mr systems .
thereby , changes in brain areas that are particularly vulnerable , such as the basal ganglia and thalamus , can be demonstrated .
in addition , chemical shift imaging enables us to measure metabolites in a matrix of voxels overlying the brain .
9 , where h - mrs chemical shift imaging is presented of a neonate with a large infarct in the territory of the right middle cerebral artery .
9chemical shift imaging a neonate with a large stroke in the territory of the right middle cerebral artery ( a ) . in the infarcted area , naa concentration
is decreased ( b , red ) , whereas lac / naa ratio is increased ( c , yellow ) chemical shift imaging a neonate with a large stroke in the territory of the right middle cerebral artery ( a ) . in the infarcted area , naa concentration is decreased ( b , red ) , whereas lac / naa ratio is increased ( c , yellow ) one of the limitations of h - mrs is the loss of quality by susceptibility .
we and others have shown decreases in naa / cho or naa / cr and elevations of lac / naa to predict a poor neurodevelopmental outcome [ 75 , 80 ] .
measurements performed after the second week of life may show normalised pcr / pi ratios .
mr imaging performed before the fourth day after the insult may not yet demonstrate changes on t1- and t2-weighted images .
diffusion - weighted images may show pseudo - normalisation , which complicates assessment of the severity of the insult .
abnormalities using h - mrs may only be missed , when this technique is performed very early , i.e. during the first day of life , before the development of secondary energy failure .
based on these aspects , it has been suggested that h - mrs is the best mr biomarker to predict neurodevelopmental outcome in asphyxiated full - term neonates .
however , since brain metabolite ratios may vary between mr systems and coils , development of normal values in one s own setting is required , and support of physicists is mandatory .
the limiting factor in the development of these normal values may be the lack of
overall , there has been tremendous progress in the mri technique over the last few decades .
both mri and proton mrs can help in detecting different patterns of brain injury in ( full - term ) human neonates following hypoxic
we recommend to perform mri and mrs in every full - term infant with encephalopathy and/or seizures admitted to a neonatal intensive care unit . | enormous progress has been made in assessing the neonatal brain , using magnetic resonance imaging ( mri ) . in this review
, we will describe the use of mri and proton magnetic resonance spectroscopy in detecting different patterns of brain injury in ( full - term ) human neonates following hypoxic
ischaemic brain injury and indicate the relevance of these findings in predicting neurodevelopmental outcome . | Introduction
MRI
Timing of insult
Main patterns of injury
MRS
None
None
Conclusion | fifty years ago , experiments have demonstrated that perinatal asphyxia could induce brain injury in primates . since then
, different patterns of brain injury have been established , which were dependent on the severity and duration of the hypoxic
these findings in animal studies were comparable to postmortem findings in asphyxiated human neonates . during the 1960s to 1980s ,
imaging of brain injury was performed in human neonates using positron emission tomography scans and ultrasound . since the introduction of magnetic resonance imaging ( mri )
, the knowledge of localisation and severity of brain injury following perinatal asphyxia ( hypoxic ischaemic brain lesions ) in surviving neonates has expanded tremendously [ 35 ] . in addition , phosphorus and proton mr spectroscopy ( mrs ) have enabled us to detect metabolic changes in the neonatal brain following hypoxia ischaemia [ 6 , 7 ] . in this review
, we will describe the use of mri and proton mrs in detecting different patterns of brain injury in ( full - term ) human neonates following hypoxic
although cranial ultrasound can still play a role in the full - term infant with hypoxic
ischaemic brain injury , mri is the method of choice to obtain more detailed and accurate information . we do , however , recommend the use of mri in all full - term infants who present with neonatal encephalopathy and/or seizures . the use of ct is restricted to infants who present with a large intracranial haemorrhage with a shift of the midline seen on cranial ultrasound . the increased use of neuroimaging techniques and mri , in particular , has been a tremendous help in timing of brain injury and recognising patterns of injury [ 4 , 12 , 13 ] . were able to show that more than 90% of affected newborns had evidence of perinatally acquired lesions on their mri , with a very low rate of established antenatal brain injury . these findings may be important for genetic counselling as well as for medicolegal issues ( fig . the child died and was subsequently diagnosed to have molybdenum cofactor deficiency the use of diffusion - weighted imaging ( dwi ) has also greatly improved our ability to time the onset of brain lesions . comparable to studies in a primate model , two main patterns of injury can be distinguished with mri in the full - term neonate :
basal ganglia
thalamus pattern ( bgt ) predominantly affecting bilaterally the central grey nuclei ( ventrolateral thalami and posterior putamina ) and perirolandic cortex . measured adc values within the plic in 28 term infants with a clinical diagnosis of hypoxic ischaemic encephalopathy ( hie ) at a mean age of 5.6 days . measuring fractional anisotropy ( fa ) was noted to be superior to measuring adc values in predicting outcome . studied 48 children at a mean age of 9 years ( range 413 years ) with dyskinetic cp mostly due to bgt injury and found that most children had gross motor function classification system levels of level iv , n = 10 , and level v , n = 28 . 2full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . severe motor impairment is uncommon in this group of infants , and they are often considered to have an early normal outcome , when seen at 1218 months . the 677c > t mutation in mothers , either in a homozygous or heterozygous state , together with poor nutritional status ( probable folate deficiency ) may represent a risk factor for irreversible brain injury in the offspring . 4full - term infant with white brain pattern of injury . note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d)another pattern of brain injury consists of lesions restricted to the periventricular white matter
, not dissimilar from the so - called punctate white matter lesions in the preterm infant ( fig . diagnosed this pattern of injury in 23% of their infants and pointed out that infants with this type of injury are significantly less mature with a milder degree of encephalopathy and fewer clinical seizures relative to other newborns in their cohort , who were diagnosed to have the two more common patterns of injury . this pattern of brain injury is also seen in newborn infants with congenital heart defects . 5full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi . measured adc values within the plic in 28 term infants with a clinical diagnosis of hypoxic ischaemic encephalopathy ( hie ) at a mean age of 5.6 days . studied 48 children at a mean age of 9 years ( range 413 years ) with dyskinetic cp mostly due to bgt injury and found that most children had gross motor function classification system levels of level iv , n = 10 , and level v , n = 28 . 2full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . also note involvement of the hippocampi full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . b ) the adc map shows low - signal intensity in the posterior watershed areas , as well as the splenium of the corpus callosum and the optic radiation full - term infant with watershed pattern of injury . the 677c > t mutation in mothers , either in a homozygous or heterozygous state , together with poor nutritional status ( probable folate deficiency ) may represent a risk factor for irreversible brain injury in the offspring . 4full - term infant with white brain pattern of injury . note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d ) full - term infant with white brain pattern of injury . note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d ) another pattern of brain injury consists of lesions restricted to the periventricular white matter , not dissimilar from the so - called punctate white matter lesions in the preterm infant ( fig . diagnosed this pattern of injury in 23% of their infants and pointed out that infants with this type of injury are significantly less mature with a milder degree of encephalopathy and fewer clinical seizures relative to other newborns in their cohort , who were diagnosed to have the two more common patterns of injury . this pattern of brain injury is also seen in newborn infants with congenital heart defects . there is also mild involvement of the corpus callosum and plic , seen on dwi full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi . diffusion tensor tractography shows loss of fibres with the corticospinal tract of the affected hemisphere recently developed techniques , such as diffusion tensor imaging ( dti ) , will allow quantification and visualisation of white matter pathways in vivo . a fa map can show asymmetry of the plic as early as the neonatal period . the increased use of neuroimaging techniques and mri , in particular , has been a tremendous help in timing of brain injury and recognising patterns of injury [ 4 , 12 , 13 ] . were able to show that more than 90% of affected newborns had evidence of perinatally acquired lesions on their mri , with a very low rate of established antenatal brain injury . performing cranial ultrasound on admission is also of value , as most of these lesions suggestive of an antenatal insult or an underlying problem will be recognised with ultrasound . the child died and was subsequently diagnosed to have molybdenum cofactor deficiency the use of diffusion - weighted imaging ( dwi ) has also greatly improved our ability to time the onset of brain lesions . comparable to studies in a primate model , two main patterns of injury can be distinguished with mri in the full - term neonate :
basal ganglia
thalamus pattern ( bgt ) predominantly affecting bilaterally the central grey nuclei ( ventrolateral thalami and posterior putamina ) and perirolandic cortex . measured adc values within the plic in 28 term infants with a clinical diagnosis of hypoxic ischaemic encephalopathy ( hie ) at a mean age of 5.6 days . measuring fractional anisotropy ( fa ) was noted to be superior to measuring adc values in predicting outcome . studied 48 children at a mean age of 9 years ( range 413 years ) with dyskinetic cp mostly due to bgt injury and found that most children had gross motor function classification system levels of level iv , n = 10 , and level v , n = 28 . 2full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . the 677c > t mutation in mothers , either in a homozygous or heterozygous state , together with poor nutritional status ( probable folate deficiency ) may represent a risk factor for irreversible brain injury in the offspring . note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d)another pattern of brain injury consists of lesions restricted to the periventricular white matter
, not dissimilar from the so - called punctate white matter lesions in the preterm infant ( fig . diagnosed this pattern of injury in 23% of their infants and pointed out that infants with this type of injury are significantly less mature with a milder degree of encephalopathy and fewer clinical seizures relative to other newborns in their cohort , who were diagnosed to have the two more common patterns of injury . this pattern of brain injury is also seen in newborn infants with congenital heart defects . 5full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi . measuring fractional anisotropy ( fa ) was noted to be superior to measuring adc values in predicting outcome . studied 48 children at a mean age of 9 years ( range 413 years ) with dyskinetic cp mostly due to bgt injury and found that most children had gross motor function classification system levels of level iv , n = 10 , and level v , n = 28 . 2full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . also note involvement of the hippocampi full - term infant with acute sentinel event ( ruptured uterus ) with mri pattern suggestive of acute near total asphyxia . severe motor impairment is uncommon in this group of infants , and they are often considered to have an early normal outcome , when seen at 1218 months . b ) the adc map shows low - signal intensity in the posterior watershed areas , as well as the splenium of the corpus callosum and the optic radiation full - term infant with watershed pattern of injury . the 677c > t mutation in mothers , either in a homozygous or heterozygous state , together with poor nutritional status ( probable folate deficiency ) may represent a risk factor for irreversible brain injury in the offspring . note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d ) full - term infant with white brain pattern of injury . note high - signal intensity of the mesencephalon ( c ) on t2se and symmetrical restricted diffusion in the cerebral peduncles and also in the cerebellum ( d ) another pattern of brain injury consists of lesions restricted to the periventricular white matter
, not dissimilar from the so - called punctate white matter lesions in the preterm infant ( fig . diagnosed this pattern of injury in 23% of their infants and pointed out that infants with this type of injury are significantly less mature with a milder degree of encephalopathy and fewer clinical seizures relative to other newborns in their cohort , who were diagnosed to have the two more common patterns of injury . this pattern of brain injury is also seen in newborn infants with congenital heart defects . 5full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi . there is also mild involvement of the corpus callosum and plic , seen on dwi full - term infant with punctate white matter lesions seen as low signal intensity changes on t2se ( tr 6284/te 120 ) and as areas of restricted diffusion on dwi . diffusion tensor tractography shows loss of fibres with the corticospinal tract of the affected hemisphere recently developed techniques , such as diffusion tensor imaging ( dti ) , will allow quantification and visualisation of white matter pathways in vivo . a fa map can show asymmetry of the plic as early as the neonatal period . mrs has been used to study changes in cerebral metabolism of neonates following ( perinatal ) hypoxia ischaemia . it has been shown that these metabolite ratios change during development , especially during the first years of life . in animal experiments
full - term neonates with perinatal asphyxia have also been studied [ 64 , 65 ] . the concept of secondary energy failure has been elaborated in animal models , in particular in newborn piglets [ 6870 ] . using p - mrs in these animal models ,
an example of the changes in p - mrs during cerebral hypoxia ischaemia in the newborn piglet is presented in ( fig . p - mrs was found to correlate with long - term neurodevelopmental outcome , especially when performed during the first few weeks after the insult [ 66 , 67 ] . due to these limitations , p - mrs has not become a routine clinical tool to assess asphyxiated full - term neonates . with shorter echo times , myo - inositol and the combined glutamate
given the large amounts of water in the neonatal brain , high - quality water suppression is essential for h - mrs . as with p - mrs ,
although some have used water as an internal standard , this may not be appropriate in asphyxiated full - term neonates with changing amounts of intracellular and extracellular brain water . with selective loss of neurons ,
8h - mrs of a full - term neonate , 4 days after perinatal asphyxia . naa / cho is still within the normal range for full - term neonates , but a very large lactate resonance can be identified at1.33 ppm
. a original spectrum of te 35 ms , b original spectrum of te 144 ms , c fitted spectrum of te 35 ms , and d fitted spectrum of te 144 ms h - mrs of a full - term neonate , 4 days after perinatal asphyxia . naa / cho is still within the normal range for full - term neonates , but a very large lactate resonance can be identified at1.33 ppm
. in a previous study , we have shown an increase in the naa / cho ratio between preterm at a gestational age of 32 weeks and term equivalent age . we have demonstrated lactate in the brain of the asphyxiated full - term neonate days after the insult . volumes of brain tissue as small as 1 cc can be assessed in 1.5-t mr systems . based on these aspects , it has been suggested that h - mrs is the best mr biomarker to predict neurodevelopmental outcome in asphyxiated full - term neonates . the limiting factor in the development of these normal values may be the lack of
p - mrs was one of the first mrs techniques to be used in neonates more than two decades ago [ 6 , 7 ] . it has been shown that these metabolite ratios change during development , especially during the first years of life . full - term neonates with perinatal asphyxia have also been studied [ 64 , 65 ] . the concept of secondary energy failure has been elaborated in animal models , in particular in newborn piglets [ 6870 ] . using p - mrs in these animal models , neuroprotective strategies like hypothermia or 2-iminobiotin
an example of the changes in p - mrs during cerebral hypoxia ischaemia in the newborn piglet is presented in ( fig . p - mrs was found to correlate with long - term neurodevelopmental outcome , especially when performed during the first few weeks after the insult [ 66 , 67 ] . due to these limitations , p - mrs has not become a routine clinical tool to assess asphyxiated full - term neonates . with shorter echo times , myo - inositol and the combined glutamate glutamine
given the large amounts of water in the neonatal brain , high - quality water suppression is essential for h - mrs . as with p - mrs ,
although some have used water as an internal standard , this may not be appropriate in asphyxiated full - term neonates with changing amounts of intracellular and extracellular brain water . with selective loss of neurons ,
8h - mrs of a full - term neonate , 4 days after perinatal asphyxia . naa / cho is still within the normal range for full - term neonates , but a very large lactate resonance can be identified at1.33 ppm
. a original spectrum of te 35 ms , b original spectrum of te 144 ms , c fitted spectrum of te 35 ms , and d fitted spectrum of te 144 ms h - mrs of a full - term neonate , 4 days after perinatal asphyxia . naa / cho is still within the normal range for full - term neonates , but a very large lactate resonance can be identified at1.33 ppm
. in a previous study , we have shown an increase in the naa / cho ratio between preterm at a gestational age of 32 weeks and term equivalent age . we have demonstrated lactate in the brain of the asphyxiated full - term neonate days after the insult . based on these aspects , it has been suggested that h - mrs is the best mr biomarker to predict neurodevelopmental outcome in asphyxiated full - term neonates . the limiting factor in the development of these normal values may be the lack of
overall , there has been tremendous progress in the mri technique over the last few decades . both mri and proton mrs can help in detecting different patterns of brain injury in ( full - term ) human neonates following hypoxic
we recommend to perform mri and mrs in every full - term infant with encephalopathy and/or seizures admitted to a neonatal intensive care unit . | [
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] |
this cross - sectional study was conducted between april 2014 and june 2014 in bandirma state hospital , balikesir , turkey .
the population of the present study comprised 585 patients diagnosed with physical illness and hospitalized between april 2014 and june 2014 in a state hospital . in the study ,
the sample of the study consisted of 470 patients who met the inclusion criteria and agreed to participate in the study .
criteria for inclusion were as follows : agreeing to participate in the study , being older than 18 years , being at least literate , being diagnosed with physical illness , being hospitalized in the internal disease clinics or surgery clinics .
criteria for exclusion were as follows : perception disorders , being hospitalized at the psychiatry , pediatrics , or intensive care services due to illness features and age .
the study data were collected with the personal information form , suicide probability scale , brief symptom inventory ( bsi ) and ways of coping with stress inventory ( wcsi ) .
the form prepared by the researchers through the literature review consists of 19 items questioning some of the socio - demographic characteristics ( age , gender , education , marital status , occupation , economic status , residence ) , psychosocial characteristics ( alcohol use , immigration status , previous psychiatric diagnosis , family history of mental disorders , history of suicide attempts , history of suicide in the family ) , the clinic where the person received treatment , and medical diagnosis of the participants .
this self - report , 36-item , and 4-point likert - type scale was developed by cull and gill in order to assess suicide risk in adolescents and adults . the turkish validity and reliability of the scale was conducted by atli et al16 in 2009 .
the scale includes 4 dimensions : hopelessness , suicidal ideation , negative self - evaluation , and hostility .
each of the sub dimensions receives a total score and the sum of all scores gives the overall suicide probability score .
in addition , total scores are divided into 4 categories in order to assess suicide risk : 0 - 24 points correspond to the normal group , 25 - 49 to the mild risk group , 50 - 74 to the medium risk group , and 75 - 100 to the high risk group.16 in this study , cronbach s alpha coefficient of the scale was calculated as 0.85 .
the validity and reliability study of the turkish version of the scale was conducted by sahin and durak20 in 1994 .
the 53-item likert - type scale is scored between 0 and 4 and consists of five subscales : anxiety , depression , negative self , somatization and hostility .
total scores range from 0 to 212.20 in this study , cronbach s alpha coefficient of the scale was calculated as 0.96 .
the validity and reliability study of the turkish version of the scale was conducted by sahin and durak21 in 1995 .
the 30-item likert - type scale is scored between 0 and 3 and consists of five subscales : self - confident approach , optimistic approach , helpless approach , submissive approach , and social support seeking approach . the self - confident , optimistic and social support seeking approaches
are considered as effective ways of coping with problems ; helpless approach and submissive approach are considered as ineffective / emotion focused ways of coping.21 the cronbach a reliability of the scale in this study was calculated as 0.74 .
the personal information form , sps , bsi , and wcsi were filled in through one - to - one interviews .
prior to data collection , necessary approvals and permissions were obtained from the cumhuriyet university ethics committee ( decision number : 04/11 ) and general secretariat of the public hospitals union , respectively .
the aim of the study was explained to all the study participants and oral consent of the participants was taken .
statistical analyses were performed using the statistical package for social sciences version 16.0 ( spss inc . ,
correlations between psychological symptoms , coping characteristics , and suicide probability were computed through the pearson s correlation analysis .
correlation between continuous variables were categorized as low ( correlation coefficient was between 0.100.29 ) , moderate ( between 0.30 - 0.49 ) and high ( > 0.50 ) according to their correlation coefficient values . to identify the independent variables ( psychological symptoms , coping strategies ) that contribute to suicide probability
this cross - sectional study was conducted between april 2014 and june 2014 in bandirma state hospital , balikesir , turkey .
the population of the present study comprised 585 patients diagnosed with physical illness and hospitalized between april 2014 and june 2014 in a state hospital . in the study ,
the sample of the study consisted of 470 patients who met the inclusion criteria and agreed to participate in the study .
criteria for inclusion were as follows : agreeing to participate in the study , being older than 18 years , being at least literate , being diagnosed with physical illness , being hospitalized in the internal disease clinics or surgery clinics .
criteria for exclusion were as follows : perception disorders , being hospitalized at the psychiatry , pediatrics , or intensive care services due to illness features and age .
the study data were collected with the personal information form , suicide probability scale , brief symptom inventory ( bsi ) and ways of coping with stress inventory ( wcsi ) .
the form prepared by the researchers through the literature review consists of 19 items questioning some of the socio - demographic characteristics ( age , gender , education , marital status , occupation , economic status , residence ) , psychosocial characteristics ( alcohol use , immigration status , previous psychiatric diagnosis , family history of mental disorders , history of suicide attempts , history of suicide in the family ) , the clinic where the person received treatment , and medical diagnosis of the participants .
this self - report , 36-item , and 4-point likert - type scale was developed by cull and gill in order to assess suicide risk in adolescents and adults . the turkish validity and reliability of the scale was conducted by atli et al16 in 2009 .
the scale includes 4 dimensions : hopelessness , suicidal ideation , negative self - evaluation , and hostility .
each of the sub dimensions receives a total score and the sum of all scores gives the overall suicide probability score .
in addition , total scores are divided into 4 categories in order to assess suicide risk : 0 - 24 points correspond to the normal group , 25 - 49 to the mild risk group , 50 - 74 to the medium risk group , and 75 - 100 to the high risk group.16 in this study , cronbach s alpha coefficient of the scale was calculated as 0.85 .
the validity and reliability study of the turkish version of the scale was conducted by sahin and durak20 in 1994 .
the 53-item likert - type scale is scored between 0 and 4 and consists of five subscales : anxiety , depression , negative self , somatization and hostility .
total scores range from 0 to 212.20 in this study , cronbach s alpha coefficient of the scale was calculated as 0.96 .
the validity and reliability study of the turkish version of the scale was conducted by sahin and durak21 in 1995 .
the 30-item likert - type scale is scored between 0 and 3 and consists of five subscales : self - confident approach , optimistic approach , helpless approach , submissive approach , and social support seeking approach . the self - confident , optimistic and social support seeking approaches
are considered as effective ways of coping with problems ; helpless approach and submissive approach are considered as ineffective / emotion focused ways of coping.21 the cronbach a reliability of the scale in this study was calculated as 0.74 .
the personal information form , sps , bsi , and wcsi were filled in through one - to - one interviews .
the study protocol was designed in compliance with the declaration of helsinki . prior to data collection ,
necessary approvals and permissions were obtained from the cumhuriyet university ethics committee ( decision number : 04/11 ) and general secretariat of the public hospitals union , respectively .
the aim of the study was explained to all the study participants and oral consent of the participants was taken .
statistical analyses were performed using the statistical package for social sciences version 16.0 ( spss inc . ,
correlations between psychological symptoms , coping characteristics , and suicide probability were computed through the pearson s correlation analysis .
correlation between continuous variables were categorized as low ( correlation coefficient was between 0.100.29 ) , moderate ( between 0.30 - 0.49 ) and high ( > 0.50 ) according to their correlation coefficient values . to identify the independent variables ( psychological symptoms , coping strategies ) that contribute to suicide probability
the mean age of the participants was 47.1417.58 . of the participants , 52.8% were female ,
64.9% were married , 41.5% graduated from elementary school , and 90.4% lived with their families . whereas , 48.9% of the participants were receiving treatment at the surgery clinics , 51.1% were receiving treatment at the internal disease clinics .
of the participants , 78.7% had moderate income levels , 67.7% lived in a county , 86% had not consulted a psychiatrist before , 14% were admitted to a psychiatry clinic before , 91.5% had never considered committing suicide , 8.5% had considered it , 96.6% had not attempted to commit suicide and 3.4% had attempted .
the mean suicide probability score of the participants ( 66.5411.64 ) was below average level and the mean score obtained from the negative self subscale ( 22.775.14 ) was higher than the mean scores obtained from the other subscales .
when we categorized the scores obtained from the sps , we determined that 74.7% of the participants were at moderate risk for suicide and that 20.4% were at high risk . according to the bsi ,
the psychological condition of the participants was good ( 40.6629.32 ) , however , their depression scores ( 10.667.88 ) were higher than their scores for other psychological symptoms . regarding the wcsi , the participants mostly used the self - confident approach ( 13.553.82 ) and the optimistic approach ( 9.212.84 ) ( table 1 ) .
distribution of suicide probability scale , brief symptom inventory and ways of coping with stress inventory total and subscale scores . in table 2 , the relationship between suicide probability scores and wcsi and bsi subscale scores were shown .
there was a strong positive correlation ( r ranging from 0.40 to 0.66 ) between the mean total score of suicide probability and bsi subscale scores ( p<0.001 )
. the mean total score of suicide probability positively correlated with the mean scores for the helpless and submissive coping approaches ( r ranging from 0.38 to 0.23 ) and negatively correlated with the optimistic approach and social support seeking subscale scores ( r ranging from -0.13 to -0.15 ) .
the hopelessness , suicidal ideation , and hostility subscale scores of the sps negatively correlated ( r ranging from -0.15 to -0.36 ) with the self - confident approach , optimistic approach , and social support seeking subscale scores of the wcsi ; whereas they positively correlated ( r ranging from 0.15 to 0.48 ) with the mean scores for the helpless approach and submissive approach . all subscales of the bsi negatively correlated ( r ranging from -0.11 to -0.31 ) with the self - confident approach , optimistic approach and social support seeking subscales of the wcsi while they positively correlated ( r ranging from 0.11 to 0.42 ) with the submissive and helpless coping approaches .
correlations between suicide probability scale , ways of coping with stress inventory and brief symptom inventory . *
1 - sps total , 2 - hopelessness , 3 - suicidal ideation , 4 - negative self - evaluation , 5 - hostility , 6 - self - confident approach , 7 - optimistic approach , 8 - helpless approach , 9 - submissive approach , 10 - social support seeking approach , 11 - anxiety , 12 - depression , 13 - negative self , 14 - somatization , 15 - hostility , p<0.01 , sps - suicide probability scale , wcsi - coping with stress inventory , bsi - brief symptom inventory the stepwise multiple linear regression analysis , which examined suicide probability according to the bsi and the wcsi , is summarized in table 3 .
the t - test results , which indicate the significance of regression coefficients , revealed that the wcsi and the bsi subscale variables were significant predictors of suicide probability ( r=0.74 , r=0.55 , f=71.45 , p<0.001 ) .
wcsi and the bsi subscale variables , together , explain 55% of the variance in suicide probability . according to the standardized regression coefficient ( ) , variables that affect suicide probability are the negative self subscale ( =0.35 ) of the bsi , the hostility subscale ( =0.25 ) , the anxiety subscale ( =0.24 ) , the self - confident approach subscale ( =0.22 ) of the wcsi , the somatization subscale ( =-0.14 ) of the bsi , the helpless approach subscale ( =0.09 ) of the wcsi , the optimistic approach ( =-0.08 ) , and the submissive approach ( =0.08 ) respectively .
variables that were excluded from the stepwise multiple linear regression analysis were the depression subscale of the bsi and the social support seeking subscale of the wcsi . stepwise regression analysis of suicide probability .
* r=0.55 , durbin - watson , 1.752 ( p<0.001 ) , wcsi - coping with stress inventory , bsi - brief symptom inventory , se - standard error
the results of the current study were discussed from 2 perspectives : suicide probability , psychological condition , and coping status .
people with physical illness are at high risk for suicide.2,19 in this study , it was determined that the suicide probability of the participants was at moderate levels and that the majority of the participants were at high risk for suicide . in a study by marusic and goodwin10 conducted with 415 people with physical illness
also , in studies that examine completed or attempted suicides , it was reported that having physical illness is a risk factor for suicide.4,19,22 previous studies pointed to the suicide rates at general hospitals where people with physical illness receive treatment . in a study conducted in finland ,
the rate of suicides committed at 26 general hospitals was 1.9% compared to all suicide cases.19 in people with physical illness , reasons including increased length and frequency of hospitalization , relapses , and deterioration of mental health and quality of life can increase suicidal ideation . in this context , it is important to assess individuals who are newly diagnosed for psychological symptoms and suicide risk starting from the onset of illness . in a study conducted in the united kingdom ,
approximately half of the elderly patients who committed suicide visited their family doctors prior to their death and more than 5% of these visits were due to physical complaints.22 therefore , nurses who are on duty for 24 hours should monitor patients with physical illness for suicide risk during their hospitalization and should prevent suicide by taking the necessary precautions in order to provide patient safety and to fulfill professional responsibilities .
short - term or long - term medicine use , organ damage , or comorbid diseases cause physical illnesses which affect one s mental health.7 this condition may also have a negative impact on disease prognosis . in the present study ,
it was determined that depression was the most frequently observed psychological symptom among people with physical illness .
other studies demonstrated that people with a chronic disease are at high risk for mental disorders.1 - 3 since better psychiatric conditions will have a positive effect on recovering from physical illness as well as preventing the development of physical and psychiatric complications , it is important for nurses to detect and meet psychiatric needs of patients in addition to physical needs .
individuals who face illness develop various coping approaches in order to cope with the illness and to minimize the negative aspects of the illness.23 each patient s personality , illness perceptions , coping strategies , and responses to illness vary . in this study
this is a positive finding and can be explained by the fact that most of the participants were past the middle ages and therefore had more experience regarding illness .
in addition , most participants lived with their families , which provided a social support network for them .
the use of effective coping styles is important in terms of disease management . in a study conducted with hemodialysis patients ,
the patients were determined to use positive coping styles.18 in another study , the participants used support systems for coping more frequently.6 the relationships between suicide probability , psychological condition , and coping .
the development of psychiatric disorders in people with physical illness facilitates suicide.19 in the study , suicide probability increased as psychiatric disorders intensified . in a study by qin
et al,1 physical and psychiatric disorders are important risk factors for suicide and suicide rates of people with physical illness and comorbid psychiatric disorders are high . in other studies ,
levels of suicidal ideation increased as the severity of depression increased.18,24 retrospective studies also revealed that most of the suicide cases in general hospitals had psychiatric disorders.19,25 in a meta - analytic study , the suicide rate was 10 times more common among people with psychiatric disorders compared to the general population.26 hospitalized people show increases in physical symptoms , limitations , and the need for medicine . this condition can have a negative impact on the psychological state of people with physical illness and can increase suicidal ideation .
the idea of ending one s life can be a source of stress for some people and can be a way for relieving stress for others.27 detecting the coping styles of people with physical illness helps determine treatment goals and therapeutic effectiveness and also helps prevent the emergence of additional problems such as suicide.23 in the current study , it was determined that people who use helpless and submissive coping styles had increased suicide probability and that those who used an optimistic style and those who sought social support had decreased levels of suicide probability . in a study by marusic and
goodwin,10 the use of coping strategies such as avoidance and emotional procrastination was determined to be associated with suicidal ideation . in another study ,
active coping , planning , and positive reframing were found to be negatively correlated with suicidal ideation.18 other studies also determined that people who attempted suicide used effective coping styles to a lesser extent8,9,27,28 and that they had inadequate problem - solving skills.29 according to the study results , improving one s coping behaviors is an important indicator of suicide prevention .
in addition , in the study , those who exhibited hopelessness , suicidal ideation , and hostility adopted helpless and submissive coping styles more frequently . in this context
, nurses should evaluate the psychosocial characteristics of individuals during the treatment process . in this study , psychological symptoms and coping styles were determine to have significant effects on suicide probability .
half of the individuals considered suicide due to the presence of psychological symptoms and ineffective coping styles . in a study by ozguven et al30 people with high levels of anxiety symptoms and with precipitous problem - solving skills
were at high risk for suicide attempts . in a study by doucet and letourneau,9 emotion and
avoidance - focused coping styles precipitated suicidal ideation among people with postpartum depression and problem - focused coping strategies did not precipitate suicidal ideation .
the study results underline the importance of nurses roles in suicide prevention , which includes detecting psychological symptoms and promoting effective coping styles in patients .
one of its limitations is that because the study was conducted with a relatively small group of patients receiving inpatient treatment at one health center and the results obtained from this study are applicable only to the study population and can not be generalized to all patients .
another limitation of the study is that the study results are limited to the data obtained from the scales based on self - expression . in conclusion ,
approximately all individuals with physical illness are at risk for suicide probability . people who exhibit psychological symptoms and use inadequate coping styles have higher suicide probability .
therefore , nurses should monitor patients for psychological symptoms beginning from the onset of physical illness and should promote effective coping styles among these patients . in patient care provided by nurses , spending enough time with patients and talking to them can facilitate the detection of their psychological condition and suicide probability .
in addition , providing regular in - service training composed of detection of psychological symptoms , promotion of effective coping styles and detection of suicide probability for health professionals and particularly for nurses can prevent adverse events . it is suggested that , in addition to psychological symptoms and coping styles , other factors that can cause suicide should be investigated in larger populations in order to prevent suicide among people with physical illness .
physical disorders can cause physical , mental , social and economic losses by affecting the entire life of an individual .
therefore , the presence of illness is a risk factor for suicide across all age groups .
however , in the literature , the number of studies which examine suicide risk in people with physical illness is limited , and studies only investigated affecting factors of completed suicides . in this context , conducting holistic evaluations of people with physical illness , determining risk factors and preventive factors for suicide and conducting research with large samples in the field as well as treatment centers will contribute to taking precautions against suicide .
one of its limitations is that because the study was conducted with a relatively small group of patients receiving inpatient treatment at one health center and the results obtained from this study are applicable only to the study population and can not be generalized to all patients .
another limitation of the study is that the study results are limited to the data obtained from the scales based on self - expression . in conclusion ,
approximately all individuals with physical illness are at risk for suicide probability . people who exhibit psychological symptoms and use inadequate coping styles have higher suicide probability .
therefore , nurses should monitor patients for psychological symptoms beginning from the onset of physical illness and should promote effective coping styles among these patients . in patient care provided by nurses , spending enough time with patients and talking to them can facilitate the detection of their psychological condition and suicide probability .
in addition , providing regular in - service training composed of detection of psychological symptoms , promotion of effective coping styles and detection of suicide probability for health professionals and particularly for nurses can prevent adverse events . it is suggested that , in addition to psychological symptoms and coping styles , other factors that can cause suicide should be investigated in larger populations in order to prevent suicide among people with physical illness .
physical disorders can cause physical , mental , social and economic losses by affecting the entire life of an individual .
therefore , the presence of illness is a risk factor for suicide across all age groups .
however , in the literature , the number of studies which examine suicide risk in people with physical illness is limited , and studies only investigated affecting factors of completed suicides . in this context , conducting holistic evaluations of people with physical illness , determining risk factors and preventive factors for suicide and conducting research with large samples in the field as well as treatment centers will contribute to taking precautions against suicide . | objective : to investigate the relationship between suicide probability and psychological symptoms and coping strategies in hospitalized patients with physical illness.methods:this cross - sectional study was conducted from april to june 2014 in bandirma state hospital , balikesir , turkey .
the sample of the study consisted of 470 inpatients who met the inclusion criteria and agreed to participate in the study .
the data were collected with the personal information form , suicide probability scale , brief symptom inventory and ways of coping with stress inventory.results:in the study , 74.7% were at moderate risk for suicide , whereas 20.4% were at high risk for suicide . according to the stepwise multiple linear regression analysis ,
sub - dimensions of the ways of coping with stress inventory and brief symptom inventory were the significant predictors of suicide probability.conclusions:the majority of the patients with physical illness were at risk for suicide probability .
individuals who had psychological symptoms and used maladaptive coping ways obtained significantly higher suicide probability scores . | Methods
Study design
Sample
Data collection instruments
Personal information form
Suicide probability scale (SPS)
Brief symptom inventory
Ways of coping with stress inventory
Application
Ethical approval
Statistical analysis
Results
Discussion
This study has some limitations | this cross - sectional study was conducted between april 2014 and june 2014 in bandirma state hospital , balikesir , turkey . the population of the present study comprised 585 patients diagnosed with physical illness and hospitalized between april 2014 and june 2014 in a state hospital . in the study ,
the sample of the study consisted of 470 patients who met the inclusion criteria and agreed to participate in the study . criteria for inclusion were as follows : agreeing to participate in the study , being older than 18 years , being at least literate , being diagnosed with physical illness , being hospitalized in the internal disease clinics or surgery clinics . the study data were collected with the personal information form , suicide probability scale , brief symptom inventory ( bsi ) and ways of coping with stress inventory ( wcsi ) . the form prepared by the researchers through the literature review consists of 19 items questioning some of the socio - demographic characteristics ( age , gender , education , marital status , occupation , economic status , residence ) , psychosocial characteristics ( alcohol use , immigration status , previous psychiatric diagnosis , family history of mental disorders , history of suicide attempts , history of suicide in the family ) , the clinic where the person received treatment , and medical diagnosis of the participants . the turkish validity and reliability of the scale was conducted by atli et al16 in 2009 . each of the sub dimensions receives a total score and the sum of all scores gives the overall suicide probability score . in addition , total scores are divided into 4 categories in order to assess suicide risk : 0 - 24 points correspond to the normal group , 25 - 49 to the mild risk group , 50 - 74 to the medium risk group , and 75 - 100 to the high risk group.16 in this study , cronbach s alpha coefficient of the scale was calculated as 0.85 . the validity and reliability study of the turkish version of the scale was conducted by sahin and durak20 in 1994 . total scores range from 0 to 212.20 in this study , cronbach s alpha coefficient of the scale was calculated as 0.96 . the validity and reliability study of the turkish version of the scale was conducted by sahin and durak21 in 1995 . the self - confident , optimistic and social support seeking approaches
are considered as effective ways of coping with problems ; helpless approach and submissive approach are considered as ineffective / emotion focused ways of coping.21 the cronbach a reliability of the scale in this study was calculated as 0.74 . the personal information form , sps , bsi , and wcsi were filled in through one - to - one interviews . prior to data collection , necessary approvals and permissions were obtained from the cumhuriyet university ethics committee ( decision number : 04/11 ) and general secretariat of the public hospitals union , respectively . the aim of the study was explained to all the study participants and oral consent of the participants was taken . ,
correlations between psychological symptoms , coping characteristics , and suicide probability were computed through the pearson s correlation analysis . correlation between continuous variables were categorized as low ( correlation coefficient was between 0.100.29 ) , moderate ( between 0.30 - 0.49 ) and high ( > 0.50 ) according to their correlation coefficient values . to identify the independent variables ( psychological symptoms , coping strategies ) that contribute to suicide probability
this cross - sectional study was conducted between april 2014 and june 2014 in bandirma state hospital , balikesir , turkey . the population of the present study comprised 585 patients diagnosed with physical illness and hospitalized between april 2014 and june 2014 in a state hospital . in the study ,
the sample of the study consisted of 470 patients who met the inclusion criteria and agreed to participate in the study . criteria for inclusion were as follows : agreeing to participate in the study , being older than 18 years , being at least literate , being diagnosed with physical illness , being hospitalized in the internal disease clinics or surgery clinics . the study data were collected with the personal information form , suicide probability scale , brief symptom inventory ( bsi ) and ways of coping with stress inventory ( wcsi ) . the form prepared by the researchers through the literature review consists of 19 items questioning some of the socio - demographic characteristics ( age , gender , education , marital status , occupation , economic status , residence ) , psychosocial characteristics ( alcohol use , immigration status , previous psychiatric diagnosis , family history of mental disorders , history of suicide attempts , history of suicide in the family ) , the clinic where the person received treatment , and medical diagnosis of the participants . the turkish validity and reliability of the scale was conducted by atli et al16 in 2009 . each of the sub dimensions receives a total score and the sum of all scores gives the overall suicide probability score . in addition , total scores are divided into 4 categories in order to assess suicide risk : 0 - 24 points correspond to the normal group , 25 - 49 to the mild risk group , 50 - 74 to the medium risk group , and 75 - 100 to the high risk group.16 in this study , cronbach s alpha coefficient of the scale was calculated as 0.85 . the validity and reliability study of the turkish version of the scale was conducted by sahin and durak20 in 1994 . total scores range from 0 to 212.20 in this study , cronbach s alpha coefficient of the scale was calculated as 0.96 . the validity and reliability study of the turkish version of the scale was conducted by sahin and durak21 in 1995 . the self - confident , optimistic and social support seeking approaches
are considered as effective ways of coping with problems ; helpless approach and submissive approach are considered as ineffective / emotion focused ways of coping.21 the cronbach a reliability of the scale in this study was calculated as 0.74 . the personal information form , sps , bsi , and wcsi were filled in through one - to - one interviews . the study protocol was designed in compliance with the declaration of helsinki . prior to data collection ,
necessary approvals and permissions were obtained from the cumhuriyet university ethics committee ( decision number : 04/11 ) and general secretariat of the public hospitals union , respectively . the aim of the study was explained to all the study participants and oral consent of the participants was taken . ,
correlations between psychological symptoms , coping characteristics , and suicide probability were computed through the pearson s correlation analysis . correlation between continuous variables were categorized as low ( correlation coefficient was between 0.100.29 ) , moderate ( between 0.30 - 0.49 ) and high ( > 0.50 ) according to their correlation coefficient values . to identify the independent variables ( psychological symptoms , coping strategies ) that contribute to suicide probability
the mean age of the participants was 47.1417.58 . of the participants , 78.7% had moderate income levels , 67.7% lived in a county , 86% had not consulted a psychiatrist before , 14% were admitted to a psychiatry clinic before , 91.5% had never considered committing suicide , 8.5% had considered it , 96.6% had not attempted to commit suicide and 3.4% had attempted . the mean suicide probability score of the participants ( 66.5411.64 ) was below average level and the mean score obtained from the negative self subscale ( 22.775.14 ) was higher than the mean scores obtained from the other subscales . when we categorized the scores obtained from the sps , we determined that 74.7% of the participants were at moderate risk for suicide and that 20.4% were at high risk . according to the bsi ,
the psychological condition of the participants was good ( 40.6629.32 ) , however , their depression scores ( 10.667.88 ) were higher than their scores for other psychological symptoms . distribution of suicide probability scale , brief symptom inventory and ways of coping with stress inventory total and subscale scores . in table 2 , the relationship between suicide probability scores and wcsi and bsi subscale scores were shown . there was a strong positive correlation ( r ranging from 0.40 to 0.66 ) between the mean total score of suicide probability and bsi subscale scores ( p<0.001 )
. the mean total score of suicide probability positively correlated with the mean scores for the helpless and submissive coping approaches ( r ranging from 0.38 to 0.23 ) and negatively correlated with the optimistic approach and social support seeking subscale scores ( r ranging from -0.13 to -0.15 ) . the hopelessness , suicidal ideation , and hostility subscale scores of the sps negatively correlated ( r ranging from -0.15 to -0.36 ) with the self - confident approach , optimistic approach , and social support seeking subscale scores of the wcsi ; whereas they positively correlated ( r ranging from 0.15 to 0.48 ) with the mean scores for the helpless approach and submissive approach . all subscales of the bsi negatively correlated ( r ranging from -0.11 to -0.31 ) with the self - confident approach , optimistic approach and social support seeking subscales of the wcsi while they positively correlated ( r ranging from 0.11 to 0.42 ) with the submissive and helpless coping approaches . correlations between suicide probability scale , ways of coping with stress inventory and brief symptom inventory . *
1 - sps total , 2 - hopelessness , 3 - suicidal ideation , 4 - negative self - evaluation , 5 - hostility , 6 - self - confident approach , 7 - optimistic approach , 8 - helpless approach , 9 - submissive approach , 10 - social support seeking approach , 11 - anxiety , 12 - depression , 13 - negative self , 14 - somatization , 15 - hostility , p<0.01 , sps - suicide probability scale , wcsi - coping with stress inventory , bsi - brief symptom inventory the stepwise multiple linear regression analysis , which examined suicide probability according to the bsi and the wcsi , is summarized in table 3 . the t - test results , which indicate the significance of regression coefficients , revealed that the wcsi and the bsi subscale variables were significant predictors of suicide probability ( r=0.74 , r=0.55 , f=71.45 , p<0.001 ) . wcsi and the bsi subscale variables , together , explain 55% of the variance in suicide probability . according to the standardized regression coefficient ( ) , variables that affect suicide probability are the negative self subscale ( =0.35 ) of the bsi , the hostility subscale ( =0.25 ) , the anxiety subscale ( =0.24 ) , the self - confident approach subscale ( =0.22 ) of the wcsi , the somatization subscale ( =-0.14 ) of the bsi , the helpless approach subscale ( =0.09 ) of the wcsi , the optimistic approach ( =-0.08 ) , and the submissive approach ( =0.08 ) respectively . variables that were excluded from the stepwise multiple linear regression analysis were the depression subscale of the bsi and the social support seeking subscale of the wcsi . stepwise regression analysis of suicide probability . * r=0.55 , durbin - watson , 1.752 ( p<0.001 ) , wcsi - coping with stress inventory , bsi - brief symptom inventory , se - standard error
the results of the current study were discussed from 2 perspectives : suicide probability , psychological condition , and coping status . people with physical illness are at high risk for suicide.2,19 in this study , it was determined that the suicide probability of the participants was at moderate levels and that the majority of the participants were at high risk for suicide . in a study by marusic and goodwin10 conducted with 415 people with physical illness
also , in studies that examine completed or attempted suicides , it was reported that having physical illness is a risk factor for suicide.4,19,22 previous studies pointed to the suicide rates at general hospitals where people with physical illness receive treatment . in a study conducted in finland ,
the rate of suicides committed at 26 general hospitals was 1.9% compared to all suicide cases.19 in people with physical illness , reasons including increased length and frequency of hospitalization , relapses , and deterioration of mental health and quality of life can increase suicidal ideation . in this context , it is important to assess individuals who are newly diagnosed for psychological symptoms and suicide risk starting from the onset of illness . in a study conducted in the united kingdom ,
approximately half of the elderly patients who committed suicide visited their family doctors prior to their death and more than 5% of these visits were due to physical complaints.22 therefore , nurses who are on duty for 24 hours should monitor patients with physical illness for suicide risk during their hospitalization and should prevent suicide by taking the necessary precautions in order to provide patient safety and to fulfill professional responsibilities . in the present study ,
it was determined that depression was the most frequently observed psychological symptom among people with physical illness . other studies demonstrated that people with a chronic disease are at high risk for mental disorders.1 - 3 since better psychiatric conditions will have a positive effect on recovering from physical illness as well as preventing the development of physical and psychiatric complications , it is important for nurses to detect and meet psychiatric needs of patients in addition to physical needs . individuals who face illness develop various coping approaches in order to cope with the illness and to minimize the negative aspects of the illness.23 each patient s personality , illness perceptions , coping strategies , and responses to illness vary . in this study
this is a positive finding and can be explained by the fact that most of the participants were past the middle ages and therefore had more experience regarding illness . in a study conducted with hemodialysis patients ,
the patients were determined to use positive coping styles.18 in another study , the participants used support systems for coping more frequently.6 the relationships between suicide probability , psychological condition , and coping . the development of psychiatric disorders in people with physical illness facilitates suicide.19 in the study , suicide probability increased as psychiatric disorders intensified . in a study by qin
et al,1 physical and psychiatric disorders are important risk factors for suicide and suicide rates of people with physical illness and comorbid psychiatric disorders are high . in other studies ,
levels of suicidal ideation increased as the severity of depression increased.18,24 retrospective studies also revealed that most of the suicide cases in general hospitals had psychiatric disorders.19,25 in a meta - analytic study , the suicide rate was 10 times more common among people with psychiatric disorders compared to the general population.26 hospitalized people show increases in physical symptoms , limitations , and the need for medicine . this condition can have a negative impact on the psychological state of people with physical illness and can increase suicidal ideation . the idea of ending one s life can be a source of stress for some people and can be a way for relieving stress for others.27 detecting the coping styles of people with physical illness helps determine treatment goals and therapeutic effectiveness and also helps prevent the emergence of additional problems such as suicide.23 in the current study , it was determined that people who use helpless and submissive coping styles had increased suicide probability and that those who used an optimistic style and those who sought social support had decreased levels of suicide probability . in a study by marusic and
goodwin,10 the use of coping strategies such as avoidance and emotional procrastination was determined to be associated with suicidal ideation . in another study ,
active coping , planning , and positive reframing were found to be negatively correlated with suicidal ideation.18 other studies also determined that people who attempted suicide used effective coping styles to a lesser extent8,9,27,28 and that they had inadequate problem - solving skills.29 according to the study results , improving one s coping behaviors is an important indicator of suicide prevention . in addition , in the study , those who exhibited hopelessness , suicidal ideation , and hostility adopted helpless and submissive coping styles more frequently . in this study , psychological symptoms and coping styles were determine to have significant effects on suicide probability . half of the individuals considered suicide due to the presence of psychological symptoms and ineffective coping styles . in a study by ozguven et al30 people with high levels of anxiety symptoms and with precipitous problem - solving skills
were at high risk for suicide attempts . in a study by doucet and letourneau,9 emotion and
avoidance - focused coping styles precipitated suicidal ideation among people with postpartum depression and problem - focused coping strategies did not precipitate suicidal ideation . the study results underline the importance of nurses roles in suicide prevention , which includes detecting psychological symptoms and promoting effective coping styles in patients . one of its limitations is that because the study was conducted with a relatively small group of patients receiving inpatient treatment at one health center and the results obtained from this study are applicable only to the study population and can not be generalized to all patients . another limitation of the study is that the study results are limited to the data obtained from the scales based on self - expression . in conclusion ,
approximately all individuals with physical illness are at risk for suicide probability . people who exhibit psychological symptoms and use inadequate coping styles have higher suicide probability . therefore , nurses should monitor patients for psychological symptoms beginning from the onset of physical illness and should promote effective coping styles among these patients . in addition , providing regular in - service training composed of detection of psychological symptoms , promotion of effective coping styles and detection of suicide probability for health professionals and particularly for nurses can prevent adverse events . it is suggested that , in addition to psychological symptoms and coping styles , other factors that can cause suicide should be investigated in larger populations in order to prevent suicide among people with physical illness . therefore , the presence of illness is a risk factor for suicide across all age groups . however , in the literature , the number of studies which examine suicide risk in people with physical illness is limited , and studies only investigated affecting factors of completed suicides . in this context , conducting holistic evaluations of people with physical illness , determining risk factors and preventive factors for suicide and conducting research with large samples in the field as well as treatment centers will contribute to taking precautions against suicide . one of its limitations is that because the study was conducted with a relatively small group of patients receiving inpatient treatment at one health center and the results obtained from this study are applicable only to the study population and can not be generalized to all patients . another limitation of the study is that the study results are limited to the data obtained from the scales based on self - expression . in conclusion ,
approximately all individuals with physical illness are at risk for suicide probability . people who exhibit psychological symptoms and use inadequate coping styles have higher suicide probability . therefore , nurses should monitor patients for psychological symptoms beginning from the onset of physical illness and should promote effective coping styles among these patients . in patient care provided by nurses , spending enough time with patients and talking to them can facilitate the detection of their psychological condition and suicide probability . in addition , providing regular in - service training composed of detection of psychological symptoms , promotion of effective coping styles and detection of suicide probability for health professionals and particularly for nurses can prevent adverse events . it is suggested that , in addition to psychological symptoms and coping styles , other factors that can cause suicide should be investigated in larger populations in order to prevent suicide among people with physical illness . therefore , the presence of illness is a risk factor for suicide across all age groups . however , in the literature , the number of studies which examine suicide risk in people with physical illness is limited , and studies only investigated affecting factors of completed suicides . in this context , conducting holistic evaluations of people with physical illness , determining risk factors and preventive factors for suicide and conducting research with large samples in the field as well as treatment centers will contribute to taking precautions against suicide . | [
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the pituitary gland is comprised of two dominant lobes , the neurohypophysis and the adenohypophysis .
it is situated within the sella turcica , a depression in the sphenoid bone which is covered by the diaphragm sella with a defect for the passage of the infundibulum .
the pituitary stalk extends inferiorly as a continuation of the hypothalamic infundibulum ( fig .
1normal pituitary anatomy normal pituitary anatomy the neurohypophysis receives its hormonal supply through direct contiguity with the hypothalamus , with axons extending from the hypothalamic supraoptic and paraventricular nuclei into the neurohypophysis .
the adenohypophysis does not have axonal contiguity between the hypothalamus and the anterior gland and relies on the hypothalamic - hypophyseal portal system .
parvocellular messengers are transported from the hypothalamic median eminence into the capillaries surrounding the anterior gland .
hormones from the adenohypophysis include : follicle stimulating hormone , luteinising hormone , corticotropin ( acth ) , thyroid - stimulating hormone , prolactin and growth hormone .
the pituitary gland develops through a complex mechanism involving the developing diencephalon and the primitive oral stomodeum .
the adenohypophysis derives from rathke s pouch , which originates rostral to the oropharyngeal membrane .
this begins at approximately 24 days of gestation , in which rathke s pouch forms as an ectodermal outpouching of stomodeum and the infundibulum which forms in the floor of the diencephalon .
rathke s pouch eventually loses its connection with the stomodeum and forms a sac which adheres to the infundibular process .
this sac continues to differentiate into the adenophyophysis and the distal aspect of the infundibulum differentiates to form the neurohypophysis ( fig .
the pituitary gland exhibits a wide range of appearances during the first 2 years of life , with considerable variability in size , shape , and signal . at birth ,
both the anterior and posterior pituitary gland have high signal intensity on t1-weighted images ( fig . 3 ) .
the anterior pituitary progressively decreases in signal intensity on t1-weighted images with signal drop to near isointensity with the posterior pons by 6 weeks of age [ 1 , 2 ] .
the posterior pituitary gland retains its high signal intensity on t1-weighted images throughout adulthood , with several papers supporting that this high signal results from neurosecretory granules or the protein neurophysin [ 35 ] .
the normal gland enhances symmetrically after intravenous gadolinium with the anterior gland enhancement appearing more noticeable given the posterior gland is already high signal at baseline.fig .
3normal physiological t1 sagittal ( a ) and axial ( b ) hyperintensity of the anterior pituitary in a 1-month - old baby normal physiological t1 sagittal ( a ) and axial ( b ) hyperintensity of the anterior pituitary in a 1-month - old baby at birth , the gland is physiologically enlarged with a corresponding concave superior margin , flattening with age .
the gland is traditionally symmetric in size ; however there can be asymmetry in normally functioning glands . in preterm infants ,
the gland is taller than in normal - term infants , even when correcting for gestational age .
this is thought to be the result of reduced insulin - like growth factor 1 and higher levels of growth hormone in premature infants .
the pituitary height is fairly stable for the first 2 years of life in normal - term children .
gender - associated differences in the rate of gland growth tend to occur after 5 years of age , with linear growth of the gland up to that point in both boys and girls . typically , the normal pituitary measures between 2 to 6 mm in height during childhood and up to 10 mm or slightly higher at puberty . in adults ,
standards for assessing the pituitary stalk size are not well established , and are based on experience rather than age - appropriate measurements .
the stalk is best visualised on post - contrast t1-weighted images because of the strong enhancement .
the stalk terminates into a bulbous neurohypophysis with high signal in the dorsal portion of the sella agenesis of the anterior pituitary gland ( adenohypophysis ) in an infant with hypopituitarism .
the stalk terminates into a bulbous neurohypophysis with high signal in the dorsal portion of the sella this is a rare congenital anomaly with a neonatal presentation . the neonates can present with metabolic acidosis , thyroid and adrenal insufficiency , hypoglycaemia , and/or seizures .
newborns are normal in size at birth , which has been explained by the presence of maternal growth hormone .
magnetic resonance imaging ( mri ) findings include the absence of the anterior pituitary gland with an associated shallow sella , and variable length of the infundibulum and position of the posterior pituitary , ranging from the hypothalamus to the sella . the infundibulum may be thinned and terminate into a partially formed bulbous posterior pituitary gland .
pre - contrast ( a ) and post - contrast ( b ) mr images show severe hypoplasia of the anterior pituitary gland which shows only minimal contrast enhancement along the floor of the sella .
the ectopic posterior pituitary is located adjacent to the optic chiasm severe hypoplasia of the anterior pituitary gland .
pre - contrast ( a ) and post - contrast ( b ) mr images show severe hypoplasia of the anterior pituitary gland which shows only minimal contrast enhancement along the floor of the sella .
the ectopic posterior pituitary is located adjacent to the optic chiasm this rare anomaly consists of hypoplasia of the anterior pituitary gland , and also potentially the infundibulum and posterior pituitary gland .
it can be associated with varying degrees of endocrine dysfunction , including panhypopituitarism and other congenital anomalies .
some research suggests that traumatic transection of the pituitary stalk during birth or hypoxic injury to the hypothalamus could result in hypoplasia , whereas others suggest an error during embryogenesis [ 912 ] .
mri findings include varying degrees of hypoplasia of the anterior pituitary gland with variable size and length of the infundibulum . as with agenesis of the adenohypophysis ,
pre - contrast ( a ) and post - contrast ( b ) mr images show a normal anterior pituitary gland with contrast enhancement and an ectopic posterior pituitary located adjacent to the optic chiasm ectopic posterior pituitary gland .
pre - contrast ( a ) and post - contrast ( b ) mr images show a normal anterior pituitary gland with contrast enhancement and an ectopic posterior pituitary located adjacent to the optic chiasm this congenital anomaly results in an aberrant position of the neurohypophysis , often located at the undersurface of the hypothalamus .
it is associated with pituitary dwarfism , delayed skeletal maturation , kallman syndrome , septo - optic dysplasia , dysgenesis of the corpus callosum , lobar holoprosencephaly , chiari 1 malformation or a persistent / enlarged craniopharyngeal canal .
mri may show a small anterior pituitary gland with the posterior bright spot on t1-weighted imaging ( t1wi ) located within the upper portion of the infundibulum or the undersurface of the hypothalamus instead of the dorsal portion of the sella [ 1 , 1315 ] .
coronal post - contrast images ( a and b ) show two pituitary stalks which extend inferiorly into separate duplicated pituitary glands .
sagittal t1wi ( c ) shows marked thickening of the hypothalamus , referred to as a pseudohamartoma , which has intermediate signal .
coronal post - contrast images ( a and b ) show two pituitary stalks which extend inferiorly into separate duplicated pituitary glands .
sagittal t1wi ( c ) shows marked thickening of the hypothalamus , referred to as a pseudohamartoma , which has intermediate signal .
the pseudohamartoma showed no contrast enhancement ( not shown ) this congenital anomaly is related to abnormal separation of the anterior portion of the notochord and prechordal plate during embryogenesis , likely due to teratogenic factors occurring during early embryogenesis [ 16 , 17 ] .
pituitary duplication may be associated with other anomalies such as dysgenesis of the corpus callosum , absent olfactory bulbs , cerebellar hypoplasia , midline defects , and teratomas .
mri findings include duplicated pituitary stalks and glands within separate pituitary fossa in the sphenoid bone .
abnormal thickening , pseudohamartoma , of the hypothalamus with duplication of the infundibular recess of the third ventricle is often seen [ 16 , 18 ] .
axial t2wi ( a ) and sagittal t1wi ( b ) shows a sessile type of hypothalamic hamartoma which has intermediate signal similar to grey matter .
pre - contrast ( c ) and post - contrast ( d ) sagittal t1wi in another patient shows a pedunculated hypothalamic hamartoma with intermediate signal and lacks contrast enhancementfig .
sagittal ( a ) and coronal ( b ) t1wi , and coronal post - contrast t1wi ( c ) show a non - enhancing bulky soft tissue mass in the region of the of the tuber cinerum hypothalamic hamartoma .
axial t2wi ( a ) and sagittal t1wi ( b ) shows a sessile type of hypothalamic hamartoma which has intermediate signal similar to grey matter .
pre - contrast ( c ) and post - contrast ( d ) sagittal t1wi in another patient shows a pedunculated hypothalamic hamartoma with intermediate signal and lacks contrast enhancement tuber cinerum hamartoma .
sagittal ( a ) and coronal ( b ) t1wi , and coronal post - contrast t1wi ( c ) show a non - enhancing bulky soft tissue mass in the region of the of the tuber cinerum congenital developmental non - neoplastic grey matter heterotopia involving the tuber cinereum , inferior hypothalamus and/or mammillary bodies which are composed of small neuronal cells within a neutrophil - like stroma and scattered fibrillary astrocytes .
these lesions typically occur in children with isosexual precocious puberty ( 0 - 8 years ) or seizures , both gelastic or partial complex , in the second decade [ 20 , 21 ] .
mri demonstrates sessile or pedunculated lesions at the tuber cinereum of the hypothalamus , often intermediate signal on t1wi and t2wi similar to grey matter
. lesions occasionally have slightly high signal on t2wi , with no contrast enhancement or restricted diffusion . rarely contain cystic and/or fatty portions may be seen in a small minority of cases .
the absence of long - term change in size or signal intensity supports the diagnosis .
axial ( a ) and sagittal ( b ) ct shows a markedly enlarged persistent craniopharyngeal canal .
coronal fat - suppressed t1wi shows inferior extension of enhancing pituitary tissue within the abnormally enlarged craniopharyngeal canal ( c ) persistent craniopharyngeal canal .
axial ( a ) and sagittal ( b ) ct shows a markedly enlarged persistent craniopharyngeal canal .
coronal fat - suppressed t1wi shows inferior extension of enhancing pituitary tissue within the abnormally enlarged craniopharyngeal canal ( c ) lack of involution of the craniopharyngeal canal , which can contain pituitary tissue .
the adenohypophysis develops from evagination of ectodermal cells at the upper posterior portion of the primitive oropharynx , rathke s pouch , at 4 - 5 weeks of gestation .
rathke s pouch passes between the developing presphenoid and basisphenoid chondrification centres at the upper clivus and sella , where it associates with the descending neuroectoderm of the infundibulum and will go on to form the neurohypophysis .
the pathway of rathke s pouch is the craniopharyngeal canal , which progressively involutes by 6 - 7 weeks of gestation .
the persistent craniopharyngeal canal on mri can be associated with trans - sphenoidal meningoencephaloceles , ectopic pituitary tissue , sphenoid teratoma and infrasellar craniopharyngiomas [ 23 , 24 ] . on mri
, the persistent canal has low t1 signal intensity compared with the high t1 signal of the clivus .
sagittal t1wi ( a ) shows a lesion in the central portion of the pituitary gland which has high signal .
post - contrast sagittal fat - suppressed t1wi ( b ) shows lack of enhancement of the rathke cleft cyst .
the cyst has intermediate signal on t2wi ( c ) secondary to slightly elevated protein content rathke s cleft cyst .
sagittal t1wi ( a ) shows a lesion in the central portion of the pituitary gland which has high signal .
post - contrast sagittal fat - suppressed t1wi ( b ) shows lack of enhancement of the rathke cleft cyst .
the cyst has intermediate signal on t2wi ( c ) secondary to slightly elevated protein content benign cystic lesion containing fluid and variable amounts of protein , mucopolysaccharides and/or cholesterol [ 25 , 26 ] .
they are 2 - 3 times more common in women than men , and occasionally become symptomatically related to compression of adjacent structures .
the frequency of these cysts on mri in one study was 1.2 % in childhood .
mri will show a well - circumscribed lesion with variable low , intermediate or high signal on t1wi and t2wi .
on t2wi , a small low - signal nodule may be seen within the predominant high signal of the lesion , which does not enhance after contrast administration .
the incidence of these intracystic nodules varies from 17 to 78 % [ 28 , 29 ] .
there is no contrast enhancement centrally ; however , occasionally there may be a thin peripheral rim of enhancement from associated inflammation .
the majority are located in the intrasellar region or combined intrasellar and suprasellar region with reports that only 8.3 % are exclusively suprasellar [ 30 , 31 ] .
12 ) can also form along the posterior margin of rathke s pouch , are typically less than 3 mm , with many considered rathke s cleft cysts on imaging as they are difficult to distinguish from one another and rathke s cleft cysts are more common .fig .
sagittal t1wi ( a ) and axial t2wi ( b ) shows a small cyst between the anterior and posterior pituitary which lacks contrast enhancement on axial fat - suppressed t1wi ( c ) pars intermedia cyst .
sagittal t1wi ( a ) and axial t2wi ( b ) shows a small cyst between the anterior and posterior pituitary which lacks contrast enhancement on axial fat - suppressed t1wi ( c ) fig .
coronal t2wi ( a ) and sagittal t1wi ( b ) shows a meningocele extending inferiorly into the nasopharynx through an osseous defect in the sphenoid bone posterior to the pituitary gland cephalocele in a 17-year - old girl .
coronal t2wi ( a ) and sagittal t1wi ( b ) shows a meningocele extending inferiorly into the nasopharynx through an osseous defect in the sphenoid bone posterior to the pituitary gland congenital malformation involving the lack of separation of neuroectoderm from surface ectoderm with resultant localised failure of bone formation . there is a wide range of abnormalities which affect the skull base within the sella turcica , ranging from a persistent craniopharyngeal canal to trans - sphenoidal and sphenoethmoidal cephaloceles , many presentations of which are extremely rare .
for example , the reported incidence of trans - sphenoidal meningoencephaloceles is 1 per 700,000 live births [ 13 , 32 ] .
clinical findings include difficulty in feeding and nasal obstruction in the first year , and potential for cerebrospinal fluid ( csf ) leaks and meningitis .
mri will show a defect in the skull , through which there is either herniation of meninges and csf ( meningocele ) , or meninges , csf and brain tissue ( meningoencephaloceles ) .
sagittal t1wi ( a ) shows an extra - axial lesion along the endocranial surface which has heterogeneous mostly low - intermdiate signal , and on t2wi has high signal ( b ) .
sagittal t1wi ( a ) shows an extra - axial lesion along the endocranial surface which has heterogeneous mostly low - intermdiate signal , and on t2wi has high signal ( b ) .
diffusion - weighted imaging ( dwi ) ( c ) shows restricted diffusion non - neoplastic congenital or acquired extra - axial off - midline lesion filled with desquamated cells and keratinaceous debris , usually with mild mass effect on the adjacent brain .
epidermoids grow by accumulation of desquamated epithelial cells into the centre of the cyst [ 3335 ] .
mri will show a well circumscribed spheroid or multilobulated extra - axial ectodermal inclusion cystic lesion with low - intermediate signal on t1wi , high signal on t2wi and no contrast enhancement .
they can be distinguished from arachnoid cysts , as they are typically high signal on flair and dwi sequences , with corresponding low signal on adc .
sagittal ( a ) and axial ( b ) t1wi shows an extra - axial lesion in the suprasellar cistern which has high signal .
multiple small foci with high signal are seen in the sulci and within the left lateral ventricle representing ruptured dermoid contents in the leptomeninges dermoid .
sagittal ( a ) and axial ( b ) t1wi shows an extra - axial lesion in the suprasellar cistern which has high signal .
multiple small foci with high signal are seen in the sulci and within the left lateral ventricle representing ruptured dermoid contents in the leptomeninges non - neoplastic congenital or acquired ectodermal inclusion cystic lesion , which like an epidermoid , is lined by squamous epithelium , and are filled with lipid material , cholesterol , desquamated cells and keratinaceous debris .
they are distinguished from epidermoids by containing deeper dermal elements and commonly arise from the midline .
they typically present in the 20- to 30-year age range , but can also be seen in adolescence , with males slightly more affected than females .
mri will show a well - circumscribed spheroid or multilobulated extra - axial lesion , usually with high signal on t1wi , variable signal on t2wi and no contrast enhancement .
they can cause a chemical meningitis if the dermoid cyst ruptures into the subarachnoid space [ 37 , 38 ] .
sagittal t1wi ( a ) and coronal t2wi ( b ) show a well defined cystic mass with low t1 and high t2 signal intensity with sellar and suprasellar components arachnoid cyst .
sagittal t1wi ( a ) and coronal t2wi ( b ) show a well defined cystic mass with low t1 and high t2 signal intensity with sellar and suprasellar components congenital arachnoid cysts are found in 0.16 % of all newborns with approximately 9 - 15 % occurring in the sellar region , with suprasellar cysts more commonly seen than intrasellar cysts .
most cases are asymptomatic ; however , they may cause headache , optic nerve compression , psychomotor retardation , endocrine dysfunction or hydrocephalus [ 39 , 40 ] .
the cysts have low signal intensity on t1wi , flair and dwi , high signal intensity on t2wi , no contrast enhancement , and may cause mild mass effect on adjacent brain .
sagittal t1wi ( a ) shows a lipoma with high signal along the inferior surface of the hypothalamus . the signal on the lipoma is nulled on coronal fat - suppressed t2wi ( b ) lipoma .
sagittal t1wi ( a ) shows a lipoma with high signal along the inferior surface of the hypothalamus . the signal on the lipoma is nulled on coronal fat - suppressed t2wi ( b ) benign fatty lesions resulting from a congenital malformation , often located at or near the midline . in the sellar region , they occur along the surface of the infundibulum , floor of the third ventricle , or adjacent to cranial nerves , and are usually discovered incidentally , unless they cause a mass effect [ 42 , 43 ] .
mri signal is isointense to subcutaneous fat on all sequences , with fat suppressed images useful in distinguishing from haemorrhagic or proteinaceous lesions .
sagittal post - contrast t1wi with fat - suppression show an enlarged sella mostly filled with csf .
the enhancing flattened pituitary tissue is located along the floor of the sella empty sella .
sagittal post - contrast t1wi with fat - suppression show an enlarged sella mostly filled with csf .
the enhancing flattened pituitary tissue is located along the floor of the sella common incidental finding involving the pituitary gland and sella , which can result from a weakened or fenestrated diaphragma sella allowing csf pulsations to flatten the pituitary gland .
they are associated with elevated intracranial pressure , a posteriorly placed optic chiasm , and are considered a normal variant or termed primary empty sella turcica in the absence of surgery , radiation therapy or medically treated intrasellar tumour . in children , the incidence of an empty sella is seen more often in boys and is more likely to be associated with clinical symptoms and endocrinopathies , particularly growth hormone or multiple pituitary hormone deficiencies .
mri findings include a thinned / flattened pituitary gland along the floor of the sella , with the infundibulum in its normal midline position .
fig . 19pituitary adenomas . sagittal post - contrast fat - suppressed t1wi ( a ) shows an heterogenously enhancing pituitary macroadenoma which contains a non - enhancing portion with high signal on t2wi ( b ) .
coronal post - contrast fat - suppressed t1wi ( c ) shows a pituitary microadenoma which shows less early contrast enhancement than the normal pituitary tissue pituitary adenomas .
sagittal post - contrast fat - suppressed t1wi ( a ) shows an heterogenously enhancing pituitary macroadenoma which contains a non - enhancing portion with high signal on t2wi ( b ) .
coronal post - contrast fat - suppressed t1wi ( c ) shows a pituitary microadenoma which shows less early contrast enhancement than the normal pituitary tissue in comparison to adults , pituitary adenomas are rare in the paediatric population , accounting for approximately 2.7 % of all supratentorial tumours .
most paediatric adenomas are functioning , with only 3 - 6 % non - functioning , compared to one - third of non - functioning adenomas in adults [ 4648 ] .
microadenomas may be associated with endocrine abnormalities related to over - secretion of hormones , with prolactinomas being the most common .
rarely , extensive haemorrhage can occur involving the adenoma , resulting in pituitary apoplexy ( sheehan syndrome ) .
macroadenomas with suprasellar extension can cause compression and displacement of the optic chiasm with associated visual disturbances ( bitemporal hemianiopsia ) .
most pituitary adenomas arise from sporadic mutations ; however , 5 % can be associated with inherited disorders such as mccune albright syndrome , carney complex and multiple endocrine neoplasia type 1 ( men 1 ) .
mri findings of a microadenoma ( < 10 mm ) commonly have intermediate signal on t1wi and t2wi , and may show characteristics of cysts , haemorrhage or necrosis .
they typically show less contrast enhancement than normal pituitary tissue , often best visualised with dynamic early - phase imaging . in macroadenomas ( > 10 mm ) , they commonly have intermediate signal on t1wi and t2wi similar to grey matter , and may also have cystic , haemorrhagic or necrotic components .
they can extend into the suprasellar cistern with a waist at the diaphragm sella or into the cavernous sinus , with occasional invasion into the skull base .
sagittal t1wi ( a ) shows a lobulated lesion filling the suprasellar cistern which contains zones of low , intermediate and high signal .
sagittal post - contrast t1wi ( b ) shows the lesion to have heterogeneous contrast enhancement craniopharyngioma .
sagittal t1wi ( a ) shows a lobulated lesion filling the suprasellar cistern which contains zones of low , intermediate and high signal .
sagittal post - contrast t1wi ( b ) shows the lesion to have heterogeneous contrast enhancement usually histologically benign locally aggressive lesions which arise from squamous epithelial rests along rathke s cleft , occurring in a bimodal age distribution , in children ( < 10 years ) and adults ( > 40 years ) .
the adamantinomatous type is more common and has a bimodal age distribution occurring in children and adults , whereas the squamous - papillary type usually occurs in adults .
although histologically benign , their insinuating pattern of growth makes complete surgical excision very difficult and not often possible .
the adamantinomatous type usually demonstrates circumscribed lobulated margins on mri , with the suprasellar location seen at a greater frequency than intrasellar .
they show variable low , intermediate and/or high signal on t1wi and t2wi , with or without a nodular or rim of contrast enhancement .
the cystic parts tend to predominate , and frequently contain highly proteinaceous fluid , which may be bright on t1wi .
sagittal ( a ) and axial ( b ) post contrast t1wi shows a partially enhancing lesion enlarging the optic chiasm and proximal optic nerves glioma in a 2-year - old girl with neurofibromatosis type 1 .
sagittal ( a ) and axial ( b ) post contrast t1wi shows a partially enhancing lesion enlarging the optic chiasm and proximal optic nerves lesions which may occur anywhere along the optic pathway and account for 4 - 6 % of brain tumours in children , with a median age of diagnosis from 5 - 9 years . approximately half of patients with optic pathway gliomas have neurofibromatosis type 1 ( nf1 ) , with symptomatic lesions only occurring in 1 - 5 % of patients
. most tumours are slow growing who grade i - ii astrocytomas , often the pilocytic type , however tumours may show a wide spectrum of progression . on mri , there is fusiform and/or nodular enlargement of the optic chiasm and/or optic nerves with thickening of the third ventricular floor and hypothalamus .
they usually have low - intermediate signal on t1wi , intermediate - high signal on t2wi and variable contrast enhancement .
sagittal t1wi ( a ) shows a large hypothalamic lesion with intermediate signal which extends superiorly into and filling the third ventricle , and inferiorly into the suparsellar cistern .
the tumour has mostly high signal on axial t2wi ( b ) pilomyxoid astrocytoma in a 4-month - old baby girl .
sagittal t1wi ( a ) shows a large hypothalamic lesion with intermediate signal which extends superiorly into and filling the third ventricle , and inferiorly into the suparsellar cistern .
the tumour has mostly high signal on axial t2wi ( b ) rare neoplasms ( who grade ii ) which contain prominent mucoid matrix and angiocentric pattern of bipolar neoplastic astrocytes .
typically , they occur in children in the first and second decades , with a mean age of 7 years .
they tend to occur at a younger age and are more aggressive with a higher rate of local recurrence than pilocytic astromcytomas .
they have a strong predilection for the hypothalamus and suprasellar cistern ; however , they can also occur in the thalamus , cerebellum , brainstem , temporal lobe and spinal cord .
the presence of intra - tumoural haemorrhage is the most prominent imaging characteristic to suggest a pilomyxoid astrocytoma over a pilocytic astrocytoma , 25 % versus 8 % , respectively .
mri demonstrates a solid / cystic focal lesion with low - intermediate signal on t1wi , high signal on t2wi and flair , variable contrast enhancement and may show increased adc values .
post - contrast t1wi ( a ) shows a large enhancing lesion in the suprasellar cistern extending superiorly into the third ventricle and posteriorly into the prepontine cistern .
sagittal post - contrast t1wi ( b ) in another child shows enhancing , disseminated germ - cell tumour in the suprasellar cistern , third and lateral ventricles , with involvement of the corpus callosum and optic chiasm germ cell tumours in two different children .
post - contrast t1wi ( a ) shows a large enhancing lesion in the suprasellar cistern extending superiorly into the third ventricle and posteriorly into the prepontine cistern .
sagittal post - contrast t1wi ( b ) in another child shows enhancing , disseminated germ - cell tumour in the suprasellar cistern , third and lateral ventricles , with involvement of the corpus callosum and optic chiasm extra - gonadal germ cell tumours include : germinoma ( most common ) , mature teratoma , malignant teratoma , yolk sac tumour , embryonal carcinoma and choriocarcinoma .
they account for 0.6 % of primary intracranial tumours , with an incidence of 0.09 per 100,000 .
peak incidence occurs between 10 and 14 years , with 90 % occurring in patients less than 25 years .
prognosis depends on the histological subtype , with a 10 year survival rate for germinomas of over 85 % .
other germ cell tumours have a lower survival rate , particularly those containing non - germinomatous malignant cells .
tumours often have intermediate signal on t1wi , slightly high signal on t2wi , and show contrast enhancement .
they are typically centred on the pituitary stalk with the posterior pituitary bright spot often absent .
sagittal t1wi ( a ) and axial t2wi ( b ) show a lesion at the undersurface of the hypothalamus which has mixed low , intermediate and high signal .
axial ct image ( c ) shows the lesion to contain zones of calcification , fat and intermediate soft - tissue attenuation teratoma .
sagittal t1wi ( a ) and axial t2wi ( b ) show a lesion at the undersurface of the hypothalamus which has mixed low , intermediate and high signal .
axial ct image ( c ) shows the lesion to contain zones of calcification , fat and intermediate soft - tissue attenuation the second most common type of germ cell tumour behind a germinoma .
they occur in children , with a greater frequency in boys , accounting for 0.5 - 1.5 % of all childhood brain tumours , and > 50 % of intracranial tumours in infants less than 2 months [ 54 , 55 ] .
histologically , they demonstrate benign or malignant types , composed of derivatives of ectoderm , mesoderm and/or endoderm . on mri
, they are relatively circumscribed lesions , most common in decreasing order frequency within the pineal region , suprasellar region , and third ventricle . on mri
, they demonstrate heterogeneous signal intensity on t1wi , t2wi and post - contrast sequences , with suppression of signal on fat - saturated imaging .
marked enhancement of the solid portions of the tumour is a key feature in distinguishing a mature versus a malignant teratoma , with malignant tumours demonstrating enhancement .
additionally , they typically present without perilesional high t2 signal due to intact capsules and an undamaged blood - brain barrier .
axial ( a , b ) and coronal ( c ) post - contrast t1wi shows prominent extra - axial contrast - enhancement in the basal meninges , surrounding the brainstem , cerebellar sulci and sylvian fissures .
the leptomeningeal infection invades the left temporal lobe a 4-month - old baby boy with tuberculous meningitis .
axial ( a , b ) and coronal ( c ) post - contrast t1wi shows prominent extra - axial contrast - enhancement in the basal meninges , surrounding the brainstem , cerebellar sulci and sylvian fissures .
the leptomeningeal infection invades the left temporal lobe mycobacterium tuberculosis infection remains a major international concern , with increasing burden of disease in developed nations and young children being the most susceptible [ 56 , 57 ] .
mri is increasingly being used to evaluate patients with suspected tuberculous meningitis , to aid in rapid early diagnosis .
. the granulomas can be either non - caseating with homogeneous enhancement or caseating with rim enhancement ( both t1 hypointense and t2 hyperintense ) .
rare malignant bone tumour comprised of proliferating neoplastic spindle - cells which produce osteoid and/or immature tumoural bone .
they usually involve the endochondral bone - forming portions of the skull - base and are locally invasive with high metastatic potential . over 75 % of osteosarcomas are diagnosed in patients between 8 and 25 years , and account for less than 1 % of all head and neck malignant tumours in the paediatric population .
they occur in children as primary tumours and adults associated with paget s disease , irradiated bone , chronic osteomyelitis , osteoblastoma , giant cell tumour and fibrous dysplasia .
mri will show a destructive lesion involving the skull base , with low - intermediate signal on t1wi , mixed low , intermediate or high signal on t2wi .
usually contains matrix mineralisation / ossification with low signal on t2wi and heterogeneous contrast enhancement .
bone tumour usually occurring between ages 5 and 30 years , with a greater prevalence in males and is the second most common primary bone malignancy affecting adolescents and young adults .
it most commonly arises in the trunk and diaphysis of long bones , with only 4 % of cases within the head and neck region .
mri will show a destructive lesion involving the skull base , with low - intermediate signal on t1wi , mixed low , intermediate or high signal on t2wi , with or without matrix mineralisation showing low signal on t2wi and heterogeneous contrast enhancement .
sagittal t1wi ( a ) and post - contrast t1wi ( b ) show a destructive mass lesion involving the sphenoid bone which invades the sphenoid sinus and sella .
sagittal t1wi ( a ) and post - contrast t1wi ( b ) show a destructive mass lesion involving the sphenoid bone which invades the sphenoid sinus and sella .
the tumour shows prominent , mildly heterogeneous contrast enhancement these malignant neoplasms of neuroectodermal origin , arise from olfactory epithelium in the upper nasal cavity and cribiform region .
there is a bimodal age of occurrence in adolescents ( 11 - 20 years ) and adults ( 50 - 60 years ) , with a greater prevalence in males . on mri , they present as a locally destructive lesion with low - intermediate signal on t1wi , intermediate - high signal on t2wi and prominent contrast enhancement .
they are commonly located within the superior nasal cavity , ethmoid air cells with occasional extension into other paranasal sinuses , orbits , anterior cranial fossa , and cavernous sinuses .
positron emission tomography ( pet)/computed tomography ( ct ) can be useful for staging of disease and detection of metastases .
axial ct ( a ) shows multiple expansile lesions involving the skull which have a ground glass appearance representing sites of polyostotic fibrous dysplasia .
sagittal t1wi shows expansion of the clivus which has diffuse low signal ( b ) with corresponding diffuse enhancement on sagittal post - contrast fat - suppressed t1wi ( c ) fibrous dysplasia in a 5-year - old girl with mccune albright syndrome .
axial ct ( a ) shows multiple expansile lesions involving the skull which have a ground glass appearance representing sites of polyostotic fibrous dysplasia .
sagittal t1wi shows expansion of the clivus which has diffuse low signal ( b ) with corresponding diffuse enhancement on sagittal post - contrast fat - suppressed t1wi ( c ) benign medullary fibro - osseous lesion , which can involve a single site ( mono - ostotic ) or multiple locations ( poly - ostotic ) , results from developmental failure in the normal process of remodelling primitive bone to mature lamellar bone with zones of immature trabeculae within dysplastic fibrous tissue .
craniofacial bones are involved in 10 - 20 % of patients with the monostotic disease and in 50 % of patients with the polyostotic form .
patients range in age from < 1 year to the eighth or ninth decades of life with 75 % occurring before the age of 30 years .
mri features depend on the proportions of bony spicules , collagen , fibroblastic spindle cells , haemorrhagic and/or cystic changes , and if associated pathological fractures are present .
lesions are usually well - circumscribed and have low - intermediate signal on t1wi and proton - density weighted imaging ( pdwi ) . on t2wi , lesions have variable mixtures of low , intermediate and/or high signal often surrounding by a low signal rim of variable thickness . internal septations and cystic changes are seen in a minority of lesions .
. refers to an uncommon disorder in which polyostotic fibrous dysplasia is associated with pigmented cutaneous macules ( caf - au - lait spots ) with irregular indented borders , precocious puberty , and/or other endocrine disorders such as acromegaly , hyperthyroidism , hyperparathyroidism , and/or cushing s syndrome .
mccune - albright syndrome is uncommon and occurs in 2 - 3 % of patients with polyostotic fibrous dysplasia .
coronal t1wi shows a lesion involving the upper pituitary gland and stalk which has intermediate signal ( a ) .
the lesion shows prominent enhancement on post - contrast coronal t1wi ( b ) langerhans cell histiocytosis in a 6-year - old girl with headaches and diabetes insipidus .
coronal t1wi shows a lesion involving the upper pituitary gland and stalk which has intermediate signal ( a ) .
the lesion shows prominent enhancement on post - contrast coronal t1wi ( b ) disorder of the reticuloendothelial system in which bone - marrow - derived dendritic langerhans cells infiltrate various organs as focal lesions or in diffuse patterns .
prevalence of 2 per 100,000 children less than 15 years , with only a third of the lesions occurring in adults .
localised lesions ( eosinophilic granuloma ) can be single or multiple in the skull , usually at the skull base .
there is a striking preference for lch to involve regions without a blood brain barrier , with the hypothalamic - pituitary axis the most frequently involved intracranial region , with patients often presenting with diabetes insipidus .
mri will typically show a fusiform or lobulated lesion with intermediate signal on t1wi and t2wi involving the pituitary stalk .
the pituitary stalk is usually > 3 mm in thickness , and is often associated with a loss of high signal on t1wi of the posterior pituitary .
29lymphocytic hypophysitis in a 16-year - old boy who presented with frequent urination , which started when he was 9 .
coronal t1wi ( a ) shows thickening of the pituitary stalk which has intermdiate signal .
post - contrast axial ( b ) and sagittal ( c ) t1wi shows thickening of the pituitary stalk with homogeneous enhancement lymphocytic hypophysitis in a 16-year - old boy who presented with frequent urination , which started when he was 9 .
coronal t1wi ( a ) shows thickening of the pituitary stalk which has intermdiate signal .
post - contrast axial ( b ) and sagittal ( c ) t1wi shows thickening of the pituitary stalk with homogeneous enhancement rare autoimmune inflammatory process involving the pituitary gland confirmed by biopsy showing varying degrees of lymphocytic infiltration and plasma cells with fibrotic changes without multinucleated giant cells .
they are more prevalent in women ( 80 % ) who are in late pregnancy or postpartum but can also occur in children .
clinical findings include headaches and pituitary hormonal dysfunction , with deficiency of acth in adults and growth hormone in children .
lesions can be associated with hashimoto thyroiditis , polymyositis , pernicious anaemia , atrophic gastritis , psoriasis , systemic lupus erythematosus , adrenalitis and/or ovarian failure .
mri shows a slightly lobulated lesion with intermediate signal on t1wi , heterogeneous low - intermediate and high signal on t2wi involving the pituitary lobe with thickened pituitary stalk , and prominent homogeneous or heterogeneous contrast enhancement involving the pituitary gland , pituitary stalk and dura .
a characteristic finding includes a parasellar t2 dark sign , which helps in determining lymphocytic hypophysitis from a pituitary adenoma .
it usually occurs in children and young adults , with a male - female ratio of 3:2 .
when there is intracranial involvement , patients may present with headaches , seizures , visual disturbances , numbness and/or paraplegia .
involvement of the central nervous system ( cns ) occurs in 4 % and usually involves the intracranial or spinal dura , rarely occurring in the sella and suprasellar region . on mri ,
suprasellar / sellar masses tend to be isointense on t1wi and hypointense / isointense on t2wi with homogeneous enhancement , and may be mistaken as a meningioma .
a central hypointensiity on t2wi , thought to be related to free radicals release by inflammatory macrophages , may help distinguish from a meningioma [ 74 , 75 ] .
sagittal post contrast t1wi demonstrates diffuse intracranial and spinal leptomeningeal enhancement in a 22-month - old infant boy with lymphoma including the sella and parasellar subarachnoid space leptomeningeal spread of lymphoma .
sagittal post contrast t1wi demonstrates diffuse intracranial and spinal leptomeningeal enhancement in a 22-month - old infant boy with lymphoma including the sella and parasellar subarachnoid space primary cns lymphoma is extremely rare in children , and usually occurs as intra - axial lesions and infrequently as leptomeningeal tumour .
leptomeningeal metastases can also be seen with acute lymphoblastic leukaemia , medulloblastomas , germ - cell tumours , ependymomas and malignant gliomas .
31cavernous malformation : coronal t2wi ( a ) and axial gradient echo ( gre ) ( b ) images in a 5-year - old girl show a large suprasellar lesion with mixed low , intermediate and high signal intensity with a thin rim of low signal on t2wi and foci of low t2 * signal from susceptibility effects of blood products on gre cavernous malformation : coronal t2wi ( a ) and axial gradient echo ( gre ) ( b ) images in a 5-year - old girl show a large suprasellar lesion with mixed low , intermediate and high signal intensity with a thin rim of low signal on t2wi and foci of low t2 * signal from susceptibility effects of blood products on gre numerous vascular malformations can be seen in the sellar / juxtasellar regions , including arterial aneurysms ( saccular or giant aneurysms of the internal carotid artery [ ica ] ) , cavernous carotid fistulas , arterial - venous malformations , haemangiomas or cavernous malformations .
cavernous malformations are the most common cns vascular lesions in children , although the incidence is lower than in adults . compared to adults , these lesions are more aggressive in children with high rates of growth and haemorrhage , and a mean age of presentation of 9 - 10 years .
mri findings will show single or multiple multilobulated intra - axial lesions with a peripheral or irregular zone of low signal on t2wi and t2 * secondary to haemosiderin , with a central zone of variable signal on t1wi and t2wi depending on ages of haemorrhagic components .
imaging of the sellar and juxtasellar region in the paediatric population is ideally performed with mri , and relies on a strong fundamental knowledge of the anatomy and signal characteristics of a normal gland and surrounding structures .
the knowledge of key differentiating mri characteristics in common and uncommon disease entities of the paediatric sellar and juxtasellar regions , in conjunction with the clinical findings , undoubtedly aids in alerting and guiding the clinician down the appropriate path of management .
it is best to approach a suspected sellar or juxtasellar abnormality with an open mind and a broad differential , with mri being the gold standard of imaging to accurately characterise these lesions . | the sellar and juxtasellar regions in the paediatric population are complex both anatomically and pathologically , with magnetic resonance imaging ( mri ) being the gold standard imaging modality due to the high contrast of detail .
assessment requires a detailed understanding of the anatomy , embryology , pathophysiology and normal signal characteristics of the pituitary gland and surrounding structures in order to appropriately characterise abnormalities .
this article aims to provide an overview of the imaging characteristics of developmental / congenital and acquired disease processes which affect the sellar and juxtasellar region in the paediatric population.main messages the sellar region is anatomically complex and covers a wide pathology spectrum. mri is the key imaging modality to assess sellar and juxtasellar pathology. numerous developmental anomalies may not be discovered until adulthood. knowledge of pathology alerts and guides the clinician towards appropriate management . | Pituitary function and anatomy
Embryology
Imaging characteristics in the paediatric population
Developmental anomalies of the pituitary gland
Pituitary lesions
Juxtasellar lesions
Conclusions | the pituitary gland is comprised of two dominant lobes , the neurohypophysis and the adenohypophysis . it is situated within the sella turcica , a depression in the sphenoid bone which is covered by the diaphragm sella with a defect for the passage of the infundibulum . the pituitary stalk extends inferiorly as a continuation of the hypothalamic infundibulum ( fig . the pituitary gland develops through a complex mechanism involving the developing diencephalon and the primitive oral stomodeum . the adenohypophysis derives from rathke s pouch , which originates rostral to the oropharyngeal membrane . this begins at approximately 24 days of gestation , in which rathke s pouch forms as an ectodermal outpouching of stomodeum and the infundibulum which forms in the floor of the diencephalon . this sac continues to differentiate into the adenophyophysis and the distal aspect of the infundibulum differentiates to form the neurohypophysis ( fig . the pituitary gland exhibits a wide range of appearances during the first 2 years of life , with considerable variability in size , shape , and signal . the posterior pituitary gland retains its high signal intensity on t1-weighted images throughout adulthood , with several papers supporting that this high signal results from neurosecretory granules or the protein neurophysin [ 35 ] . 3normal physiological t1 sagittal ( a ) and axial ( b ) hyperintensity of the anterior pituitary in a 1-month - old baby normal physiological t1 sagittal ( a ) and axial ( b ) hyperintensity of the anterior pituitary in a 1-month - old baby at birth , the gland is physiologically enlarged with a corresponding concave superior margin , flattening with age . gender - associated differences in the rate of gland growth tend to occur after 5 years of age , with linear growth of the gland up to that point in both boys and girls . in adults ,
standards for assessing the pituitary stalk size are not well established , and are based on experience rather than age - appropriate measurements . the stalk is best visualised on post - contrast t1-weighted images because of the strong enhancement . the stalk terminates into a bulbous neurohypophysis with high signal in the dorsal portion of the sella agenesis of the anterior pituitary gland ( adenohypophysis ) in an infant with hypopituitarism . the stalk terminates into a bulbous neurohypophysis with high signal in the dorsal portion of the sella this is a rare congenital anomaly with a neonatal presentation . magnetic resonance imaging ( mri ) findings include the absence of the anterior pituitary gland with an associated shallow sella , and variable length of the infundibulum and position of the posterior pituitary , ranging from the hypothalamus to the sella . pre - contrast ( a ) and post - contrast ( b ) mr images show severe hypoplasia of the anterior pituitary gland which shows only minimal contrast enhancement along the floor of the sella . the ectopic posterior pituitary is located adjacent to the optic chiasm severe hypoplasia of the anterior pituitary gland . pre - contrast ( a ) and post - contrast ( b ) mr images show severe hypoplasia of the anterior pituitary gland which shows only minimal contrast enhancement along the floor of the sella . the ectopic posterior pituitary is located adjacent to the optic chiasm this rare anomaly consists of hypoplasia of the anterior pituitary gland , and also potentially the infundibulum and posterior pituitary gland . some research suggests that traumatic transection of the pituitary stalk during birth or hypoxic injury to the hypothalamus could result in hypoplasia , whereas others suggest an error during embryogenesis [ 912 ] . mri findings include varying degrees of hypoplasia of the anterior pituitary gland with variable size and length of the infundibulum . as with agenesis of the adenohypophysis ,
pre - contrast ( a ) and post - contrast ( b ) mr images show a normal anterior pituitary gland with contrast enhancement and an ectopic posterior pituitary located adjacent to the optic chiasm ectopic posterior pituitary gland . pre - contrast ( a ) and post - contrast ( b ) mr images show a normal anterior pituitary gland with contrast enhancement and an ectopic posterior pituitary located adjacent to the optic chiasm this congenital anomaly results in an aberrant position of the neurohypophysis , often located at the undersurface of the hypothalamus . mri may show a small anterior pituitary gland with the posterior bright spot on t1-weighted imaging ( t1wi ) located within the upper portion of the infundibulum or the undersurface of the hypothalamus instead of the dorsal portion of the sella [ 1 , 1315 ] . sagittal t1wi ( c ) shows marked thickening of the hypothalamus , referred to as a pseudohamartoma , which has intermediate signal . the pseudohamartoma showed no contrast enhancement ( not shown ) this congenital anomaly is related to abnormal separation of the anterior portion of the notochord and prechordal plate during embryogenesis , likely due to teratogenic factors occurring during early embryogenesis [ 16 , 17 ] . abnormal thickening , pseudohamartoma , of the hypothalamus with duplication of the infundibular recess of the third ventricle is often seen [ 16 , 18 ] . sagittal ( a ) and coronal ( b ) t1wi , and coronal post - contrast t1wi ( c ) show a non - enhancing bulky soft tissue mass in the region of the of the tuber cinerum hypothalamic hamartoma . sagittal ( a ) and coronal ( b ) t1wi , and coronal post - contrast t1wi ( c ) show a non - enhancing bulky soft tissue mass in the region of the of the tuber cinerum congenital developmental non - neoplastic grey matter heterotopia involving the tuber cinereum , inferior hypothalamus and/or mammillary bodies which are composed of small neuronal cells within a neutrophil - like stroma and scattered fibrillary astrocytes . these lesions typically occur in children with isosexual precocious puberty ( 0 - 8 years ) or seizures , both gelastic or partial complex , in the second decade [ 20 , 21 ] . lesions occasionally have slightly high signal on t2wi , with no contrast enhancement or restricted diffusion . the adenohypophysis develops from evagination of ectodermal cells at the upper posterior portion of the primitive oropharynx , rathke s pouch , at 4 - 5 weeks of gestation . on mri
, the persistent canal has low t1 signal intensity compared with the high t1 signal of the clivus . sagittal t1wi ( a ) shows a lesion in the central portion of the pituitary gland which has high signal . sagittal t1wi ( a ) shows a lesion in the central portion of the pituitary gland which has high signal . the majority are located in the intrasellar region or combined intrasellar and suprasellar region with reports that only 8.3 % are exclusively suprasellar [ 30 , 31 ] . coronal t2wi ( a ) and sagittal t1wi ( b ) shows a meningocele extending inferiorly into the nasopharynx through an osseous defect in the sphenoid bone posterior to the pituitary gland cephalocele in a 17-year - old girl . coronal t2wi ( a ) and sagittal t1wi ( b ) shows a meningocele extending inferiorly into the nasopharynx through an osseous defect in the sphenoid bone posterior to the pituitary gland congenital malformation involving the lack of separation of neuroectoderm from surface ectoderm with resultant localised failure of bone formation . there is a wide range of abnormalities which affect the skull base within the sella turcica , ranging from a persistent craniopharyngeal canal to trans - sphenoidal and sphenoethmoidal cephaloceles , many presentations of which are extremely rare . multiple small foci with high signal are seen in the sulci and within the left lateral ventricle representing ruptured dermoid contents in the leptomeninges non - neoplastic congenital or acquired ectodermal inclusion cystic lesion , which like an epidermoid , is lined by squamous epithelium , and are filled with lipid material , cholesterol , desquamated cells and keratinaceous debris . they typically present in the 20- to 30-year age range , but can also be seen in adolescence , with males slightly more affected than females . sagittal t1wi ( a ) and coronal t2wi ( b ) show a well defined cystic mass with low t1 and high t2 signal intensity with sellar and suprasellar components arachnoid cyst . sagittal t1wi ( a ) and coronal t2wi ( b ) show a well defined cystic mass with low t1 and high t2 signal intensity with sellar and suprasellar components congenital arachnoid cysts are found in 0.16 % of all newborns with approximately 9 - 15 % occurring in the sellar region , with suprasellar cysts more commonly seen than intrasellar cysts . sagittal t1wi ( a ) shows a lipoma with high signal along the inferior surface of the hypothalamus . in the sellar region , they occur along the surface of the infundibulum , floor of the third ventricle , or adjacent to cranial nerves , and are usually discovered incidentally , unless they cause a mass effect [ 42 , 43 ] . mri signal is isointense to subcutaneous fat on all sequences , with fat suppressed images useful in distinguishing from haemorrhagic or proteinaceous lesions . the enhancing flattened pituitary tissue is located along the floor of the sella common incidental finding involving the pituitary gland and sella , which can result from a weakened or fenestrated diaphragma sella allowing csf pulsations to flatten the pituitary gland . mri findings include a thinned / flattened pituitary gland along the floor of the sella , with the infundibulum in its normal midline position . coronal post - contrast fat - suppressed t1wi ( c ) shows a pituitary microadenoma which shows less early contrast enhancement than the normal pituitary tissue in comparison to adults , pituitary adenomas are rare in the paediatric population , accounting for approximately 2.7 % of all supratentorial tumours . microadenomas may be associated with endocrine abnormalities related to over - secretion of hormones , with prolactinomas being the most common . mri findings of a microadenoma ( < 10 mm ) commonly have intermediate signal on t1wi and t2wi , and may show characteristics of cysts , haemorrhage or necrosis . they show variable low , intermediate and/or high signal on t1wi and t2wi , with or without a nodular or rim of contrast enhancement . approximately half of patients with optic pathway gliomas have neurofibromatosis type 1 ( nf1 ) , with symptomatic lesions only occurring in 1 - 5 % of patients
. typically , they occur in children in the first and second decades , with a mean age of 7 years . sagittal post - contrast t1wi ( b ) in another child shows enhancing , disseminated germ - cell tumour in the suprasellar cistern , third and lateral ventricles , with involvement of the corpus callosum and optic chiasm germ cell tumours in two different children . sagittal post - contrast t1wi ( b ) in another child shows enhancing , disseminated germ - cell tumour in the suprasellar cistern , third and lateral ventricles , with involvement of the corpus callosum and optic chiasm extra - gonadal germ cell tumours include : germinoma ( most common ) , mature teratoma , malignant teratoma , yolk sac tumour , embryonal carcinoma and choriocarcinoma . sagittal t1wi ( a ) and axial t2wi ( b ) show a lesion at the undersurface of the hypothalamus which has mixed low , intermediate and high signal . they occur in children , with a greater frequency in boys , accounting for 0.5 - 1.5 % of all childhood brain tumours , and > 50 % of intracranial tumours in infants less than 2 months [ 54 , 55 ] . on mri
, they demonstrate heterogeneous signal intensity on t1wi , t2wi and post - contrast sequences , with suppression of signal on fat - saturated imaging . marked enhancement of the solid portions of the tumour is a key feature in distinguishing a mature versus a malignant teratoma , with malignant tumours demonstrating enhancement . the leptomeningeal infection invades the left temporal lobe mycobacterium tuberculosis infection remains a major international concern , with increasing burden of disease in developed nations and young children being the most susceptible [ 56 , 57 ] . over 75 % of osteosarcomas are diagnosed in patients between 8 and 25 years , and account for less than 1 % of all head and neck malignant tumours in the paediatric population . mri will show a destructive lesion involving the skull base , with low - intermediate signal on t1wi , mixed low , intermediate or high signal on t2wi . bone tumour usually occurring between ages 5 and 30 years , with a greater prevalence in males and is the second most common primary bone malignancy affecting adolescents and young adults . it most commonly arises in the trunk and diaphysis of long bones , with only 4 % of cases within the head and neck region . mri will show a destructive lesion involving the skull base , with low - intermediate signal on t1wi , mixed low , intermediate or high signal on t2wi , with or without matrix mineralisation showing low signal on t2wi and heterogeneous contrast enhancement . the tumour shows prominent , mildly heterogeneous contrast enhancement these malignant neoplasms of neuroectodermal origin , arise from olfactory epithelium in the upper nasal cavity and cribiform region . there is a bimodal age of occurrence in adolescents ( 11 - 20 years ) and adults ( 50 - 60 years ) , with a greater prevalence in males . sagittal t1wi shows expansion of the clivus which has diffuse low signal ( b ) with corresponding diffuse enhancement on sagittal post - contrast fat - suppressed t1wi ( c ) fibrous dysplasia in a 5-year - old girl with mccune albright syndrome . sagittal t1wi shows expansion of the clivus which has diffuse low signal ( b ) with corresponding diffuse enhancement on sagittal post - contrast fat - suppressed t1wi ( c ) benign medullary fibro - osseous lesion , which can involve a single site ( mono - ostotic ) or multiple locations ( poly - ostotic ) , results from developmental failure in the normal process of remodelling primitive bone to mature lamellar bone with zones of immature trabeculae within dysplastic fibrous tissue . coronal t1wi shows a lesion involving the upper pituitary gland and stalk which has intermediate signal ( a ) . coronal t1wi shows a lesion involving the upper pituitary gland and stalk which has intermediate signal ( a ) . prevalence of 2 per 100,000 children less than 15 years , with only a third of the lesions occurring in adults . the pituitary stalk is usually > 3 mm in thickness , and is often associated with a loss of high signal on t1wi of the posterior pituitary . coronal t1wi ( a ) shows thickening of the pituitary stalk which has intermdiate signal . post - contrast axial ( b ) and sagittal ( c ) t1wi shows thickening of the pituitary stalk with homogeneous enhancement lymphocytic hypophysitis in a 16-year - old boy who presented with frequent urination , which started when he was 9 . coronal t1wi ( a ) shows thickening of the pituitary stalk which has intermdiate signal . post - contrast axial ( b ) and sagittal ( c ) t1wi shows thickening of the pituitary stalk with homogeneous enhancement rare autoimmune inflammatory process involving the pituitary gland confirmed by biopsy showing varying degrees of lymphocytic infiltration and plasma cells with fibrotic changes without multinucleated giant cells . mri shows a slightly lobulated lesion with intermediate signal on t1wi , heterogeneous low - intermediate and high signal on t2wi involving the pituitary lobe with thickened pituitary stalk , and prominent homogeneous or heterogeneous contrast enhancement involving the pituitary gland , pituitary stalk and dura . it usually occurs in children and young adults , with a male - female ratio of 3:2 . involvement of the central nervous system ( cns ) occurs in 4 % and usually involves the intracranial or spinal dura , rarely occurring in the sella and suprasellar region . 31cavernous malformation : coronal t2wi ( a ) and axial gradient echo ( gre ) ( b ) images in a 5-year - old girl show a large suprasellar lesion with mixed low , intermediate and high signal intensity with a thin rim of low signal on t2wi and foci of low t2 * signal from susceptibility effects of blood products on gre cavernous malformation : coronal t2wi ( a ) and axial gradient echo ( gre ) ( b ) images in a 5-year - old girl show a large suprasellar lesion with mixed low , intermediate and high signal intensity with a thin rim of low signal on t2wi and foci of low t2 * signal from susceptibility effects of blood products on gre numerous vascular malformations can be seen in the sellar / juxtasellar regions , including arterial aneurysms ( saccular or giant aneurysms of the internal carotid artery [ ica ] ) , cavernous carotid fistulas , arterial - venous malformations , haemangiomas or cavernous malformations . imaging of the sellar and juxtasellar region in the paediatric population is ideally performed with mri , and relies on a strong fundamental knowledge of the anatomy and signal characteristics of a normal gland and surrounding structures . the knowledge of key differentiating mri characteristics in common and uncommon disease entities of the paediatric sellar and juxtasellar regions , in conjunction with the clinical findings , undoubtedly aids in alerting and guiding the clinician down the appropriate path of management . it is best to approach a suspected sellar or juxtasellar abnormality with an open mind and a broad differential , with mri being the gold standard of imaging to accurately characterise these lesions . | [
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numerous studies over the last two decades , in other countries as well as in the united states , have found wide variations in the per capita utilization rates of many medical and surgical procedures .
though many have contributed , this variations phenomenon has come to be most closely associated with the work of wennberg and his coinvestigators ( wennberg and gittlesohn , 1980 ; wennberg and gittlesohn , 1982 ; mcpherson et al . , 1982 ) .
wennberg has argued that the substantial variations can not be adequately explained by differences in population characteristics such as age or morbidity ( wennberg , 1987 ) and has proposed that the most important factor is the style of medical practice ( wennberg and gittlesohn , 1982 ) . a wide variety of acceptable practice style is made possible by the substantial uncertainty physicians face in diagnosing disease and prescribing appropriate treatment ( wennberg , barnes , and zubkoff , 1982 ) .
the proposition that variations in per capita utilization are important because of such factors may be referred to as the practice style hypothesis . in a recent article , stano examined wennberg 's practice style hypothesis by analyzing not only intermarket variations in per capita utilization , the standard measure in studies of this kind , but also variations in the intensity with which physicians treat their patients ( stano , 1986 ) . for a large sample of procedures provided to regular michigan blue shield subscribers in 1980 , it confirmed the wide intermarket differences in per capita utilization rates that were expected on the basis of previous research .
however , the study also revealed substantial intramarket variations in the intensity of care rendered by physicians to their patients .
more important , no clear relationships were found between per capita utilization and either the average intensity at which physicians treat their patients or the degree of intramarket variation in this intensity .
this evidence suggests that practice style may not be as important in explaining the variations phenomenon as has been advanced in the literature .
the article also suggests that patients in high - care areas tend to see a higher number of providers , a potentially significant alternative factor in explaining differences in per capita utilization .
the present study applies the stano ( 1986 ) methodology to data for michigan 's medicare enrollees and further investigates the practice style hypothesis in the medicare context .
this analysis both illuminates the practice style issue and bears on public policy concerns over medicare cost containment . with especially rapid rates of increase in health care spending by the elderly ( waldo and lazenby , 1984 ) and the consequent budgetary pressures on the medicare program , the search for cost - saving strategies has led to a growing concern over the extent to which the variations phenomenon also applies to medical services provided to medicare beneficiaries .
for example , a recent contribution on medicare part b utilization ( chassin et al . , 1986 ) found wide variation between markets in the per capita utilization rates of many procedures .
across 13 large areas of 8 states , the per capita utilization rate in the highest use area was at least three times the rate in the lowest area for 67 of the 123 medical and surgical procedures that were examined .
not surprisingly , the variation between areas , as measured by the coefficient of variation , was high for many procedures .
the value of this index was greater than 30 for 76 of the 123 procedures , and averaged 44 for the set of 117 procedures that excluded physician office visits .
it uses geographic areas of analysis designed to represent physician market areas as opposed to the larger areas used in the chassin et al .
the per capita utilization variable is an aggregate across 311 procedures based on a relative value scale , which not only standardizes heterogeneous procedures into a single scale but also permits an assessment of the degree to which variations in individual procedures wash out in the aggregate .
more importantly , in addition to per capita utilization , it presents direct evidence on variations in a practice style indicator intensity of patient care .
this intensity is measured by the average number of relative value units a physician provided per patient for those patients treated in 1980 .
this measure permits an assessment of the degree to which practice style accounts for variations in per capita utilization .
the results show that per capita utilization rates for medicare part b , when aggregated through a relative value scale , are more uniform than one might expect from the existing literature .
the medicare utilization rates are considerably more uniform than a comparable index of use by regular blue shield members .
moreover , as is the case for blue shield beneficiaries , there are substantial within - market variations in intensity of care . finally , unlike the suggestions by wennberg and others , we find that the relationship between intensity of care and per capita utilization is weak to insignificant .
to permit comparisons with the data reported in the michigan blue shield study , we identified 311 medicare procedures as being comparable in scope to the 424 high - frequency or high - charge procedures used in that analysis .
the medicare files define groups of procedures somewhat differently from the blue shield files so that the 311 medicare procedures represent the same set of procedures as the 424 blue shield procedures .
thus , the utilization rates for medicare can be directly compared with those for blue shield .
the 311 procedures were dominated by surgical procedures and accounted for 35 percent of all medicare part b charges in michigan in 1980 . to standardize the heterogeneous services into a single measure
, the 311 procedures were aggregated on the basis of relative value units ( rvu 's ) . by this method ,
an office visit for an established patient - brief service was chosen as the base unit and was arbitrarily assigned the value of 1 .
then the weight for the ith procedure was taken as the statewide relative charge for the ith procedure .
for example , a procedure whose average statewide charge was 10 times the charge for a brief office visit was given the weight of 10 .
thus , the total rvu 's in each utilization measure represents the total brief office visit equivalents .
we examine this rvu aggregate , not because it is inherently always superior to analysis of individual procedures , but because it complements previous research in several useful ways .
first , by reducing the data to a single measure , the data become easily understood and the index is readily applied to various analyses .
second , the data provide a basis for assessing the average cost implications of inter - area variations . finally , if an area tends to have higher than average utilization for one set of procedures but lower for another , the variation in rvu 's ( and consequently in expenditures ) will be less than that reported for many individual procedures .
thus , the rvu measure permits some assessment of the tendency for variation in practice style to wash out . two measures of utilization were derived using the 1980 rvu data .
one measure , the per capita utilization , is defined as the total number of rvu 's generated by physicians in an area for that year divided by the area 's effective eligible population .
it is defined as the total number of rvu 's the physician provided divided by the total number of different patients seen by the physician in that year .
this figure represents the average number of rvu 's provided by physicians to their patients in 1980 . by computing this value for every physician in the sample , both the average intensity with which physicians render care in each area as well as
the intensity measure is used in this study as an index of the standard of practice .
its intramarket variation is taken as an indicator of the extent to which that standard varies .
we stress that , although the two utilization measures derive from the same rvu data , they are not equivalent measures .
ceteris paribus , the per capita utilization rate will be a direct function of the average intensity of care , but it will also vary directly with the proportion of eligible enrollees that visit physicians and the average number of physicians that treat those patients who have sought care .
thus , when all things are considered , per capita utilization and intensity are not necessarily positively correlated .
the data for both measures are presented by physician market areas derived on the basis of the economics of location and adjusted for patient bordercrossing . to devise the market areas , data on patient origin and place of service
were arrayed for each of michigan 's 83 counties forming an 83 83 matrix .
the matrix recorded the percentage of relative value units the residents of each county received from every other county .
the major weight in determining central places was placed on the ability of the county to serve its own patients . in most cases ,
the central place counties were those that provided at least 75 percent of the care received by patients originating from that county .
noncentral place counties were then assigned to contiguous central places based on the percentage of care provided in a manner that generated groups of counties identified as physician market areas . each market area formed provided at least 80 percent of the patient visits originating from its own population .
in addition , a correction for patient bordercrossing was made by reassigning population to calculate each area 's effective population . with this adjustment , if , for example , residents of market a received 10 percent of their rvu 's in market b , the latter 's effective population was increased by 10 percent of market a 's population , and market a 's population was correspondingly reduced .
thus , each market received 14 adjustments to account for net export ( import ) of services to the other market areas .
although there are exceptions , high - unadjusted - use areas tend to be net exporters of patient services . a method that ignores bordercrossing would seriously distort the utilization rates and indicate greater intermarket variation than actually exists .
however , we did find that medicare patients received a greater portion of their care in their own market areas than blue shield patients . in each of the following analyses ,
we have also scaled the coefficient by multiplying the ratio by 100 . in dividing by the mean , the coefficient standardizes the measure of variation to account for the scale of the series ; thus , two series with different means may be compared .
however , most area studies have reported the cv and that evidence offers some basis for comparison .
presented in table 1 are the 15 market areas , their physician and medicare populations , and their utilization rates for both blue shield subscribers and medicare eligibles .
the coefficient of variation for the two utilization series is also discussed in table 1 .
as one would expect , medicare utilization is substantially higher , 134 percent higher for the state , than the blue shield utilization .
however , the patterns for both populations are similar , with the contiguous detroit , flint , pontiac , and warren markets showing substantially higher utilization than the rest of the state .
the populations in these market areas have the highest per capita incomes and are among the most comprehensively insured in michigan .
other medical resources , such as hospitals and physician specialists , are also concentrated in these areas .
the data also suggest that there are roughly two groups of utilization rates across the state .
one is the approximately 19 rvu 's per medicare enrollee found in the high - use areas noted earlier .
the other is the approximately 12 - 15 rvu range found for most of the remaining markets .
however , these two small population areas are probably anomalies in that they are the two areas in the state that are most sensitive to uncaptured out - of - state service .
most noteworthy , the variation among the medicare population is much smaller than the variation among the blue shield population .
the coefficient of variation ( cv ) for medicare is 30 percent lower than that for blue shield , i.e. , 21.3 versus 30.3 .
the difference in variation shows up in the difference between the higher - and lower - use area .
joseph areas , medicare utilization in the highest area ( detroit ) is 62 percent higher than the utilization in the lowest area ( petoskey ) . by comparison ,
blue shield utilization in the highest area ( pontiac ) is 86 percent higher than utilization in the lowest area ( kalamazoo ) .
the result for michigan suggests that the variations phenomenon may be less of a concern for the medicare population than for the non - medicare population . the relatively lower level of variation for medicare utilization also suggests that michigan 's medicare enrollees have not been greatly disadvantaged by uneven access to care .
it was possible that variations in medicare utilization could be exacerbated by variations in the rates at which physicians accept assignment and variations in financial abilities of those patients whose physicians do not accept assignment .
however , the relatively low coefficient of variation for medicare utilization suggests this is not a problem .
physicians accepted assignment on claims accounting for 73 percent of medicare part b charges in 1980 , a figure that was much higher than the rates in most other states as well as the national average . in 1978 ,
we estimated that blue cross payments per rvu were only 9 percent higher than medicare payments per rvu .
this figure is considerably less than the 20 to 30 percent differences found in one national study ( cromwell and burstein , 1985 ) .
so the relative financial difference to physicians of treating medicare versus blue shield patients was small .
secondly , the coefficient of variation for the medicare population appears relatively low in comparison to other reported figures . as noted previously , the coefficients of variation reported by chassin et al .
although the differences could result from underlying differences in the populations and the physicians , another plausible explanation is at hand in the process of aggregation .
if levels of utilization of individual procedures are not highly correlated , the variations will tend to be dampened in an aggregate measure .
it has been reported elsewhere that areas that are high in one set of procedures tend to be low in others ( chassin et al . , 1986 ) .
thus , the aggregate rvu measure will be less volatile . in consequence , and to this degree , variations in average costs will tend to be less than might have been expected based on previous reports for individual procedures .
this result for rvu 's helps to illuminate one aspect of the variations phenomenon , but it does not invalidate wennberg 's ( 1984 ) policy proposals aimed at curbing the degree of variation in individual procedures . if some procedures are indeed excessively used in some areas , a reduction in their utilization could be important for cost savings .
however , we underscore chassin 's ( 1986 ) warning that the variations phenomenon is easily misinterpreted in this regard .
it is too tempting to conclude that inappropriately high levels of care are being rendered in high - use markets .
the observed differences might be explained by insufficient use in some markets , differences in the incidence of illness , or other unknown effects .
a unique feature of this data set is that it contains a direct indicator of practice style .
as previously defined , intensity of care measures the number of rvu 's provided to an average patient over a period of time by each physician .
variations in intensity may be interpreted as reflecting variations in a physician 's propensity to utilize various medical procedures for a given set of indications , hence practice style . because intensity is calculated for each physician
intensity data for the 15 markets for both primary care physicians and general surgeons are reported in table 2 . the variation in intensity
is measured by the coefficient of variation and is reported for both within markets ( note the cv columns ) and between markets ( note the cv row ) .
the physician samples consist of all physicians and doctors of osteopathy who had practiced in the same market area from 1975 to 1980 , provided at least 25 rvu 's each year , and treated at least 5 different medicare patients per year . as is apparent in table 2 , variations in intensity within markets are quite substantial .
coefficients of variation for primary care range from a low of 18.1 to a high of 76.0 .
variation in intensity between markets is relatively low , 14.8 and 18.9 for primary care physicians and general surgeons , respectively . that these coefficients of variation are lower than those for within market variation is to be expected because the market intensity values are themselves sample means
the more important issue examined here is whether the differences in physician practice style account significantly for the observed intermarket variations in per capita utilization rates .
this issue is pursued by conducting simple bivariate regressions with the dependent variable taken as the medicare per capita utilization rate and the independent variable the intensity level .
these estimates and regressions of intensity level on the respective intramarket coefficient of variation are reported in table 3 . for primary care physicians ,
the coefficient of the intensity variable is positive but the equation overall is not significant at the 5-percent level .
likewise , in the third regression , the primary care intensity level is not significantly related to the primary care intramarket coefficient of variation .
therefore , there is no clear evidence that a more intense style of practice by primary care physicians , the dominant specialty in terms of size , is associated with higher per capita utilization .
as argued elsewhere , higher per capita utilization rates must result from a higher proportion of beneficiaries who seek care , or larger numbers of physicians seen by a typical patient ( stano , 1985 ) .
the insignificance of primary care intensity suggests a simple but clear warning that these other sources of variation should not be overlooked .
furthermore , these other sources of variation are not in any obvious sense physician - caused or practice style phenomena , and consequently suggest very different policy responses . because many variations studies have focused on surgical procedures , the results for general surgeons are of special interest . for general surgeons ,
furthermore , the coefficient for general surgeon intensity ( gsint ) is positive and significant as is suggested by the practice style hypothesis .
second , and more important , the significance of the general surgeon equations is largely the result of two outlier observations , pontiac and warren .
both pontiac and warren have high per capita utilization that are not only the highest rvu 's per patient , but also have higher values for the coefficient of variation of the intensity variable . without these two observations ,
the f value for the regression of utilization on general surgeon intensity is only 0.26 and insignificant .
although there is no reason to exclude these observations , the dependence on so few observations for significance indicates that the relationship is tenuous .
when the two outliers are excluded , the equation f value drops from 15.14 to 0.70 .
finally , though not reported in table 3 , we also developed equations to predict the individual physician values of the intensity variable for both primary care and general surgeons .
independent variables included medicare fees , a large set of market and economic and demographic variables , indicators of the health status of the population , the availability of physicians , and the availability of hospital beds .
in particular , the average intensity with which general surgeons practiced was not related to the availability of general surgeons or hospital beds .
the average intensity for primary care physicians also was unrelated to their availability or to the availability of other specialists and hospital beds .
we have examined the extent of intermarket variations in per capita utilization of physician services by michigan 's medicare population . the analysis employed an aggregation of individual procedures by a relative value unit index that reflects relative costs .
in addition , the analysis employs physician market areas devised through the economics of location with populations adjusted to reflect patient bordercrossing .
the results indicate that intermarket variation in utilization for medicare is substantially less than that for blue shield .
this suggests , for michigan at least , that the variations phenomenon is less of a public policy worry for the medicare population than for the non - medicare population .
in addition , the level of variation in medicare utilization is found to be relatively low .
the coefficient of variation value of 21 for our aggregate of 311 procedures is less than one - half of the average value reported by chassin et al .
if utilization in an area is high for one set of procedures but low for others , the variations phenomenon will tend to wash out in the aggregate .
our result indicates that substantial washing out occurs so that variations in average costs will tend to be less than is suggested by reports on individual procedures .
this result does not in itself invalidate policy designed to save costs by curbing variation .
if in fact high utilization for some procedures in some areas is unnecessary , then substantial costs may be saved by curbing variation in individual procedures .
a relatively unique feature of our data set is that it contains a direct indicator of practice style .
the indicator of practice style is the intensity with which physicians treat their patients , here measured as the number of relative value units each physician delivers to an average patient over a year .
there are wide variations within markets in intensity for both primary care physicians and general surgeons .
the within - market - area coefficients of variation for the two groups , respectively , had average values of 55 and 64 ( although in each case it was less than its previously reported blue shield counterpart ) .
the across - market - area coefficients of variation , in contrast , were relatively low , 15 and 19 , respectively .
perhaps the most wide - ranging issue addressed in this article is the relationship between intensity and per capita utilization .
we found that primary care physician intensity is not significantly related to medicare per capita utilization in michigan .
furthermore , the relationship between general surgeons ' intensity and medicare utilization is only weakly significant and depends critically on two outlier observations .
these results present the warning that the practice style hypothesis does not adequately account for the variations phenomenon , at least in michigan .
thus , the higher utilization of some market areas must be attributable to other factors , namely a higher proportion of eligible users who seek care or a larger number of physicians who provide care to patients who seek care . because there is no way of determining from these data whether these factors are physician influenced or originate largely with patients
, one can not conclude that the practice style factor accounts for much of the observed per capita variations .
one major influence on patients ' decisions to seek care or to seek out more sources of care is likely to be the extent of the availability of medical resources .
this accounts for the significant availability effects found in most aggregated studies of per capita utilization , e.g. , the classic study by fuchs and kramer ( 1972 ) .
although the practice style factor may be an element of treatment decisions that increase the number of sources of care , as manifested by greater use of referrals to specialists , laboratories , and other diagnostic tests , this is distinct from wennberg 's concept of a largely random phenomenon where physicians have a predilection to practice more intensively in one market , and less so elsewhere .
it is also distinct from the positive relationships between per capita rates of surgery and the availability of surgeons and hospital beds that wennberg and others have found .
our results do not imply that variations in physician utilization are unimportant . to the extent that one would expect higher per capita utilization of physician care , for whatever reason , to be accompanied by increases in the use of other medical resources ,
although physician care may substitute for other forms of care , hospital utilization and physician utilization are likely to be related . with hospital costs for the elderly more than twice the expenditures on physician care ( waldo and lazenby , 1984 ) , variations in hospital costs resulting from variations in physician care will be magnified .
the importance of this magnification is illustrated , for example , in one study of hospital utilization and costs that documented over threefold differences in contiguous markets in 1983 ( schramm , 1985 ) .
many , however , interpret the variations in per capita utilization as strong evidence of unnecessary utilization .
we believe that those who have reached this conclusion , or use the evidence to support a policy of controls that would impose the standards found in the more moderate use markets to other areas , are ignoring the complexity of the issue .
in particular , the evidence we have provided indicates that the variations in per capita use in a set consisting of a large number of procedures are not closely related to an index of the practice style factor that was introduced here .
elsewhere , the practice style concept is itself not well defined so that it appears as a catchall for the residual variation in per capita use rates that has not been explained .
these alternatives could include the number and types of providers seen , differences in consumer preferences with respect to physician and other forms of care , an uneven diffusion of medical technology , and the structure of public and private insurance programs .
controlling health care expenditures and reducing unnecessary utilization are legitimate priorities , but the focus on local medical standards , physician training , and other dimensions of practice style to explain the disparities in use rates has obscured the role of these potentially important factors . | variations in the utilization of physicians ' services by medicare enrollees in michigan are examined in this article . two measures of market - area utilization are estimated .
one is the standard per capita utilization rate , which has been the common focus of many small area variation studies .
the second measures the intensity with which physicians treat their patients and can be taken as an indicator of the so - called practice - style phenomenon .
the results show that , although substantial intermarket variation in per capita utilization is found , the variations are not as large as one might expect and are considerably less than the variations in per capita utilization for michigan 's blue shield population . more important
, the relationship between a market 's per capita utilization and intensity of care of primary care physicians is insignificant .
the relevance of these findings , especially within the context of the practice style hypothesis and policy proposals that would establish physician practice norms , are discussed . | Introduction
Data and methods
Variations in per capita utilization
Variations in intensity of care
Intensity and per capita utilization
Conclusions | numerous studies over the last two decades , in other countries as well as in the united states , have found wide variations in the per capita utilization rates of many medical and surgical procedures . wennberg has argued that the substantial variations can not be adequately explained by differences in population characteristics such as age or morbidity ( wennberg , 1987 ) and has proposed that the most important factor is the style of medical practice ( wennberg and gittlesohn , 1982 ) . a wide variety of acceptable practice style is made possible by the substantial uncertainty physicians face in diagnosing disease and prescribing appropriate treatment ( wennberg , barnes , and zubkoff , 1982 ) . the proposition that variations in per capita utilization are important because of such factors may be referred to as the practice style hypothesis . in a recent article , stano examined wennberg 's practice style hypothesis by analyzing not only intermarket variations in per capita utilization , the standard measure in studies of this kind , but also variations in the intensity with which physicians treat their patients ( stano , 1986 ) . for a large sample of procedures provided to regular michigan blue shield subscribers in 1980 , it confirmed the wide intermarket differences in per capita utilization rates that were expected on the basis of previous research . however , the study also revealed substantial intramarket variations in the intensity of care rendered by physicians to their patients . more important , no clear relationships were found between per capita utilization and either the average intensity at which physicians treat their patients or the degree of intramarket variation in this intensity . this evidence suggests that practice style may not be as important in explaining the variations phenomenon as has been advanced in the literature . the article also suggests that patients in high - care areas tend to see a higher number of providers , a potentially significant alternative factor in explaining differences in per capita utilization . the present study applies the stano ( 1986 ) methodology to data for michigan 's medicare enrollees and further investigates the practice style hypothesis in the medicare context . this analysis both illuminates the practice style issue and bears on public policy concerns over medicare cost containment . with especially rapid rates of increase in health care spending by the elderly ( waldo and lazenby , 1984 ) and the consequent budgetary pressures on the medicare program , the search for cost - saving strategies has led to a growing concern over the extent to which the variations phenomenon also applies to medical services provided to medicare beneficiaries . , 1986 ) found wide variation between markets in the per capita utilization rates of many procedures . across 13 large areas of 8 states , the per capita utilization rate in the highest use area was at least three times the rate in the lowest area for 67 of the 123 medical and surgical procedures that were examined . the value of this index was greater than 30 for 76 of the 123 procedures , and averaged 44 for the set of 117 procedures that excluded physician office visits . it uses geographic areas of analysis designed to represent physician market areas as opposed to the larger areas used in the chassin et al . the per capita utilization variable is an aggregate across 311 procedures based on a relative value scale , which not only standardizes heterogeneous procedures into a single scale but also permits an assessment of the degree to which variations in individual procedures wash out in the aggregate . more importantly , in addition to per capita utilization , it presents direct evidence on variations in a practice style indicator intensity of patient care . this measure permits an assessment of the degree to which practice style accounts for variations in per capita utilization . the results show that per capita utilization rates for medicare part b , when aggregated through a relative value scale , are more uniform than one might expect from the existing literature . the medicare utilization rates are considerably more uniform than a comparable index of use by regular blue shield members . moreover , as is the case for blue shield beneficiaries , there are substantial within - market variations in intensity of care . finally , unlike the suggestions by wennberg and others , we find that the relationship between intensity of care and per capita utilization is weak to insignificant . to permit comparisons with the data reported in the michigan blue shield study , we identified 311 medicare procedures as being comparable in scope to the 424 high - frequency or high - charge procedures used in that analysis . the medicare files define groups of procedures somewhat differently from the blue shield files so that the 311 medicare procedures represent the same set of procedures as the 424 blue shield procedures . thus , the utilization rates for medicare can be directly compared with those for blue shield . the 311 procedures were dominated by surgical procedures and accounted for 35 percent of all medicare part b charges in michigan in 1980 . to standardize the heterogeneous services into a single measure
, the 311 procedures were aggregated on the basis of relative value units ( rvu 's ) . then the weight for the ith procedure was taken as the statewide relative charge for the ith procedure . thus , the total rvu 's in each utilization measure represents the total brief office visit equivalents . first , by reducing the data to a single measure , the data become easily understood and the index is readily applied to various analyses . second , the data provide a basis for assessing the average cost implications of inter - area variations . finally , if an area tends to have higher than average utilization for one set of procedures but lower for another , the variation in rvu 's ( and consequently in expenditures ) will be less than that reported for many individual procedures . thus , the rvu measure permits some assessment of the tendency for variation in practice style to wash out . two measures of utilization were derived using the 1980 rvu data . one measure , the per capita utilization , is defined as the total number of rvu 's generated by physicians in an area for that year divided by the area 's effective eligible population . by computing this value for every physician in the sample , both the average intensity with which physicians render care in each area as well as
the intensity measure is used in this study as an index of the standard of practice . its intramarket variation is taken as an indicator of the extent to which that standard varies . we stress that , although the two utilization measures derive from the same rvu data , they are not equivalent measures . ceteris paribus , the per capita utilization rate will be a direct function of the average intensity of care , but it will also vary directly with the proportion of eligible enrollees that visit physicians and the average number of physicians that treat those patients who have sought care . thus , when all things are considered , per capita utilization and intensity are not necessarily positively correlated . the data for both measures are presented by physician market areas derived on the basis of the economics of location and adjusted for patient bordercrossing . to devise the market areas , data on patient origin and place of service
were arrayed for each of michigan 's 83 counties forming an 83 83 matrix . the major weight in determining central places was placed on the ability of the county to serve its own patients . in most cases ,
the central place counties were those that provided at least 75 percent of the care received by patients originating from that county . noncentral place counties were then assigned to contiguous central places based on the percentage of care provided in a manner that generated groups of counties identified as physician market areas . each market area formed provided at least 80 percent of the patient visits originating from its own population . with this adjustment , if , for example , residents of market a received 10 percent of their rvu 's in market b , the latter 's effective population was increased by 10 percent of market a 's population , and market a 's population was correspondingly reduced . a method that ignores bordercrossing would seriously distort the utilization rates and indicate greater intermarket variation than actually exists . however , we did find that medicare patients received a greater portion of their care in their own market areas than blue shield patients . in dividing by the mean , the coefficient standardizes the measure of variation to account for the scale of the series ; thus , two series with different means may be compared . presented in table 1 are the 15 market areas , their physician and medicare populations , and their utilization rates for both blue shield subscribers and medicare eligibles . as one would expect , medicare utilization is substantially higher , 134 percent higher for the state , than the blue shield utilization . however , the patterns for both populations are similar , with the contiguous detroit , flint , pontiac , and warren markets showing substantially higher utilization than the rest of the state . the populations in these market areas have the highest per capita incomes and are among the most comprehensively insured in michigan . one is the approximately 19 rvu 's per medicare enrollee found in the high - use areas noted earlier . the other is the approximately 12 - 15 rvu range found for most of the remaining markets . however , these two small population areas are probably anomalies in that they are the two areas in the state that are most sensitive to uncaptured out - of - state service . most noteworthy , the variation among the medicare population is much smaller than the variation among the blue shield population . the coefficient of variation ( cv ) for medicare is 30 percent lower than that for blue shield , i.e. joseph areas , medicare utilization in the highest area ( detroit ) is 62 percent higher than the utilization in the lowest area ( petoskey ) . by comparison ,
blue shield utilization in the highest area ( pontiac ) is 86 percent higher than utilization in the lowest area ( kalamazoo ) . the result for michigan suggests that the variations phenomenon may be less of a concern for the medicare population than for the non - medicare population . the relatively lower level of variation for medicare utilization also suggests that michigan 's medicare enrollees have not been greatly disadvantaged by uneven access to care . it was possible that variations in medicare utilization could be exacerbated by variations in the rates at which physicians accept assignment and variations in financial abilities of those patients whose physicians do not accept assignment . however , the relatively low coefficient of variation for medicare utilization suggests this is not a problem . this figure is considerably less than the 20 to 30 percent differences found in one national study ( cromwell and burstein , 1985 ) . secondly , the coefficient of variation for the medicare population appears relatively low in comparison to other reported figures . as noted previously , the coefficients of variation reported by chassin et al . if levels of utilization of individual procedures are not highly correlated , the variations will tend to be dampened in an aggregate measure . thus , the aggregate rvu measure will be less volatile . in consequence , and to this degree , variations in average costs will tend to be less than might have been expected based on previous reports for individual procedures . this result for rvu 's helps to illuminate one aspect of the variations phenomenon , but it does not invalidate wennberg 's ( 1984 ) policy proposals aimed at curbing the degree of variation in individual procedures . however , we underscore chassin 's ( 1986 ) warning that the variations phenomenon is easily misinterpreted in this regard . it is too tempting to conclude that inappropriately high levels of care are being rendered in high - use markets . the observed differences might be explained by insufficient use in some markets , differences in the incidence of illness , or other unknown effects . a unique feature of this data set is that it contains a direct indicator of practice style . as previously defined , intensity of care measures the number of rvu 's provided to an average patient over a period of time by each physician . variations in intensity may be interpreted as reflecting variations in a physician 's propensity to utilize various medical procedures for a given set of indications , hence practice style . because intensity is calculated for each physician
intensity data for the 15 markets for both primary care physicians and general surgeons are reported in table 2 . as is apparent in table 2 , variations in intensity within markets are quite substantial . variation in intensity between markets is relatively low , 14.8 and 18.9 for primary care physicians and general surgeons , respectively . that these coefficients of variation are lower than those for within market variation is to be expected because the market intensity values are themselves sample means
the more important issue examined here is whether the differences in physician practice style account significantly for the observed intermarket variations in per capita utilization rates . this issue is pursued by conducting simple bivariate regressions with the dependent variable taken as the medicare per capita utilization rate and the independent variable the intensity level . for primary care physicians ,
the coefficient of the intensity variable is positive but the equation overall is not significant at the 5-percent level . likewise , in the third regression , the primary care intensity level is not significantly related to the primary care intramarket coefficient of variation . therefore , there is no clear evidence that a more intense style of practice by primary care physicians , the dominant specialty in terms of size , is associated with higher per capita utilization . as argued elsewhere , higher per capita utilization rates must result from a higher proportion of beneficiaries who seek care , or larger numbers of physicians seen by a typical patient ( stano , 1985 ) . the insignificance of primary care intensity suggests a simple but clear warning that these other sources of variation should not be overlooked . furthermore , these other sources of variation are not in any obvious sense physician - caused or practice style phenomena , and consequently suggest very different policy responses . because many variations studies have focused on surgical procedures , the results for general surgeons are of special interest . for general surgeons ,
furthermore , the coefficient for general surgeon intensity ( gsint ) is positive and significant as is suggested by the practice style hypothesis . second , and more important , the significance of the general surgeon equations is largely the result of two outlier observations , pontiac and warren . both pontiac and warren have high per capita utilization that are not only the highest rvu 's per patient , but also have higher values for the coefficient of variation of the intensity variable . without these two observations ,
the f value for the regression of utilization on general surgeon intensity is only 0.26 and insignificant . although there is no reason to exclude these observations , the dependence on so few observations for significance indicates that the relationship is tenuous . when the two outliers are excluded , the equation f value drops from 15.14 to 0.70 . finally , though not reported in table 3 , we also developed equations to predict the individual physician values of the intensity variable for both primary care and general surgeons . independent variables included medicare fees , a large set of market and economic and demographic variables , indicators of the health status of the population , the availability of physicians , and the availability of hospital beds . in particular , the average intensity with which general surgeons practiced was not related to the availability of general surgeons or hospital beds . the average intensity for primary care physicians also was unrelated to their availability or to the availability of other specialists and hospital beds . we have examined the extent of intermarket variations in per capita utilization of physician services by michigan 's medicare population . in addition , the analysis employs physician market areas devised through the economics of location with populations adjusted to reflect patient bordercrossing . the results indicate that intermarket variation in utilization for medicare is substantially less than that for blue shield . this suggests , for michigan at least , that the variations phenomenon is less of a public policy worry for the medicare population than for the non - medicare population . in addition , the level of variation in medicare utilization is found to be relatively low . the coefficient of variation value of 21 for our aggregate of 311 procedures is less than one - half of the average value reported by chassin et al . if utilization in an area is high for one set of procedures but low for others , the variations phenomenon will tend to wash out in the aggregate . our result indicates that substantial washing out occurs so that variations in average costs will tend to be less than is suggested by reports on individual procedures . if in fact high utilization for some procedures in some areas is unnecessary , then substantial costs may be saved by curbing variation in individual procedures . a relatively unique feature of our data set is that it contains a direct indicator of practice style . the indicator of practice style is the intensity with which physicians treat their patients , here measured as the number of relative value units each physician delivers to an average patient over a year . there are wide variations within markets in intensity for both primary care physicians and general surgeons . the within - market - area coefficients of variation for the two groups , respectively , had average values of 55 and 64 ( although in each case it was less than its previously reported blue shield counterpart ) . the across - market - area coefficients of variation , in contrast , were relatively low , 15 and 19 , respectively . perhaps the most wide - ranging issue addressed in this article is the relationship between intensity and per capita utilization . we found that primary care physician intensity is not significantly related to medicare per capita utilization in michigan . furthermore , the relationship between general surgeons ' intensity and medicare utilization is only weakly significant and depends critically on two outlier observations . these results present the warning that the practice style hypothesis does not adequately account for the variations phenomenon , at least in michigan . thus , the higher utilization of some market areas must be attributable to other factors , namely a higher proportion of eligible users who seek care or a larger number of physicians who provide care to patients who seek care . because there is no way of determining from these data whether these factors are physician influenced or originate largely with patients
, one can not conclude that the practice style factor accounts for much of the observed per capita variations . one major influence on patients ' decisions to seek care or to seek out more sources of care is likely to be the extent of the availability of medical resources . this accounts for the significant availability effects found in most aggregated studies of per capita utilization , e.g. although the practice style factor may be an element of treatment decisions that increase the number of sources of care , as manifested by greater use of referrals to specialists , laboratories , and other diagnostic tests , this is distinct from wennberg 's concept of a largely random phenomenon where physicians have a predilection to practice more intensively in one market , and less so elsewhere . it is also distinct from the positive relationships between per capita rates of surgery and the availability of surgeons and hospital beds that wennberg and others have found . our results do not imply that variations in physician utilization are unimportant . to the extent that one would expect higher per capita utilization of physician care , for whatever reason , to be accompanied by increases in the use of other medical resources ,
although physician care may substitute for other forms of care , hospital utilization and physician utilization are likely to be related . with hospital costs for the elderly more than twice the expenditures on physician care ( waldo and lazenby , 1984 ) , variations in hospital costs resulting from variations in physician care will be magnified . many , however , interpret the variations in per capita utilization as strong evidence of unnecessary utilization . we believe that those who have reached this conclusion , or use the evidence to support a policy of controls that would impose the standards found in the more moderate use markets to other areas , are ignoring the complexity of the issue . in particular , the evidence we have provided indicates that the variations in per capita use in a set consisting of a large number of procedures are not closely related to an index of the practice style factor that was introduced here . elsewhere , the practice style concept is itself not well defined so that it appears as a catchall for the residual variation in per capita use rates that has not been explained . these alternatives could include the number and types of providers seen , differences in consumer preferences with respect to physician and other forms of care , an uneven diffusion of medical technology , and the structure of public and private insurance programs . controlling health care expenditures and reducing unnecessary utilization are legitimate priorities , but the focus on local medical standards , physician training , and other dimensions of practice style to explain the disparities in use rates has obscured the role of these potentially important factors . | [
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] |
myeloproliferative neoplasms ( mpn ) are clonal hematopoietic stem cell disorders characterized by abnormal proliferation and expansion of one or more myeloid lineages [ 1 , 2 ] .
the group of the common , classic mpn includes chronic myeloid leukemia ( cml ) defined by the philadelphia chromosome ( ph ) and the three ph - negative entities ' polycythemia vera ( pv ) , essential thrombocythemia ( et ) , and primary myelofibrosis ( pmf ) .
the group of nonclassic mpn includes systemic mastocytosis ( sm ) , chronic neutrophilic leukemia ( cnl ) , and chronic eosinophilic leukemia ( cel ) [ 1 , 3 ] .
aberrant tyrosine kinase ( tk ) signaling is a common hallmark in mpn and has been shown to represent a key driver of the disease . the bcr - abl1 fusion gene , which results in a constitutive activation of abl1 kinase activity , characterizes cml [ 46 ] . in a majority of patients with pv , et , and pmf , the activating v617f mutation in the receptor - associated tk jak2
in addition , mutations in exon 12 of jak2 and mutations in the thrombopoietin receptor ( mpl
w515k / l ) have been described in these entities [ 11 , 12 ] .
more recently , somatic mutations in calr were found in jak2- and mpl - negative patients with et or pmf [ 13 , 14 ] .
the activating point mutation d816v in the kit receptor tk is a diagnostic criterion for sm and is found in more than 80% of all patients with sm .
a constitutively activated fip1l1-pdgfra fusion tk has been identified in patients with cel with or without an accompanying hypereosinophilic syndrome ( he s ) [ 16 , 17 ] .
more recently , csfr3 mutations have been described as a recurrent defect in patients with cnl .
common pathogenic mechanisms are observed despite the variety of different oncogenic mutations underling specific mpn types .
aberrant expression of inflammatory cytokines has been associated with patients ' symptoms and alterations of the bone marrow ( bm ) microenvironment as well as progression of the disease .
several different studies have suggested an important role for the bm microenvironment in the pathogenesis of hematologic malignancies including mpn .
in fact , alterations in the bm microenvironment such as increased microvessel density ( angiogenesis ) , fibrosis , and thickening of bone trabeculae are typical pathological findings in mpn and may contribute to disease phenotypes and disease progression .
this review focuses on the cytokine regulation of microenvironmental cells with special emphasis on common as well as distinct pathogenetic mechanisms in various mpn .
in particular , expression and functional relevance of interleukin-6 ( il-6 ) , il-8 , vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( fgf - b ) , hepatocyte growth factor ( hgf ) , platelet derived growth factor ( pdgf ) , oncostatin m ( osm ) , tumor necrosis factor- ( tnf- ) , transforming growth factor- ( tgf- ) , and stroma derived factor-1 ( sdf-1 , cxcl-12 ) are reviewed .
evidence for increased expression of these cytokines in various mpn is summarized in table 1 .
furthermore , the effect of jak1/2 inhibitors on the cytokine storm in mpn and targeted drugs for vegf / vegfr , hgf / c - met , and sdf-1/cxcr-4 are discussed .
cml is characterized by the reciprocal chromosome translocation t(9;22 ) and the resulting bcr - abl1 fusion gene [ 5 , 6 ] .
the bcr - abl1 oncoprotein exhibits constitutive tk activity and triggers key signaling pathways , including the ras - raf - mek - erk pathway , the phosphoinositide 3-kinase - akt pathway , and stat5 [ 19 , 20 ] .
cytokines and other effector molecules downstream of these aberrant signaling cascades have been implicated in the pathogenesis of cml .
aguayo et al . investigated bm vascularity and cytokine levels in cml and other hematologic neoplasms .
cml patients reportedly have increased bm vessel density and elevated serum levels of vegf , hgf , fgf - b , and tnf- compared to controls [ 23 , 24 ] .
furthermore , high vegf levels were found to correlate with a shorter survival of patients in chronic phase cml .
immunohistochemical staining of bm sections showed that vegf is expressed primarily in myeloid progenitor cells , megakaryocytes , and mature granulomonocytic cells in chronic phase cml as well as in myeloid differentiated blast cells in the blast phase of cml .
the bcr - abl1 oncoprotein was found to upregulate expression of vegf in cml cells , and analysis of signaling pathways downstream of bcr - abl1 revealed that the mammalian target of rapamycin ( mtor ) contributes to bcr - abl1-dependent expression of vegf .
targeting of mtor by rapamycin in cml cells inhibited not only vegf expression but also the in vitro growth of leukemic cells . cd34 +
bm cells derived from cml patients secreted up to 10 times more vegf , fgf - b , hgf , and il-8 compared to normal donors ' bm cd34 + cells .
furthermore , bm mononuclear cells isolated from cml patients induced vascularization of matrigel implants in mice .
a number of additional studies described expression of hgf in cml cells [ 3033 ] .
in particular , elevated hgf levels in bm and peripheral blood and a correlation of hgf expression with microvessel density in the bm were found .
zhelyazkova and colleagues reported evaluated plasma hgf , cellular hgf , and expression of the hgf receptor c - met in cml patients . the plasma hgf level correlated with markers reflecting the tumor burden as well as with the phase of cml and overall survival in these patients .
in contrast , no prognostic relevance for vegf levels in chronic phase cml was observed in this study .
also , contrary to vegf , bcr - abl1 did not induce synthesis of hgf in vitro and targeting of bcr - abl1 with imatinib showed no effect on hgf expression .
although various cell types may express and release hgf , immunostaining of bm sections revealed that basophils are a primary source of hgf in cml .
expression of pdgf was reported to be associated with bm fibrosis in accelerated and blast phase cml .
il-2 and il-6 serum levels in patients with cml were found to be significantly elevated compared to controls . moreover , il-6 levels in cml patients were found to correlate with bm angiogenesis and reportedly increase during disease progression [ 51 , 52 , 92 ] .
the bcr - abl1 targeting tk inhibitor ( tki ) imatinib was found to downregulate il-6 and il-8 release in primary cml cells in vitro .
identified il-8 as one of the strongest downregulated genes in cml upon treatment with the tki dasatinib .
expression of bcr - abl1 resulted in a substantial upregulation of il-8 which was inhibited by dasatinib or nilotinib .
tnf- has recently been implicated in stem cell biology of mpn [ 94 , 95 ] .
a study investigated il-1 , il-6 , and tnf- serum levels in cml patients and described no significant difference for tnf- compared to controls .
however , cml stem / progenitor cells were found to produce tnf- in a kinase - independent fashion , and at higher levels relative to their normal cd34 + counterparts .
in addition , tnf- concentrations were found to be elevated in bm supernatants derived from bcr - abl1 transgenic mice compared to wild type mice .
elevated levels of inflammatory cytokines have been reported in all entities of classical mpn [ 38 , 49 , 51 , 53 , 58 , 9699 ] .
in particular in pmf , patients suffer from severe constitutional symptoms , and increasing evidence shows that several of these symptoms are mediated by proinflammatory cytokines .
tefferi et al . investigated the prognostic significance of cytokines in pmf by determining serum levels of a comprehensive cytokine panel . in this study , il-1 , il-1ra , il-2r , il-6 , il-8 , il-10 , il-12 , il-13 , il-15 , tnf- , granulocyte colony - stimulating factor ( g - csf ) , interferon ( ifn- ) , macrophage inflammatory protein 1 ( mip-1 ) , mip-1 , hgf , ifn--inducible protein 10 ( ip-10 ) , monokine induced by ifn- ( mig )
, monocyte chemotactic protein 1 ( mcp-1 ) , and vegf levels were found to be elevated in pmf patients .
in addition , the authors identified il-8 , il-2r , il-12 , and il-15 levels as independent prognostic factors for survival of patients with pmf .
these findings are in line with other studies showing elevated cytokine level in pmf , et , and pv [ 38 , 51 , 53 , 58 , 9698 ] .
however , the methods applied in these studies differed and the panels of elevated cytokines within different studies showed some inconsistences between these studies as reviewed by hasselbalch .
thus , better standardization is apparently needed to directly compare cytokine production in different mpn cohorts .
nevertheless , increasing evidence indicates that the disease burden of mpn is not only mediated by the primary neoplastic clone but also mediated by a secondary inflammation with an aberrant cytokine production and changes of the bm microenvironment .
the concept of cytokines contributing to tissue fibrosis , angiogenesis , and osteosclerosis / osteopenia in mpn has been well established .
in particular , fgf - b , il-8 , vegf , hgf , pdgfr , tgf- , tnf- , and osm have been implicated in bm microenvironment alterations in patients with mpn [ 21 , 22 , 3941 , 68 , 69 ] .
evidence for expression of these cytokines in pv , et , and pmf is discussed in the next paragraphs .
fgf - b was found to be elevated in the serum of mpn patients . while musolino et al .
reported increased fgf - b levels in pv , et , and pmf , vaidya et al .
found fgf - b together with il-1 , il-1ra , il-2r , egf , il-10 , fgf - b , il-12 , ifn- , and rantes to be particularly elevated in pmf when compared to pv patients .
moreover , high levels of il-6 and fgf - b were observed in a coculture model of jak2 v617f positive hematopoietic cells and stroma cells .
il-8 serum levels were significantly increased in patients with pmf , and il-8 expression was observed in various hematopoietic cell types , including granulocytes , monocytes , megakaryocytic cells , cd34 + progenitor cells , and platelets . increased serum levels of il-8
have also been described in patients with pv and et [ 38 , 58 , 101 ] , and il-8 was found to enhance formation of erythroid colonies in vitro . within a pmf patient cohort ,
a number of studies have described elevated vegf serum levels in mpn [ 36 , 49 , 78 ] .
immunohistochemical studies performed on bm sections of et , pv , and pmf patients revealed an increased expression of vegf and its receptor in all mpn groups compared to controls [ 79 , 80 ] .
megakaryocytes , macrophages , and immature myeloid precursors showed positive immunostaining while erythroid ( precursor ) cells stained negative for vegf .
detected elevated levels of hgf in the serum and bm plasma obtained from pv patients compared to secondary erythrocytosis patients that were employed as controls .
furthermore , bm stem cells and clonal erythroblasts were identified as the major sources of hgf in patients with pv .
further studies analyzing cytokine panels in plasma of mpn patients confirmed elevated hgf levels in pmf , pv , and et [ 38 , 49 ] .
subsequent studies found higher levels of tgf- in megakaryocytes in the bm of patients with myelofibrosis compared to controls .
in contrast , no increase in tgf- was found in bm cells of patients with et .
however , a substantial number of patients showed no detectable tnf- in peripheral blood and no association with clinical parameters and disease progression was observed .
another study identified tnf- as one of two cytokines that were differentially expressed when stratifying et and pv patients according to their jak2 v617f mutation status . in line with this finding , a murine bm transplant model for jak2 v617f showed a marked increase of tnf- serum levels .
this increased tnf- level was found to be accompanied by a decrease in erythropoietin and g - csf , which the authors discussed as a possible suppressive effect of tnf- on normal hematopoiesis .
we studied jak2 v617f - mediated gene expression and identified il-6 and the il-6 family members osm and leukemia inhibitory factor ( lif ) to be directly upregulated by v617f - mutated jak2 .
immunohistochemistry staining of bm section from patients with pmf , et , and pv showed that megakaryocytes , endothelial cells , and myeloid progenitors stain positive for osm , whereas erythroid cells were osm negative .
this pattern correlates with expression of phosphorylated stat5 , which was identified as the major signaling pathway of oncogene - dependent osm expression .
sm is mpn characterized by an abnormal accumulation of mast cells in the bm and other organs . in a substantial subset of patients , sm is accompanied by increased release of various mediators from mast cells and consecutive clinical symptoms [ 105107 ] .
kit is a receptor tk , and activation of kit signaling through its ligand stem cell factor ( scf ) mediates cell proliferation and survival in immature progenitor cells and mast cell differentiation , as well as mast cells migration , activation , and adhesion .
kit d816v shows constitutively active tk signaling and induces the recruitment of several downstream signaling pathways , including pi3-kinase / akt , mtor , and stat5 [ 109 , 111 ] .
brockow et al . measured levels of growth factors in plasma and skin blister fluid of patients with sm . il-3 and il-4 were not detectable , and scf as well as vegf levels showed no significant difference between patient samples and controls .
in contrast , il-6 was significantly increased in plasma of sm patients and correlated with serum tryptase levels .
subsequent studies confirmed increased il-6 plasma levels in sm cohorts and suggested a correlation with the severity of symptoms and the presence of osteoporosis . moreover ,
il-6 levels were found to correlate with disease category , severity of bm pathology , organomegaly , and extent of skin involvement .
thus , the authors suggested that il-6 was a useful surrogate marker of severity of disease .
high levels of il-6 were accompanied by increased levels of the osteoclast - regulating factors receptor activator of nuclear factor kappa - b ligand ( rankl ) and osteoprotegerin .
the authors argue that cytokines produced by mast cells might shift the balance of bone turnover towards increased bone resorption and decreased bone formation .
il-31 has been implicated in the induction of chronic skin inflammation and was found to be increased in patients with sm and to correlate with disease severity .
gene expression studies of purified bm mast cells in sm detected high expression of ccl-23 in indolent and aggressive sm , whereas il-1 , il-13 , or osm were particularly upregulated in aggressive sm . a number of studies used mast cell lines , in particular the kit d816v mutated human mast cell line hmc-1 , to investigate cytokine expression in sm .
selvan and colleagues described expression of mcp-1 , mip-1 , mip-1 , rantes , and il-8 in hmc-1 cells .
subsequent studies showed expression of tnf- , il-1 , and osm in hmc-1 cells .
fgf - b was found to be expressed in a number of murine mast cell lines and to be regulated by scf , tgf- , and tnf- .
immunohistochemical staining of bm sections derived from patients with sm showed expression of fgf - b and in some cases weak expression of tgf- . furthermore , mast cell infiltrates expressed vegf as determined by immunohistochemistry of bm sections . although no significant elevation of vegf levels was found in plasma of sm patients , it is likely that vegf is locally increased in the bm microenvironment and contributes to increased angiogenesis in sm .
comparative oncology studies in dogs showed expression of vegf in neoplastic mast cells [ 89 , 90 ] .
moreover , a correlation of vegf plasma levels with tumor grade and microvascular density was observed in canine mastocytoma .
we studied the effect of kit d816v on cytokine expression in various in vitro models .
the cytokine profile induced by kit d816v showed a marked overlap when compared to the profile induced by jak2 v617f and fip1l1-pdgfra .
a number of cytokines , including osm , were found to be regulated by all three oncogenes [ 59 , 61 , 115 ] .
these studies suggest that the mutant tk in mpn activate common signaling pathways resulting in overlapping effects on cytokine production . moreover , these and other data indicate that targeting of tk signaling or relevant downstream signaling molecules will reduce the aberrant inflammatory cytokine production not only in pmf , et , and pv but also in other mpn
. a comprehensive analysis of cytokine serum levels in a large cohort of sm patients would be useful to compare the expression of inflammatory cytokines in sm with the pattern observed in other mpn .
fibrosis is considered to be a reactive process that is often associated with tissue remodeling and tissue repair .
tissue fibrosis may occur in various organs and involves fibroblasts and other connective tissue cells . concerning development and characteristics of mpn ,
the process of fibrosis involves not only local fibroblasts and infiltrating leukocytes resulting in persistence of inflammation in the tissue , but also the proliferation of cells with a myofibroblast phenotype .
the pathological mechanisms underlying the development of fibrosis in mpn patients are still not fully understood .
involved cells produce different growth factors , proteolytic enzymes , angiogenic factors , and fibrogenic cytokines , which results in enhancement of connective tissue elements ' deposition .
, all mpn can develop bm fibrosis , although the likelihood for this varies considerably between the subtypes .
the fibrotic potential of mpn with predominant thrombocytosis such as et can be differentiated from pmf on the basis of morphology . in pmf ,
the stromal reaction that accompanies clonal hematopoietic stem cell proliferation is characterized by a consistent myelofibrosis associated with osteosclerosis and neoangiogenesis .
in addition , a higher fibrosis grade in patients with pmf correlated with worse prognosis . in patients with pv or et , reticulin fibrosis at the time of diagnosis is associated with an increased risk of transformation to post - pv or post - et myelofibrosis . in cml
, bm fibrosis occurs in up to 40% of patients at diagnosis and is associated with a poor prognosis .
recently , bm fibrosis in cml was proposed as an independent predictor of responses to tki therapy .
mastocytosis is also commonly associated with slight - to - moderate bm fibrosis [ 48 , 117 ] . in the bm of sm patients ,
in addition , mast cell infiltration with consecutive fibrosis may also occur in the liver , spleen , and lymph nodes [ 48 , 117 ] .
immunohistochemical studies show a close correlation between the mast cell expression of fgf - b and the reticulin fibrosis of mastocytosis lesions .
concerning pmf development , the megakaryocytic lineage seems to play an essential role in promoting myelofibrosis .
megakaryocytic cells were found to produce a variety of growth factors and cytokines leading to proliferation of fibroblast and the development of fibrosis .
pdgf is one of the first growth factors that has been implicated in the role of megakaryocytes in development of bm fibrosis .
several studies described increased levels of pdgf in patients with pmf [ 62 , 63 ] , and immunohistochemical staining showed that megakaryocytes and erythroid precursors were highly positive for pdgf .
patients with et showed increased plasma levels of pdgf ; in particular the subgroup of patients with reticulin fibrosis had higher pdgf plasma levels .
pdgf not only enhances the replication , survival , and migration of myofibroblasts but also modulates the production and secretion of pro- and anti - inflammatory mediators in the pathogenesis of fibrotic diseases .
further studies revealed that the expression and production of tgf- were increased in patients suffering from mpn .
these groups reported on quantitative alterations of tgf- and its receptors in megakaryocytic , platelet , and cd34 + progenitor cells and concluded that tgf- was involved in myelofibrosis and myeloproliferation [ 39 , 40 , 42 , 6972 , 124 , 125 ] .
tgf- is a growth factor displaying potent fibrogenic properties and is furthermore associated with not only bm fibrosis , but also clonal hematopoietic expansion and angiogenesis .
moreover , tgf- has been described to negatively regulate progenitor cell growth [ 126 , 127 ] .
in addition , tgf- reportedly promotes the deposition of extracellular matrix in different tissues [ 128 , 129 ] .
in pmf , the pathogenic relevance of tgf- is based on the ability to induce production of types i , iii , and iv collagens , fibronectin , tenascin , and proteoglycans .
furthermore , tgf- blocks matrix degradation by reducing collagenase - like protease synthesis , while enhancing protease inhibitor expression .
importantly , tgf- downstream signaling , through smad2/3 phosphorylation , has been shown to be active in megakaryocytes extending proplatelets , indicating an autocrine stimulation in megakaryocyte development .
tgf- induced pi3-kinase / akt / nf-b signaling in hemangioblasts , and activation of this pathway enhanced the production of matrix metalloproteinase-9 [ 66 , 67 ] .
apart from tgf- and pdgf , fgf - b is considered to be a cytokine with potent fibrogenic characteristics .
the levels of circulating fgf - b were significantly higher in the serum of mpn patients when compared to healthy controls , the highest levels being measured in patients with marked bm fibrosis [ 37 , 3941 , 43 , 44 , 124 ] .
further studies are required to identify the importance of fgf - b in development and progression of mpn .
they showed a significantly elevated calmodulin excretion in pmf patients when compared to pv , et , and cml . using a neutralizing antibody to calmodulin
extracellular calmodulin should also be considered a potential mitogen involved in the stroma cell reaction in patients with pmf .
fibrosis is usually detected in the endomyocardium , which is not the case in other mpn types .
one hypothesis is that eosinophils , once entering cardiac tissues , can promote local fibrosis .
eosinophil - related tissue fibrosis has been attributed to infiltration of the tissues with eosinophils and deposition of eosinophil granule proteins .
furthermore , eosinophils were shown to produce the cytokines il-1 , il-2 , il-3 , il-4 , il-5 , il-6 , il-8 , gm - csf , tgf- , tgf- , tnf- , mip-1 , rantes , eotaxin , and osm .
many of the eosinophil - derived cytokines have the potential to stimulate fibroblast proliferation and contribute to local inflammation as well as recruitment of other leukocytes [ 115 , 132 ] . however , further in vitro and in vivo models are mandatory to deeply understand the role of cytokines for organ specific fibrosis in cel .
angiogenesis , the formation of new vessels from preexisting vessels , plays an important role in development and progression of different tumor types , and targeting of angiogenesis has been successfully translated into clinical practice in various solid tumor models .
the process of angiogenesis in hematological malignancies is comparable to the process observed in solid tumors .
endothelial cells from preexisting vessels are activated in the bm by an angiogenic stimulus ( e.g. , vegf ) and proliferate , migrate , and form new vessels .
initiation of leukemia - induced angiogenesis involves secretion of angiogenic cytokines by leukemic cells and their interaction with the bm stroma .
apart from solid tumors , the importance of angiogenesis becomes increasingly evident in various mpn and other hematologic malignancies .
angiogenesis in the bm of mpn patients was described to correlate with disease burden , progression , and prognosis [ 36 , 79 ] . among the classical bcr - abl1 negative mpn ,
increased bm microvessel density ( mvd ) has been observed in a number of studies in all mpn entities but is most abundant in pmf [ 45 , 75 , 8083 , 136 ] .
not only does pmf show the highest mvd among the classical mpn , but mvd was also described as an independent adverse prognostic factor in pmf .
significantly higher mvd was also found in the bm of patients with post - pv or post - et myelofibrosis compared to pv or et .
the association of jak2 v617f mutation status with mvd showed no significant difference between jak2 wild type and mutant mpn patients in two out of three studies [ 79 , 81 , 138 ] .
in contrast to the observation that the increase in bm vascularity seems to be generally independent of the jak2 v617f status , mvd correlated with jak2 v617f mutant allele burden within the jak2 v617f+ subgroup . in chronic phase cml , the bm is hypercellular , with a prominent myeloid compartment and left shift in the granulomonocytic cell compartment . along with myeloid hyperplasia , augmented
, the bm of patients with cml shows a significant increase in mvd , functionally associated with elevated levels of angiogenic cytokines .
furthermore , the bcr - abl1 targeting tki imatinib was found to reduce the mvd in cml .
alterations of the bm microenvironment are frequently noticed not only in classical mpn but also in sm .
these alterations include angiogenesis , thickened bone trabeculae , and sometimes massive bm fibrosis [ 48 , 88 , 117 , 139 ] .
the median bm mvd was found to be significantly higher in sm compared to cutaneous mastocytosis or controls .
the process of angiogenesis is tightly controlled by a variety of angiogenic and antiangiogenic cytokines .
vegf is able to bind to three receptors : vegf receptor-1 ( vegfr-1 ; fms - like tyrosine kinase-1 , flt-1 ) , vegfr-2 ( human kinase domain region , kdr / murine fetal liver kinase-1 , flk-1 ) , and vegfr-3 ( flt-4 ) .
vegfr-2 was found to be both necessary and sufficient to mediate effects of vegf on endothelial cells , like induction of vascular permeability and angiogenesis .
in addition , vegfr-1 is expressed on hematopoietic stem cells and frequently on leukemic cells , whereas megakaryocytes express vegfr-2 , and vegfr-3 is mainly involved in the regulation of lymphangiogenesis .
vegf not only promotes bm neovascularization but was also found to signal through vegfrs expressed on the surface of neoplastic hematopoietic cells .
thus , secreted vegf has been considered to contribute to disease progression by an autocrine or paracrine mechanism .
numerous studies reported increased levels of vegf in the blood as well as expression in the bm of patients with pv , et , and pmf [ 21 , 36 , 46 , 49 , 75 , 76 , 80 , 8387 ] . increased expression of vegf was also found in cml [ 23 , 25 , 26 , 77 ] and in bm section of sm patients .
endothelial cells express the hgf receptor c - met and the role of hgf in angiogenesis is well established .
hgf enhances vascular matrix degradation and endothelial cell invasion and migration , as well as proliferation of vascular endothelial cells .
furthermore , hgf induces capillary tube formation in a matrigel assay and promotes angiogenesis in vivo .
hgf acts synergistically with vegf on endothelial growth but has also been shown to induce angiogenesis independent of vegf .
elevated levels of hgf have been described in patients with pv , et , and pmf [ 38 , 49 , 50 ] , as well as in cml [ 3033 ] .
it induces growth of fibroblasts and endothelial cells in vitro and stimulates angiogenesis and fibrosis in vivo .
elevated levels of fgf - b have been described in pv and et , but particular high levels were found in patients with pmf [ 36 , 38 ] .
in addition , cml patients showed also increased expression of fgf - b [ 24 , 29 ] .
il-6 is a proinflammatory cytokine that has been implicated in the pathogenesis of various mpn . among many other functions ,
il-6 has been reported to stimulate angiogenesis in the tumor microenvironment and to enhance proliferation and migration of endothelial cells [ 145147 ] .
a recent study reported defective pericyte coverage of vessels after il-6 stimulation compared to vegf - stimulated vessels .
we identified the il-6 family member osm as an oncoprotein - dependent cytokine in neoplastic cells of jak2 v617f , kit d816v , and fip1l1-pdgfra positive mpn [ 59 , 61 , 115 ] .
osm has been described to act as a growth factor for various mesenchymal cells , including fibroblasts , osteoblasts , and endothelial cells and to induce angiogenesis in vitro and in vivo [ 149152 ] .
thus , osm has been implicated in tissue remodeling , inflammation , and tissue fibrosis [ 151 , 153155 ] . similarly , il-8 is a multifunctional proinflammatory cytokine which is highly expressed in various mpn .
it has been implicated in tumor growth and angiogenesis . in particular , il-8 was shown to promote endothelial cell proliferation , capillary tube organization , and matrix metalloproteinase expression in endothelial cells . in summary , a number of inflammatory cytokines , abundantly expressed in various mpn , have the potential to trigger angiogenesis in the bm and other organ systems .
this pathogenetic process has therefore been proposed as a potential target in cml and other mpn and is best studied for targeted drugs against vegf / vegfr and hgf / c - met .
apart from direct effects on endothelial cells and fibroblast , neoplastic cell - derived inflammatory cytokines are also involved in autocrine and paracrine loops between neoplastic ( stem ) cells and mesenchymal ( stem ) cells ( figure 1 ) .
hematopoietic stem cells ( hsc ) rely on their interactions with the bm niche to maintain their quiescent state and to protect their integrity and functions but also to undergo asymmetrical cell division and differentiation in order to regulate and support blood cell production on demand .
similarly , disease - initiating leukemic stem cells ( lsc ) interact with the bm niche .
however , the bm niche in hematopoietic malignancies is commonly altered and the leukemia - induced remodeling of the niche may directly contribute to the aberrant function of lsc . a number of these complex interactions have been described as potentially interesting targets in mpn , of which some are exemplified in this section .
arranz et al . recently described the effect of nestin - positive mesenchymal stromal cells and sympathetic nerve fibers on the regulation of hematopoietic stem cells in jak2 v617f positive mpn .
sympathetic nerve fibers , supporting schwann cells , and nestin - positive mesenchymal stromal cells were found to be reduced in the bm of mpn patients and murine mpn models , a process that may be triggered by il-1 produced by mutated mpn cells .
depletion of nestin - positive cells or their production of stroma derived factor-1 ( sdf-1 , cxcl12 ) accelerated mpn progression .
this elegant study demonstrates how inflammatory cytokines produced by neoplastic cells alter or even damage the niche - forming mesenchymal stromal cells in mpn .
furthermore , neuroprotective or sympathomimetic drugs were described as potential therapeutic agents to target this interaction .
expanded myeloid cml cells were found to produce the proinflammatory cytokine il-6 in inducible bcr - abl1 transgenic mouse model recapitulation features of human chronic phase cml .
il-6 served as a positive feedback loop to sustain cml development in this model and reprogrammed both normal and leukemic multipotent progenitor cells towards myeloid development at the expense of lymphoid differentiation .
these results suggest that blocking of il-6 or targeting the il-6 signal transduction pathway could represent a valuable target in cml .
moreover , the authors suggested that such self - reinforcing loop involving il-6 , or other secreted proinflammatory factors might be relevant in a broad spectrum of mpn .
traer and colleagues studied the effect of the bm microenvironment on imatinib resistance in cml .
fgf released from stromal cells was found to promote growth of cml cells through the fgf receptor and the map - kinase pathway . in line with the in vitro data , cml patients resistant to imatinib without kinase domain mutation showed increased expression of fgf in the bm .
resistance could be overcome with ponatinib , a multikinase inhibitor that targets the fgf receptor in addition to bcr - abl1 .
another study focused on the effect of stromal cells on the resistance to jak2 inhibitor treatment in jak2 v617f+ disease .
cytokines were found to contribute to this protective effects of stromal cells , and neutralizing antibodies against il-6 , fgf , or cxcl-10 restored the apoptosis induced by jak2 inhibition .
we found that osm secreted by neoplastic cells did not only stimulate growth of fibroblasts , osteoblasts and microvascular endothelial cells but also induced the production of the angiogenic and profibrogenic cytokines hgf and vegf in human fibroblasts .
thus , specific tumor cell - stroma cell interactions may potentiate the cytokine storm observed in mpn , that is , by inducing the production and release of cytokines that modulate growth of stromal cells as well as their activation , with consecutive expression of additional cytokines and cytokine receptors .
schwaller et al . showed that retroviral overexpression of osm in bm cells was sufficient to induce a lethal mpn with marked bm fibrosis and polyclonal expansion of myeloid cells .
tnf- in turn was found to reduce colony formation in normal hematopoietic cells while jak2 v617f+ progenitor cells were resistant to tnf-. thus , oncogenic jak2 generates a tnf- rich environment which facilitates clonal expansion of mutant cells in mpn .
similarly , cml stem and progenitor cells were found to produce higher levels of tnf- than their normal cd34 + counterparts .
tnf- promoted survival of cml stem cells in an autocrine manner by the nuclear factor b / p65 pathway and expression of il-3 .
importantly , tnf- inhibition induced apoptosis of cml cells and acted synergistically with nilotinib . together , these findings suggest tnf- as new putative therapeutic target in mpn .
increased cytokine production was described as a hallmark of classical mpn that contributes to symptom burden of the patients and was referred to as cytokine storm .
targeting of this increased overall cytokine production has been successfully implicated in pmf , pv , and et . in particular , the identification of the jak2 v617f mutation led to the development of various jak2 inhibitors .
it targets wild type and mutant jak2 as well as jak1 and was found to induce marked and durable reductions in splenomegaly and symptoms in patients with pmf . despite having only limited effects on the jak2 v617f allele burden , significant improvements of fatigue , pain , night sweats , and pruritus were observed after ruxolitinib treatment .
in addition , a reduction of cytokine serum levels including il-6 , il-8 , tnf- , vegf , and fgf - b was found .
changes in cytokine level correlated with reduction in spleen size and coincided with symptom improvement .
thus , it is tempting to speculate that the cytokine storm observed in pmf significantly contributes to the symptom burden in pmf .
this is in line with the observation of similar activation patterns of downstream signaling pathways in jak2 mutant and wild type cases . the majority of jak2 wild type patients harbor calr mutations .
. therefore , targeting of jak1/jak2 is effective to reduce proinflammatory cytokines in pmf irrespective of the jak2/mpl / calr mutation status .
autocrine gm - csf stimulation was identified as mechanism of imatinib resistance in cml leading to bcr - abl1-independent activation of jak / stat signaling .
wang et al . used the jak2 inhibitor ag490 to target gm - csf induced activation of jak / stat signaling and could thus overcome resistance to imatinib and nilotinib in vitro .
furthermore , activated jak2/stat5 signaling has been described as a potential target in lsc in cml [ 164 , 165 ] .
in addition , bcr - abl1 was also found to activate stat5 directly and independently of jak2 , and high levels of stat5 activation contributed to imatinib resistance .
. showed that the jak1/2 inhibitor ruxolitinib synergized with nilotinib in inhibiting the proliferation of cd34 + cells in patients with cml .
these findings provide a rationale for the application of jak2 inhibitors to eradicate residual disease in cml .
targeting of vegf and/or the vegf receptors ( vegfrs ) is a widely used concept of antiangiogenesis in oncology .
neutralizing antibodies and soluble receptors are used to inhibit the interaction between vegf and its receptors ( figure 2(b ) ) .
targeting of vegfr with kinase inhibitors resulted in a reduction in stromal fibroblasts , macrophages , and endothelial cells in in vitro cultures of human bm whereas hematopoietic colony formation was not impaired .
bevacizumab is a humanized monoclonal antibody against vegf approved for antiangiogenic treatment in solid tumors .
none of the patients treated with bevacizumab had an objective response , but significant toxicity was observed .
small molecule inhibitors targeting vegfr and other kinases , for example , sorafenib and sunitinib , have been approved for treatment of patients with renal cell and hepatocellular carcinoma .
only one out of 14 patients showed clinical improvement , whereas a high rate of adverse events was observed .
vatalanib is a vegfr kinase inhibitor with greater potency against vegfr-2 than against vegfr-1 or vegfr-3 .
a phase i study in pmf showed modest activity with clinical improvement in 20% of the patients examined .
targeting of mtor by rapamycin in cml cells inhibited not only vegf expression but also the in vitro growth of leukemic cells . a clinical pilot study to evaluate the antileukemic and antiangiogenic effects of rapamycin in patients with imatinib - resistant cml showed transient antileukemic effects in a subset of cases . in summary , despite promising data in preclinical models , direct targeting of vegf resulted only in modest clinical effects on patients with mpn so far .
aberrant activation of hgf and/or its receptor c - met has been described in solid tumors as well as in acute myeloid leukemia ( aml ) , myeloma , and mpn .
production of hgf was found to be independent of bcr - abl1 in cml and independent of jak2 v617f in other mpn .
thus , blocking of the hgf / c - met function was suggested as an independent therapeutic target which could synergize with tki treatment in mpn .
c - met neutralizing antibodies bind to the extracellular domain of the receptor and prevent binding of hgf to c - met ( figure 2(c ) ) .
have shown that c - met neutralizing antibodies can effectively suppress the growth of jak2 v617f - mutated cells , including pv erythroblasts and the hel cell line , which expresses hgf at high levels .
in addition , small molecule inhibitors targeting c - met and the c - met - related ron receptor have been developed .
the c - met inhibitors su-11274 and pha-665752 decreased the survival of aml cells in a dose dependent manner .
su-11274 was found to inhibit colony formation , to reduce viability , and to induce differentiation in a9 m , u937 , and oci - aml cells .
moreover , the c - met inhibitors were found to block the response to hgf in a myeloma model .
our group tested the effects of su-11274 and pf-02341066 ( crizotinib ) on bcr - abl1 positive cells and found that both drugs induce a significant growth reduction in ku812 cells and k562 cells .
furthermore , c - met inhibitors were found to reduce proliferation of primary cml cells in vitro .
boissinot and colleagues tested the efficacy of combining c - met and jak inhibitors on the proliferation of the jak2 v167f positive hel and uke-1 cell lines .
only a weak inhibition was observed when molecules were tested separately , whereas the combination of the c - met inhibitor pf-2341066 and the jak inhibitor ruxolitinib inhibited growth of uke-1 cells . in summary ,
increasing evidence suggests an important role of the bm microenvironment in the regulation of proliferation and survival of normal and leukemic hematopoietic stem cells .
thus , targeting of the specific bm niches and stem cell - niche interactions has been suggested as a promising therapeutic strategy .
sdf-1 ( cxcl-12 ) is a chemokine produced by mesenchymal cells of the bm stroma ( e.g. , endothelial cells and osteoblasts ) with particularly high expression in perivascular , niche - forming mesenchymal stromal cells .
hematopoietic stem and progenitor cells express the sdf-1 receptor cxcr4 and migrate specifically towards sdf-1 .
plerixafor ( amd3100 ) inhibits the sdf-1/cxcr4 interaction and is clinically used to mobilize hematopoietic stem and progenitor cells in stem cell transplant donors .
the sdf-1/cxcr4 axis is one potential target in the interplay of leukemic stem cells ( lsc ) and the bm microenvironment ( figure 2(d ) ) .
cxcr4 is highly expressed on the surface of malignant cell in chronic lymphocytic leukemia ( cll ) , and sdf-1 was found to promote chemotaxis of cll cells and their interaction with stromal cells , which was shown to induce resistance of cll cells to cytotoxic agents , and was furthermore suggested to mediate persistence of minimal residual disease in the bm during therapy . in line with this concept ,
cxcr4 antagonists were successfully used to block interactions between cll and stromal cells and to mobilize cll cells from their protective microenvironments , becoming thus accessible to conventional drugs .
similar targeting concepts were applied in preclinical models for aml and acute lymphoblastic leukemia ( all ) .
cxcr4 antagonist inhibited the proliferation of aml cells and reduced protection against chemotherapy by stromal cells in vitro and in vivo [ 181183 ] .
leukemic cells in t - all were found to be in direct , stable contact with sdf-1-producing bm stroma .
furthermore , genetic targeting of cxcr4 in murine t - all led to rapid , sustained disease remission and cxcr4 antagonism suppressed human t - all in primary xenograft models .
partly ambivalent results have been published for the role of sdf-1/cxcr4 in mpn , and although increased levels of sdf-1 have been reported , this may not necessarily result in a sustained activation of cxcr4 signaling in neoplastic cells [ 185 , 186 ] . on the one hand , mobilization of cd34
+ cells in patients with pmf has been attributed to reduced cxcr4 expression and hypermethylation of the cxcr4 promoter [ 187 , 188 ] .
moreover , although elevated levels of immunoreactive forms of sdf-1 were found in the bm and peripheral blood of patients with pmf and pv , detailed studies using mass spectrometry have shown that sdf-1 was mainly truncated and thus expressed in an inactive form in these patients .
the authors of this study concluded that reduced levels of intact sdf-1 due to proteolytic degradation would contribute to the mobilization of hematopoietic stem cells in pmf . in line with these data , cd34
+ cells in cml showed an impaired chemotactic response to sdf-1 although no decrease in cxcr4 expression was observed [ 189 , 190 ] .
our group identified the cell surface enzyme dipeptidylpeptidase - iv ( cd26 ) as a marker of cml lsc .
cd26 was shown to disrupt the sdf-1/cxcr4 axis by cleaving sdf-1 , and targeting of cd26 by gliptins suppressed the expansion of bcr - abl1 + cells .
cd26 expression may explain the mobilization of lsc and the observed extramedullary spread of hematopoietic stem and progenitor cells in cml , and inhibition of cd26 may revert abnormal lsc function . on the other hand
, the sdf-1/cxcr axis between stroma and leukemic cells contributes to resistance to tki treatment in cml .
imatinib was found to enhance migration of cml cells towards stromal cell layers , which may in turn promote nonpharmacological resistance to imatinib [ 192 , 193 ] .
mechanistically , this finding was linked to cxcr4 redistribution into the lipid raft fraction , in which cxcr4 colocalized with active lyn after tki treatment .
the cxcr4 inhibitor plerixafor diminished migration of bcr - abl1 positive cells and reduced adhesion of these cells to extracellular - matrix components and to bm stromal cells in vitro .
moreover , plerixafor was also found to decrease the drug resistance of cml cells induced by coculture with bm stromal cells in vitro .
importantly , plerixafor was shown to mobilize leukemic cells in vivo and to act synergistically with nilotinib to reduce the leukemia burden in a mouse model .
the authors of this study argue that the combination of cxcr4 inhibition with tki treatment in cml might be a useful approach to override drug resistance and to achieve deeper responses in cml .
in contrast , another study tested the effects of plerixafor in combination with either imatinib or dasatinib in a murine cml bm transplant model . in this study , no beneficial effect of plerixafor over tki monotherapy was observed .
the discrepancy of these date can partly be explained by difference in the cml mouse model ( e.g. , irradiation possibly contributing to cns infiltration ) and in the tki administration .
weisberg et al . applied plerixafor after marked reduction of disease burden with nilotinib as a model of minimal residual disease and argued that the absence of significant disease burden was relevant for the beneficial effects of the combination therapy .
thus , the sdf-1/cxcr4 axis is a promising but still controversial target in cml and other types of mpn . the effect of cxcr4 inhibitors in pv , et , pmf , and sm remains to be addressed in further preclinical models .
the complex interplay between neoplastic cells and microenvironmental cells in mpn has gained increasing interest in recent years .
one important aspect is that oncogenic signaling promotes cytokine production in mpn cells and alters their interaction with the bm stroma .
a number of pathogenetic mechanisms are found to be conserved between various mpn , and lessons learned from one disease can be exploited for the other mpn types .
thus , it will be important to compare systematically the various common as well as rare mpn - variants in terms of basic and clinical science .
more recently , the pathologically altered interactions between neoplastic cells and their microenvironment have been investigated with the aim of defining new potential targets of therapy and to develop novel therapeutic approaches .
first , the increased angiogenesis and bm fibrosis may serve as novel targets of therapy in mpn . indeed
, several tki used to treat mpn may also suppress angiogenesis and/or fibrosis through inhibition of vascular target kinases .
thus , the vegf / vegfr , hgf / c - met , and sdf-1/cxcr4 axis are potential targets in mpn , and a number of other molecular targets are under investigation .
many open questions still have to be addressed in preclinical model , and so far only few of the many exciting approaches were successfully translated to the clinic .
best evidence for targeting of the inflammatory cytokine storm is derived from the clinical efficacy of jak inhibitors in mpn , which show marked benefits in patients despite their lack of specificity for mutant jak2 .
other targeting approaches for inflammatory cytokines will most likely be combined with established or experimental inhibitors of the primary oncoprotein in the given mpn .
increasing knowledge of the lsc - niche interaction will help to optimize this combined targeting approach and to establish synergistic strategies for therapy or even cure of mpn . | the term myeloproliferative neoplasms ( mpn ) refers to a heterogeneous group of diseases including not only polycythemia vera ( pv ) , essential thrombocythemia ( et ) , and primary myelofibrosis ( pmf ) , but also chronic myeloid leukemia ( cml ) , and systemic mastocytosis ( sm ) . despite the clinical and biological differences between these diseases ,
common pathophysiological mechanisms have been identified in mpn .
first , aberrant tyrosine kinase signaling due to somatic mutations in certain driver genes is common to these mpn .
second , alterations of the bone marrow microenvironment are found in all mpn types and have been implicated in the pathogenesis of the diseases .
finally , elevated levels of proinflammatory and microenvironment - regulating cytokines are commonly found in all mpn - variants . in this paper , we review the effects of mpn - related oncogenes on cytokine expression and release and describe common as well as distinct pathogenetic mechanisms underlying microenvironmental changes in various mpn .
furthermore , targeting of the microenvironment in mpn is discussed .
such novel therapies may enhance the efficacy and may overcome resistance to established tyrosine kinase inhibitor treatment in these patients .
nevertheless , additional basic studies on the complex interplay of neoplastic and stromal cells are required in order to optimize targeting strategies and to translate these concepts into clinical application . | 1. Myeloproliferative Neoplasms
2. Cytokine Expression in Classical MPN
3. Cytokine Expression in Nonclassical MPN: Systemic Mastocytosis
4. Cytokine Regulation of Microenvironmental Cells
5. Targeting the Cytokine Storm and the Microenvironment in MPN: A Novel Concept
6. Concluding Remarks and Future Perspectives | myeloproliferative neoplasms ( mpn ) are clonal hematopoietic stem cell disorders characterized by abnormal proliferation and expansion of one or more myeloid lineages [ 1 , 2 ] . the group of the common , classic mpn includes chronic myeloid leukemia ( cml ) defined by the philadelphia chromosome ( ph ) and the three ph - negative entities ' polycythemia vera ( pv ) , essential thrombocythemia ( et ) , and primary myelofibrosis ( pmf ) . the group of nonclassic mpn includes systemic mastocytosis ( sm ) , chronic neutrophilic leukemia ( cnl ) , and chronic eosinophilic leukemia ( cel ) [ 1 , 3 ] . aberrant tyrosine kinase ( tk ) signaling is a common hallmark in mpn and has been shown to represent a key driver of the disease . in a majority of patients with pv , et , and pmf , the activating v617f mutation in the receptor - associated tk jak2
in addition , mutations in exon 12 of jak2 and mutations in the thrombopoietin receptor ( mpl
w515k / l ) have been described in these entities [ 11 , 12 ] . more recently , somatic mutations in calr were found in jak2- and mpl - negative patients with et or pmf [ 13 , 14 ] . aberrant expression of inflammatory cytokines has been associated with patients ' symptoms and alterations of the bone marrow ( bm ) microenvironment as well as progression of the disease . several different studies have suggested an important role for the bm microenvironment in the pathogenesis of hematologic malignancies including mpn . in fact , alterations in the bm microenvironment such as increased microvessel density ( angiogenesis ) , fibrosis , and thickening of bone trabeculae are typical pathological findings in mpn and may contribute to disease phenotypes and disease progression . this review focuses on the cytokine regulation of microenvironmental cells with special emphasis on common as well as distinct pathogenetic mechanisms in various mpn . in particular , expression and functional relevance of interleukin-6 ( il-6 ) , il-8 , vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( fgf - b ) , hepatocyte growth factor ( hgf ) , platelet derived growth factor ( pdgf ) , oncostatin m ( osm ) , tumor necrosis factor- ( tnf- ) , transforming growth factor- ( tgf- ) , and stroma derived factor-1 ( sdf-1 , cxcl-12 ) are reviewed . evidence for increased expression of these cytokines in various mpn is summarized in table 1 . furthermore , the effect of jak1/2 inhibitors on the cytokine storm in mpn and targeted drugs for vegf / vegfr , hgf / c - met , and sdf-1/cxcr-4 are discussed . cytokines and other effector molecules downstream of these aberrant signaling cascades have been implicated in the pathogenesis of cml . immunohistochemical staining of bm sections showed that vegf is expressed primarily in myeloid progenitor cells , megakaryocytes , and mature granulomonocytic cells in chronic phase cml as well as in myeloid differentiated blast cells in the blast phase of cml . targeting of mtor by rapamycin in cml cells inhibited not only vegf expression but also the in vitro growth of leukemic cells . the plasma hgf level correlated with markers reflecting the tumor burden as well as with the phase of cml and overall survival in these patients . tnf- has recently been implicated in stem cell biology of mpn [ 94 , 95 ] . elevated levels of inflammatory cytokines have been reported in all entities of classical mpn [ 38 , 49 , 51 , 53 , 58 , 9699 ] . in this study , il-1 , il-1ra , il-2r , il-6 , il-8 , il-10 , il-12 , il-13 , il-15 , tnf- , granulocyte colony - stimulating factor ( g - csf ) , interferon ( ifn- ) , macrophage inflammatory protein 1 ( mip-1 ) , mip-1 , hgf , ifn--inducible protein 10 ( ip-10 ) , monokine induced by ifn- ( mig )
, monocyte chemotactic protein 1 ( mcp-1 ) , and vegf levels were found to be elevated in pmf patients . nevertheless , increasing evidence indicates that the disease burden of mpn is not only mediated by the primary neoplastic clone but also mediated by a secondary inflammation with an aberrant cytokine production and changes of the bm microenvironment . in particular , fgf - b , il-8 , vegf , hgf , pdgfr , tgf- , tnf- , and osm have been implicated in bm microenvironment alterations in patients with mpn [ 21 , 22 , 3941 , 68 , 69 ] . evidence for expression of these cytokines in pv , et , and pmf is discussed in the next paragraphs . il-8 serum levels were significantly increased in patients with pmf , and il-8 expression was observed in various hematopoietic cell types , including granulocytes , monocytes , megakaryocytic cells , cd34 + progenitor cells , and platelets . immunohistochemical studies performed on bm sections of et , pv , and pmf patients revealed an increased expression of vegf and its receptor in all mpn groups compared to controls [ 79 , 80 ] . detected elevated levels of hgf in the serum and bm plasma obtained from pv patients compared to secondary erythrocytosis patients that were employed as controls . further studies analyzing cytokine panels in plasma of mpn patients confirmed elevated hgf levels in pmf , pv , and et [ 38 , 49 ] . kit is a receptor tk , and activation of kit signaling through its ligand stem cell factor ( scf ) mediates cell proliferation and survival in immature progenitor cells and mast cell differentiation , as well as mast cells migration , activation , and adhesion . il-3 and il-4 were not detectable , and scf as well as vegf levels showed no significant difference between patient samples and controls . high levels of il-6 were accompanied by increased levels of the osteoclast - regulating factors receptor activator of nuclear factor kappa - b ligand ( rankl ) and osteoprotegerin . il-31 has been implicated in the induction of chronic skin inflammation and was found to be increased in patients with sm and to correlate with disease severity . although no significant elevation of vegf levels was found in plasma of sm patients , it is likely that vegf is locally increased in the bm microenvironment and contributes to increased angiogenesis in sm . we studied the effect of kit d816v on cytokine expression in various in vitro models . these studies suggest that the mutant tk in mpn activate common signaling pathways resulting in overlapping effects on cytokine production . moreover , these and other data indicate that targeting of tk signaling or relevant downstream signaling molecules will reduce the aberrant inflammatory cytokine production not only in pmf , et , and pv but also in other mpn
. concerning development and characteristics of mpn ,
the process of fibrosis involves not only local fibroblasts and infiltrating leukocytes resulting in persistence of inflammation in the tissue , but also the proliferation of cells with a myofibroblast phenotype . the pathological mechanisms underlying the development of fibrosis in mpn patients are still not fully understood . in the bm of sm patients ,
in addition , mast cell infiltration with consecutive fibrosis may also occur in the liver , spleen , and lymph nodes [ 48 , 117 ] . pdgf is one of the first growth factors that has been implicated in the role of megakaryocytes in development of bm fibrosis . pdgf not only enhances the replication , survival , and migration of myofibroblasts but also modulates the production and secretion of pro- and anti - inflammatory mediators in the pathogenesis of fibrotic diseases . tgf- is a growth factor displaying potent fibrogenic properties and is furthermore associated with not only bm fibrosis , but also clonal hematopoietic expansion and angiogenesis . the levels of circulating fgf - b were significantly higher in the serum of mpn patients when compared to healthy controls , the highest levels being measured in patients with marked bm fibrosis [ 37 , 3941 , 43 , 44 , 124 ] . further studies are required to identify the importance of fgf - b in development and progression of mpn . eosinophil - related tissue fibrosis has been attributed to infiltration of the tissues with eosinophils and deposition of eosinophil granule proteins . furthermore , eosinophils were shown to produce the cytokines il-1 , il-2 , il-3 , il-4 , il-5 , il-6 , il-8 , gm - csf , tgf- , tgf- , tnf- , mip-1 , rantes , eotaxin , and osm . many of the eosinophil - derived cytokines have the potential to stimulate fibroblast proliferation and contribute to local inflammation as well as recruitment of other leukocytes [ 115 , 132 ] . angiogenesis , the formation of new vessels from preexisting vessels , plays an important role in development and progression of different tumor types , and targeting of angiogenesis has been successfully translated into clinical practice in various solid tumor models . angiogenesis in the bm of mpn patients was described to correlate with disease burden , progression , and prognosis [ 36 , 79 ] . not only does pmf show the highest mvd among the classical mpn , but mvd was also described as an independent adverse prognostic factor in pmf . alterations of the bm microenvironment are frequently noticed not only in classical mpn but also in sm . vegf not only promotes bm neovascularization but was also found to signal through vegfrs expressed on the surface of neoplastic hematopoietic cells . numerous studies reported increased levels of vegf in the blood as well as expression in the bm of patients with pv , et , and pmf [ 21 , 36 , 46 , 49 , 75 , 76 , 80 , 8387 ] . elevated levels of hgf have been described in patients with pv , et , and pmf [ 38 , 49 , 50 ] , as well as in cml [ 3033 ] . elevated levels of fgf - b have been described in pv and et , but particular high levels were found in patients with pmf [ 36 , 38 ] . il-6 is a proinflammatory cytokine that has been implicated in the pathogenesis of various mpn . osm has been described to act as a growth factor for various mesenchymal cells , including fibroblasts , osteoblasts , and endothelial cells and to induce angiogenesis in vitro and in vivo [ 149152 ] . thus , osm has been implicated in tissue remodeling , inflammation , and tissue fibrosis [ 151 , 153155 ] . it has been implicated in tumor growth and angiogenesis . in summary , a number of inflammatory cytokines , abundantly expressed in various mpn , have the potential to trigger angiogenesis in the bm and other organ systems . hematopoietic stem cells ( hsc ) rely on their interactions with the bm niche to maintain their quiescent state and to protect their integrity and functions but also to undergo asymmetrical cell division and differentiation in order to regulate and support blood cell production on demand . a number of these complex interactions have been described as potentially interesting targets in mpn , of which some are exemplified in this section . recently described the effect of nestin - positive mesenchymal stromal cells and sympathetic nerve fibers on the regulation of hematopoietic stem cells in jak2 v617f positive mpn . sympathetic nerve fibers , supporting schwann cells , and nestin - positive mesenchymal stromal cells were found to be reduced in the bm of mpn patients and murine mpn models , a process that may be triggered by il-1 produced by mutated mpn cells . this elegant study demonstrates how inflammatory cytokines produced by neoplastic cells alter or even damage the niche - forming mesenchymal stromal cells in mpn . another study focused on the effect of stromal cells on the resistance to jak2 inhibitor treatment in jak2 v617f+ disease . cytokines were found to contribute to this protective effects of stromal cells , and neutralizing antibodies against il-6 , fgf , or cxcl-10 restored the apoptosis induced by jak2 inhibition . we found that osm secreted by neoplastic cells did not only stimulate growth of fibroblasts , osteoblasts and microvascular endothelial cells but also induced the production of the angiogenic and profibrogenic cytokines hgf and vegf in human fibroblasts . thus , specific tumor cell - stroma cell interactions may potentiate the cytokine storm observed in mpn , that is , by inducing the production and release of cytokines that modulate growth of stromal cells as well as their activation , with consecutive expression of additional cytokines and cytokine receptors . targeting of this increased overall cytokine production has been successfully implicated in pmf , pv , and et . it targets wild type and mutant jak2 as well as jak1 and was found to induce marked and durable reductions in splenomegaly and symptoms in patients with pmf . therefore , targeting of jak1/jak2 is effective to reduce proinflammatory cytokines in pmf irrespective of the jak2/mpl / calr mutation status . used the jak2 inhibitor ag490 to target gm - csf induced activation of jak / stat signaling and could thus overcome resistance to imatinib and nilotinib in vitro . none of the patients treated with bevacizumab had an objective response , but significant toxicity was observed . targeting of mtor by rapamycin in cml cells inhibited not only vegf expression but also the in vitro growth of leukemic cells . aberrant activation of hgf and/or its receptor c - met has been described in solid tumors as well as in acute myeloid leukemia ( aml ) , myeloma , and mpn . thus , blocking of the hgf / c - met function was suggested as an independent therapeutic target which could synergize with tki treatment in mpn . su-11274 was found to inhibit colony formation , to reduce viability , and to induce differentiation in a9 m , u937 , and oci - aml cells . boissinot and colleagues tested the efficacy of combining c - met and jak inhibitors on the proliferation of the jak2 v167f positive hel and uke-1 cell lines . in summary ,
increasing evidence suggests an important role of the bm microenvironment in the regulation of proliferation and survival of normal and leukemic hematopoietic stem cells . thus , targeting of the specific bm niches and stem cell - niche interactions has been suggested as a promising therapeutic strategy . cxcr4 is highly expressed on the surface of malignant cell in chronic lymphocytic leukemia ( cll ) , and sdf-1 was found to promote chemotaxis of cll cells and their interaction with stromal cells , which was shown to induce resistance of cll cells to cytotoxic agents , and was furthermore suggested to mediate persistence of minimal residual disease in the bm during therapy . in line with this concept ,
cxcr4 antagonists were successfully used to block interactions between cll and stromal cells and to mobilize cll cells from their protective microenvironments , becoming thus accessible to conventional drugs . furthermore , genetic targeting of cxcr4 in murine t - all led to rapid , sustained disease remission and cxcr4 antagonism suppressed human t - all in primary xenograft models . partly ambivalent results have been published for the role of sdf-1/cxcr4 in mpn , and although increased levels of sdf-1 have been reported , this may not necessarily result in a sustained activation of cxcr4 signaling in neoplastic cells [ 185 , 186 ] . on the one hand , mobilization of cd34
+ cells in patients with pmf has been attributed to reduced cxcr4 expression and hypermethylation of the cxcr4 promoter [ 187 , 188 ] . moreover , although elevated levels of immunoreactive forms of sdf-1 were found in the bm and peripheral blood of patients with pmf and pv , detailed studies using mass spectrometry have shown that sdf-1 was mainly truncated and thus expressed in an inactive form in these patients . on the other hand
, the sdf-1/cxcr axis between stroma and leukemic cells contributes to resistance to tki treatment in cml . the cxcr4 inhibitor plerixafor diminished migration of bcr - abl1 positive cells and reduced adhesion of these cells to extracellular - matrix components and to bm stromal cells in vitro . the authors of this study argue that the combination of cxcr4 inhibition with tki treatment in cml might be a useful approach to override drug resistance and to achieve deeper responses in cml . in contrast , another study tested the effects of plerixafor in combination with either imatinib or dasatinib in a murine cml bm transplant model . applied plerixafor after marked reduction of disease burden with nilotinib as a model of minimal residual disease and argued that the absence of significant disease burden was relevant for the beneficial effects of the combination therapy . the complex interplay between neoplastic cells and microenvironmental cells in mpn has gained increasing interest in recent years . a number of pathogenetic mechanisms are found to be conserved between various mpn , and lessons learned from one disease can be exploited for the other mpn types . thus , it will be important to compare systematically the various common as well as rare mpn - variants in terms of basic and clinical science . more recently , the pathologically altered interactions between neoplastic cells and their microenvironment have been investigated with the aim of defining new potential targets of therapy and to develop novel therapeutic approaches . first , the increased angiogenesis and bm fibrosis may serve as novel targets of therapy in mpn . best evidence for targeting of the inflammatory cytokine storm is derived from the clinical efficacy of jak inhibitors in mpn , which show marked benefits in patients despite their lack of specificity for mutant jak2 . increasing knowledge of the lsc - niche interaction will help to optimize this combined targeting approach and to establish synergistic strategies for therapy or even cure of mpn . | [
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] |
mental health is regarded as an essential component of health by the world health organization .
a person could be termed depressed if he / she shows a variable combination of low mood ; loss of interest or pleasure ; feelings of guilt ; low self - esteem ; disturbed appetite ; disturbed sleep ; or disturbed concentration.1 the american psychological association characterizes anxiety and stress2,3 by feelings of tension , worried thoughts , and physical changes .
anxiety is more related to autonomic arousal , skeletal muscle tension , and situational aspects , whereas stress is more related to irritability , impatience , and difficulty in relaxing .
the curricular objectives are dynamic due to expanding knowledge and evolving therapies . during this period
, medical students should acquire adequate professional knowledge , skill , and attitudes in order to prepare themselves to deal with life - long professional challenges independently .
however , the demands of the learning and training might adversely affect the student s physical and mental health .
it has been reported that medical students consequently suffer from depression , anxiety , and stress.46 yusoff et al7 have previously reported that healthy students develop depression and stress after commencing their medical education .
the competition for getting postgraduate training and job opportunities could be an additional trigger for psychological illness .
it has also been reported that physicians tend to have a higher suicidal rate than the general population.8 the subject has been studied in many countries ; however , the student population in middle east is still understudied .
the government in saudi arabia puts emphasis on observing morality in society as well as promoting and facilitating the practice of religion .
islam , the predominant religion in the country , advocates honesty and hard work , and educates its followers to remain optimistic and peaceful in all circumstances .
the crime rate is also among the lowest in the world.9 thus , it could be assumed that the prevalence of depression , anxiety , or stress should be lower than other contemporary societies .
the saudi arabian economy is largely tax - free and has a high gross domestic product .
thus , it draws skilled and non - skilled labor from all parts of the world .
hence , non - saudi expatriate workers living in the kingdom only have the provision of private - sector institutions , such as ours , should they wish their children to pursue higher education .
since the students get admission after rigorous competition and can afford to pay for their fees , this could translate into a sound academic record and being financially well - off .
presumably , this may further favor lower levels of depression , anxiety , or stress .
the students are generally supported as well as supervised by their parents or guardians in terms of sustenance , nurture , and education .
this trend is further consolidated by prohibition of work permits to dependent children of foreign workers in the country . like many societies in the middle east and south asia , youth , especially females ,
presumably , this could confer a sense of security and thus psychological well - being .
a recent report has suggested that cigarette smoking is on the rise among youth in saudi arabia .
a number of studies have identified the pattern , predictors , and characteristics of tobacco smoking among local youth.10 smoking , the largest cause of preventable death , has also been associated with stress , anxiety , and depression .
conversely , it has been noted that periods of stress lead to increased smoking prevalence.11 similarly , smoking also increases stress , anxiety , and depression1214 as well as externalizing
behavior.14 thus , it potentially leads to a vicious cycle of escalating psychological illness and increased smoking .
some recent large studies conducted in various cities of saudi arabia1518 have suggested that stress , anxiety , or depression exists among medical students . although these studies have inducted a large number of students ( total 2,448 ) , the subject is only partially addressed .
further , the participants were only saudis and different tools were used to measure the mental well - being .
thus , the status of mental well - being as influenced by nationality , living conditions , sex , and smoking at a competitive , multiethnic setting like ours in saudi arabia remains unclear .
the college of medicine at alfaisal university , riyadh , saudi arabia is a relatively new institution which offers medical training to all sexes and ethnicities .
although there are no international students , the institution consists of a vibrant mixture of nationalities from many countries and backgrounds owing to expatriates residing in the country .
thus , to draw meaningful conclusions , all expatriate students were grouped as a single group to compare them with their saudi counterparts . the medical curriculum at alfaisal consists of basic and clinical subjects complimenting each other in a gradient fashion .
the learning methods include lectures , problem - based learning , laboratory sessions , and clinical rotations .
a number of faculty members and students are also supported financially to undertake research at local , national , and international levels .
the first batch of graduates is already seeking suitable residency and postgraduate positions in north america and europe , and some students have already found suitable placements .
the medical students of this university scored consistently higher than students from other saudi medical colleges in recently conducted nationwide progress tests
( organized by qassim college of medicine , qassim university , qassim , saudi arabia ) .
there has been concern regarding the mental health and well - being of the students as raised by a number of students and faculty at the college of medicine , alfaisal university at different fora .
the hectic schedule , course burden , and ancillary courses are being considered as potential reasons for this perceived psychological pressure .
this led us to design the present study to explore mental health status in our students .
there are several tools to appraise mental health . among them , a short , reliable , and validated questionnaire called depression , anxiety , and stress scale-21 ( dass-21 ) is of particular importance.19 this tool can identify traits of depression , anxiety , and stress rather than actual diagnosis.20 the dass-21 questionnaire21 categorizes each condition into five subcategories , namely , normal , mild , moderate , severe , and extremely severe , as given in table 1 .
we designed this study to assess the level of depression , anxiety , and stress among medical students at alfaisal university , and to identify possible correlates such as sex , nationality , year of scholarship , housing , and smoking .
all medical students in basic ( years 1 , 2 , and 3 ) as well as clerkship ( years 4 and 5 ) phases of the academic year 20122013 were administered the dass-21 questionnaire .
the responses were sought in a self - reported , anonymous fashion . since admission to medical college was open only for male students in the senior batches ( years 3 , 4 , and 5 in the current study ) , the female students were available only in years 1 and 2 .
the students were given printed written information and procedure of consent along with the dass-21 questionnaire . since the identity of the students remained undisclosed , no signature of participants was necessary .
rather , filling out the questionnaire and submission by the student himself / herself was considered as a declaration of willingness to participate .
the ethical approval was obtained from the committee for medical and bioethics , office of research and graduate studies , alfaisal university . to facilitate meaningful analysis , we categorized the students into two broad groups .
normal and mild positive scores for each category of dass-21were considered as a single group , whereas the moderate and severe positive scores for each category were grouped together .
in addition to dass-21 , information was sought regarding sex ; year of study ; attendance of a premedical university preparatory program ( upp ) ; housing and living ; smoking ; and use of any recreational drugs .
an additional open - ended section was included to allow students to reflect on the reason / reasons , if any , for their choices in dass-21 .
the questionnaire was administered at two time - points for all students , as follows : 1 ) 23 weeks before a major terminal examination at the end of a semester ( thus referred to as pre - exam ) ; and 2 ) during regular classes in the following semester ( thus referred to as post - exam ) .
the independent factors included sex , nationality , year of scholarship , housing , and smoking .
the dependent factors were pre - exam dass-21 scores , post - exam dass-21 scores , and the difference between them .
the results were analyzed through one - way analysis of variance , chi - square tests , or multiple linear regression where appropriate , using spss version 21 ( ibm corporation , armonk , ny , usa ) .
the medical students in all 5 years were administered the dass-21 questionnaire in a pre - exam and post - exam fashion .
a total of 442 ( 76.8% ) students participated in the pre - exam group whereas 431 ( 74.9% ) participated in post - exam group .
since student admissions had increased over recent years , the most senior batch had the least absolute numbers , but they had a proportionate representation .
the expatriates largely ( > 90% ) included citizens from neighboring arab states ( syria , jordan , palestine , and egypt ) , south asia ( pakistan , bangladesh , and india ) , and north america ( canada and usa ) .
the scores for depression , anxiety , and stress were significantly higher in the pre - exam group .
students in year 4 had the highest level of depression , anxiety , and stress .
the dass-21 scores were also higher among saudis , students who attended a year of upp before commencing their medical curriculum , and the regular smokers .
housing and sex did not appear to be associated with depression , anxiety , or stress scores .
table 4 presents the prevalence ( percent , frequency ) of depression , anxiety , and stress and its variation with an impending examination .
although the depression and stress also increased with an approaching examination , statistical significance was not achieved .
however , it is noteworthy that the baseline levels ( post - exam ) of all three were already high .
the study population was divided in two , ie , pre - exam and post - exam groups .
the total dass-21 score was considered as the dependent variable while the independent variables included sex ( male or female ) , nationality ( saudi , non - saudi ) , attendance of a upp ( attended or not ) , academic year in mbbs program ( year 15 ) , housing ( with parents , with relatives , or alone ) , and smoking status .
the f statistic was 2.84 ( p=0.01 ) and 2.71 ( p=0.01 ) , for the pre - exam and post - exam groups , respectively .
the standardized coefficient beta , t statistic , significance level ( p - value ) , and 95% confidence intervals are given .
the pre - exam group showed that dass-21 scores increase ( predicts 21.5% of this phenomenon ) with advancing years in mbbs program , while in the post - exam group , dass-21 score is mainly influenced by regular smoking ( 16% ) and female sex ( 11.3% ) .
it should be noted that dass-21 scores are lower in the post - exam group .
only 12.3% and 9% of students from pre - exam and post - exam groups , respectively , wrote reasons which they perceived to be responsible for their feelings .
the data shows frequency ( n ) of the responses falling under a theme . in the majority of instances ,
the examinations , course burden , and hectic schedule was regarded as the reason for their depression , anxiety , or stress .
the main results of this study suggest that among medical students at alfaisal university , there is a high baseline level of depression , anxiety , and stress , which tends to remain much higher if a major examination is near . smoking and female sex appears to predict higher levels of depression , anxiety , or stress in the absence of an impending examination .
the students perceive the course burden and hectic schedule as the most important reasons underlying their high dass-21 scores . to the best of our knowledge ,
this study was the largest in the kingdom where both saudi and expatriate students were included .
further , we studied traits of depression , anxiety , and stress while most of the others had reported fewer parameters .
we also studied the dass-21 scores at two different time - points to see the variation among medical students .
we found that students would be highly stressed , anxious , and depressed if an examination was approaching .
it appears that method and assessment grading systems affects student well - being more than any other component of the curriculum .
further , it has been reported that the greater the number of grading scale categories , the higher the level of stress and emotional exhaustion . a similar effect is seen if examinations take place frequently.22 also , in the case of our students , the grading system comprised multiple categories , ie , grade a+ , a , a , b+ , b , b , and so on .
a number of students are awarded scholarships annually , which is tied to their consistent high performance in examinations .
it is possible that this setup is contributing to their high level of depression , anxiety , or stress . as presented in table 5 , many students have attributed their condition either to difficulty in coping with their studies , or fear of failure or losing their scholarship .
it is quite possible that the high prevalence of depression , anxiety , or stress could lead to burnout among them and thereby affect their performance , progress , or personal life in the long - term , thus requiring a follow up study . like many other countries , in saudi arabia ,
transition from a high school or college to a professional career is a big jump .
it is no wonder 1st year medical students feel stress due to greater work load.23 similarly , the commencement of a hospital or practical phase of their career has its own demands .
higher levels of depression have been reported during internship and residency due to non - supportive and highly challenging environments as compared to other undergraduate years.23,24 in the current study , although there was high stress among 1st year students , it peaked in year 4 , where the clerkship phase starts and students rotate through various disciplines of hospital as their major method of training .
thus , this agrees with other similar studies elsewhere.23,24 mental health has been studied in various parts of the world .
the baseline levels of stress , anxiety , and depression could vary due to different social , economic , and personal factors , and thus , these factors should affect the results of such studies .
however , studies from the middle east in general , and saudi arabia in particular , are a recent phenomenon . in a recent study15 conducted simultaneously in saudi arabia and egypt by a single team
, it was concluded that saudi students had lower anxiety and depression than their egyptian counterparts .
however , the students were studied in their respective home countries . in our study of students in the same setting
, we found that saudi students were more anxious and stressed than their expatriate counterparts .
a number of studies done in various state - run medical colleges in the kingdom have reported high prevalence of depression , anxiety , or stress , where the students are primarily saudi nationals.1518 the results are compared in table 7 .
those students who were admitted to medicine after a year - long upp had higher anxiety levels . at alfaisal university ,
a student may get direct admission if his / her grades are above a certain threshold .
thus , it could be inferred , albeit indirectly , that among those who had better preadmission qualifying grades , enabling a direct admission to the medical college ( without upp ) , there were lower anxiety levels . out of the direct admissions ,
whether this is the reason for higher dass-21 scores among saudi students , needs further research .
some previous studies have concluded that female students are more likely to experience depression2325 and female physicians tend to have higher suicide rates than their male colleagues.8 in our study , the logistic regression ( table 4 ) suggested that females tends to have a significantly higher dass-21 score only in the post - exam group . it could be possible that an overall higher dass-21 score masked this phenomenon in the pre - exam group . in our study
however , there were students ( 15% ) who either lived alone or with relatives .
although the levels of depression , anxiety , and stress were highest among those who lived alone , it did not reach statistical significance . in our study , there was a strong positive relationship of regular smoking with depression and anxiety . however , a cause
effect relationship can not be drawn from this study . while it could be considered to confirm earlier observations , the proportion of reported smokers among the study sample was small .
depression is considered a stigma for medical students as this could be perceived as detrimental to their professional and personal life .
it is observed that the medical students suffering from depression feel guilty about sharing their emotional state with others , or are reluctant to receive treatment.26,27 a recent review suggested that ignoring one s psychological problem could lead to serious consequences.28 unfortunately , medical students experience suicidal thoughts and ideas because of depression , anxiety , and other reasons.29 further , burnout and chronic stress have also been found to correlate strongly with professional misconduct or reduced altruistic values among physicians.30 this is a serious consequence not only for the health professionals but also for society . at present , we can not confirm or reject such extreme ideation and their consequences in our setting . whether religious teachings and culture play any preventive role in this regard is also difficult to ascertain .
at least a few of the students comments hinted that they tend to remain content and patient in accordance with religious teachings .
secondly , it gauges the prevalence of depression , anxiety , or stress among medical students in all 5 years at alfaisal university , which is a leading institution in the kingdom of saudi arabia .
thirdly , it compares simultaneously the dass-21 scores in relation to sex , nationality , smoking status , and other possible underlying reasons in this culture .
fourthly , the sample size is large and adequately represents the students in all 5 years from basic sciences to clerkship phases .
firstly , the data on depression , anxiety , or stress among the study cohort before the commencement of medical education is not available . secondly , the prevalence of depression , anxiety , or stress in the general population is also not available .
fourthly , the majority of students did not report their status for smoking and recreational drug use ; nor did they provide any descriptive comments about their perceived underlying reasons for their depression , anxiety , or stress . finally , the anonymity of students made it impossible to check for any change of individual dass-21 scores .
first , the unnecessary course burden and busy schedule could be rectified by devising a justified list of observable specific course objectives in all courses .
the number of lectures could be reduced as continuous lecturing may not be helpful in retention of information.31 second , as there is much emphasis on problem - based learning , all possible care should be taken to avoid overlap of objectives to be covered in lectures and problem - based learning sessions .
this could free some space in the schedule for self - directed learning and reflection , which is an important aspect of learning .
third , regular feedback from faculty as well as students regarding the academics should be encouraged and considered .
we found that students would be highly stressed , anxious , and depressed if an examination was approaching .
it appears that method and assessment grading systems affects student well - being more than any other component of the curriculum .
further , it has been reported that the greater the number of grading scale categories , the higher the level of stress and emotional exhaustion . a similar effect is seen if examinations take place frequently.22 also , in the case of our students , the grading system comprised multiple categories , ie , grade a+ , a , a , b+ , b , b , and so on .
a number of students are awarded scholarships annually , which is tied to their consistent high performance in examinations .
it is possible that this setup is contributing to their high level of depression , anxiety , or stress . as presented in table 5 , many students have attributed their condition either to difficulty in coping with their studies , or fear of failure or losing their scholarship .
it is quite possible that the high prevalence of depression , anxiety , or stress could lead to burnout among them and thereby affect their performance , progress , or personal life in the long - term , thus requiring a follow up study .
like many other countries , in saudi arabia , the students are admitted to medical college after completing 12 years of school education .
transition from a high school or college to a professional career is a big jump .
it is no wonder 1st year medical students feel stress due to greater work load.23 similarly , the commencement of a hospital or practical phase of their career has its own demands .
higher levels of depression have been reported during internship and residency due to non - supportive and highly challenging environments as compared to other undergraduate years.23,24 in the current study , although there was high stress among 1st year students , it peaked in year 4 , where the clerkship phase starts and students rotate through various disciplines of hospital as their major method of training .
the baseline levels of stress , anxiety , and depression could vary due to different social , economic , and personal factors , and thus , these factors should affect the results of such studies .
however , studies from the middle east in general , and saudi arabia in particular , are a recent phenomenon . in a recent study15 conducted simultaneously in saudi arabia and egypt by a single team
, it was concluded that saudi students had lower anxiety and depression than their egyptian counterparts .
however , the students were studied in their respective home countries . in our study of students in the same setting
, we found that saudi students were more anxious and stressed than their expatriate counterparts .
a number of studies done in various state - run medical colleges in the kingdom have reported high prevalence of depression , anxiety , or stress , where the students are primarily saudi nationals.1518 the results are compared in table 7 .
those students who were admitted to medicine after a year - long upp had higher anxiety levels . at alfaisal university , a student may get direct admission if his / her grades are above a certain threshold . thus , it could be inferred , albeit indirectly , that among those who had better preadmission qualifying grades , enabling a direct admission to the medical college ( without upp ) , there were lower anxiety levels . out of the direct admissions ,
whether this is the reason for higher dass-21 scores among saudi students , needs further research .
some previous studies have concluded that female students are more likely to experience depression2325 and female physicians tend to have higher suicide rates than their male colleagues.8 in our study , the logistic regression ( table 4 ) suggested that females tends to have a significantly higher dass-21 score only in the post - exam group .
it could be possible that an overall higher dass-21 score masked this phenomenon in the pre - exam group . in our study
however , there were students ( 15% ) who either lived alone or with relatives .
although the levels of depression , anxiety , and stress were highest among those who lived alone , it did not reach statistical significance .
in our study , there was a strong positive relationship of regular smoking with depression and anxiety .
however , a cause effect relationship can not be drawn from this study . while it could be considered to confirm earlier observations , the proportion of reported smokers among the study sample was small .
depression is considered a stigma for medical students as this could be perceived as detrimental to their professional and personal life .
it is observed that the medical students suffering from depression feel guilty about sharing their emotional state with others , or are reluctant to receive treatment.26,27 a recent review suggested that ignoring one s psychological problem could lead to serious consequences.28 unfortunately , medical students experience suicidal thoughts and ideas because of depression , anxiety , and other reasons.29 further , burnout and chronic stress have also been found to correlate strongly with professional misconduct or reduced altruistic values among physicians.30 this is a serious consequence not only for the health professionals but also for society . at present ,
we can not confirm or reject such extreme ideation and their consequences in our setting .
whether religious teachings and culture play any preventive role in this regard is also difficult to ascertain .
at least a few of the students comments hinted that they tend to remain content and patient in accordance with religious teachings .
secondly , it gauges the prevalence of depression , anxiety , or stress among medical students in all 5 years at alfaisal university , which is a leading institution in the kingdom of saudi arabia .
thirdly , it compares simultaneously the dass-21 scores in relation to sex , nationality , smoking status , and other possible underlying reasons in this culture .
fourthly , the sample size is large and adequately represents the students in all 5 years from basic sciences to clerkship phases .
firstly , the data on depression , anxiety , or stress among the study cohort before the commencement of medical education is not available . secondly , the prevalence of depression , anxiety , or stress in the general population is also not available .
fourthly , the majority of students did not report their status for smoking and recreational drug use ; nor did they provide any descriptive comments about their perceived underlying reasons for their depression , anxiety , or stress .
finally , the anonymity of students made it impossible to check for any change of individual dass-21 scores .
first , the unnecessary course burden and busy schedule could be rectified by devising a justified list of observable specific course objectives in all courses .
the number of lectures could be reduced as continuous lecturing may not be helpful in retention of information.31 second , as there is much emphasis on problem - based learning , all possible care should be taken to avoid overlap of objectives to be covered in lectures and problem - based learning sessions .
this could free some space in the schedule for self - directed learning and reflection , which is an important aspect of learning .
third , regular feedback from faculty as well as students regarding the academics should be encouraged and considered .
thus , we conclude that the medical students had high levels of traits of depression , anxiety , and stress . anticipated examinations , attending upp , smoking , and female sex were associated with higher levels of depression , anxiety , or stress among them .
students suggested that study burden and busy schedule were major reasons for their high scores . | backgroundcontemporary literature suggests that medical education might adversely affect students mental health .
alfaisal university in riyadh , saudi arabia is a developing institution ; hence , there has been a concern regarding the mental well - being of the students.objectivesthis study was designed to assess the traits of depression , anxiety , and stress among students in relation to potential underlying reasons.methodsall 575 medical students across the 5 years of study participated by filling out the depression , anxiety , and stress scale-21 ( dass-21 ) questionnaire anonymously twice .
firstly , 23 weeks before a major examination ( pre - examination ) , and secondly , during regular classes ( post - examination ) .
correlation was sought regarding sex , year of scholarship , attendance of a premedical university preparatory program ( upp ) , housing , and smoking .
subjective comments from students were also obtained.resultsa total of 76.8% and 74.9% of students participated in pre - and post - examination groups , respectively .
the majority were the children of expatriate workers in saudi arabia , and included arabs , south asians , and north americans .
prevalence of depression , anxiety , and stress was high ( 43% , 63% , and 41% , respectively ) which reduced ( to 30% , 47% , and 30% , respectively ) to some extent after examinations .
saudis and those who had attended upp had higher dass-21 scores .
smoking and female sex predicted higher levels of baseline depression , anxiety , or stress .
the students perceived the curriculum and schedule to be the primary causes of their high dass-21 scores.conclusionthe students had high baseline
traits of depression , anxiety , and stress , and these were higher if an examination was near , especially among saudis and those who had attended upp .
smoking and female sex predicted higher levels of baseline depression , anxiety , or stress .
students suggested that study burden and a busy schedule were the major reasons for their high dass-21 scores . | Introduction
Materials and methods
Results
Discussion
Relationship of DASS-21 scores with assessment
Relationship of DASS-21 scores with level of scholarship
Relationship of DASS-21 scores with nationality
Relationship of DASS-21 scores with preadmission grades
Relationship of DASS-21 scores with sex and living conditions
Relationship of DASS-21 scores with tobacco smoking
Long-term effects of psychological problems among health professionals
Advantages of this study
Limitations of this study
Implications of this study
Conclusion | anxiety is more related to autonomic arousal , skeletal muscle tension , and situational aspects , whereas stress is more related to irritability , impatience , and difficulty in relaxing . during this period
, medical students should acquire adequate professional knowledge , skill , and attitudes in order to prepare themselves to deal with life - long professional challenges independently . however , the demands of the learning and training might adversely affect the student s physical and mental health . it has been reported that medical students consequently suffer from depression , anxiety , and stress.46 yusoff et al7 have previously reported that healthy students develop depression and stress after commencing their medical education . the government in saudi arabia puts emphasis on observing morality in society as well as promoting and facilitating the practice of religion . islam , the predominant religion in the country , advocates honesty and hard work , and educates its followers to remain optimistic and peaceful in all circumstances . the crime rate is also among the lowest in the world.9 thus , it could be assumed that the prevalence of depression , anxiety , or stress should be lower than other contemporary societies . thus , it draws skilled and non - skilled labor from all parts of the world . hence , non - saudi expatriate workers living in the kingdom only have the provision of private - sector institutions , such as ours , should they wish their children to pursue higher education . since the students get admission after rigorous competition and can afford to pay for their fees , this could translate into a sound academic record and being financially well - off . presumably , this may further favor lower levels of depression , anxiety , or stress . the students are generally supported as well as supervised by their parents or guardians in terms of sustenance , nurture , and education . this trend is further consolidated by prohibition of work permits to dependent children of foreign workers in the country . like many societies in the middle east and south asia , youth , especially females ,
presumably , this could confer a sense of security and thus psychological well - being . a recent report has suggested that cigarette smoking is on the rise among youth in saudi arabia . a number of studies have identified the pattern , predictors , and characteristics of tobacco smoking among local youth.10 smoking , the largest cause of preventable death , has also been associated with stress , anxiety , and depression . conversely , it has been noted that periods of stress lead to increased smoking prevalence.11 similarly , smoking also increases stress , anxiety , and depression1214 as well as externalizing
behavior.14 thus , it potentially leads to a vicious cycle of escalating psychological illness and increased smoking . some recent large studies conducted in various cities of saudi arabia1518 have suggested that stress , anxiety , or depression exists among medical students . although these studies have inducted a large number of students ( total 2,448 ) , the subject is only partially addressed . further , the participants were only saudis and different tools were used to measure the mental well - being . thus , the status of mental well - being as influenced by nationality , living conditions , sex , and smoking at a competitive , multiethnic setting like ours in saudi arabia remains unclear . the college of medicine at alfaisal university , riyadh , saudi arabia is a relatively new institution which offers medical training to all sexes and ethnicities . the medical students of this university scored consistently higher than students from other saudi medical colleges in recently conducted nationwide progress tests
( organized by qassim college of medicine , qassim university , qassim , saudi arabia ) . there has been concern regarding the mental health and well - being of the students as raised by a number of students and faculty at the college of medicine , alfaisal university at different fora . the hectic schedule , course burden , and ancillary courses are being considered as potential reasons for this perceived psychological pressure . this led us to design the present study to explore mental health status in our students . among them , a short , reliable , and validated questionnaire called depression , anxiety , and stress scale-21 ( dass-21 ) is of particular importance.19 this tool can identify traits of depression , anxiety , and stress rather than actual diagnosis.20 the dass-21 questionnaire21 categorizes each condition into five subcategories , namely , normal , mild , moderate , severe , and extremely severe , as given in table 1 . we designed this study to assess the level of depression , anxiety , and stress among medical students at alfaisal university , and to identify possible correlates such as sex , nationality , year of scholarship , housing , and smoking . all medical students in basic ( years 1 , 2 , and 3 ) as well as clerkship ( years 4 and 5 ) phases of the academic year 20122013 were administered the dass-21 questionnaire . since admission to medical college was open only for male students in the senior batches ( years 3 , 4 , and 5 in the current study ) , the female students were available only in years 1 and 2 . the students were given printed written information and procedure of consent along with the dass-21 questionnaire . since the identity of the students remained undisclosed , no signature of participants was necessary . rather , filling out the questionnaire and submission by the student himself / herself was considered as a declaration of willingness to participate . in addition to dass-21 , information was sought regarding sex ; year of study ; attendance of a premedical university preparatory program ( upp ) ; housing and living ; smoking ; and use of any recreational drugs . the questionnaire was administered at two time - points for all students , as follows : 1 ) 23 weeks before a major terminal examination at the end of a semester ( thus referred to as pre - exam ) ; and 2 ) during regular classes in the following semester ( thus referred to as post - exam ) . the independent factors included sex , nationality , year of scholarship , housing , and smoking . the dependent factors were pre - exam dass-21 scores , post - exam dass-21 scores , and the difference between them . the medical students in all 5 years were administered the dass-21 questionnaire in a pre - exam and post - exam fashion . a total of 442 ( 76.8% ) students participated in the pre - exam group whereas 431 ( 74.9% ) participated in post - exam group . the expatriates largely ( > 90% ) included citizens from neighboring arab states ( syria , jordan , palestine , and egypt ) , south asia ( pakistan , bangladesh , and india ) , and north america ( canada and usa ) . the scores for depression , anxiety , and stress were significantly higher in the pre - exam group . students in year 4 had the highest level of depression , anxiety , and stress . the dass-21 scores were also higher among saudis , students who attended a year of upp before commencing their medical curriculum , and the regular smokers . housing and sex did not appear to be associated with depression , anxiety , or stress scores . table 4 presents the prevalence ( percent , frequency ) of depression , anxiety , and stress and its variation with an impending examination . although the depression and stress also increased with an approaching examination , statistical significance was not achieved . however , it is noteworthy that the baseline levels ( post - exam ) of all three were already high . the study population was divided in two , ie , pre - exam and post - exam groups . the total dass-21 score was considered as the dependent variable while the independent variables included sex ( male or female ) , nationality ( saudi , non - saudi ) , attendance of a upp ( attended or not ) , academic year in mbbs program ( year 15 ) , housing ( with parents , with relatives , or alone ) , and smoking status . the f statistic was 2.84 ( p=0.01 ) and 2.71 ( p=0.01 ) , for the pre - exam and post - exam groups , respectively . the standardized coefficient beta , t statistic , significance level ( p - value ) , and 95% confidence intervals are given . the pre - exam group showed that dass-21 scores increase ( predicts 21.5% of this phenomenon ) with advancing years in mbbs program , while in the post - exam group , dass-21 score is mainly influenced by regular smoking ( 16% ) and female sex ( 11.3% ) . it should be noted that dass-21 scores are lower in the post - exam group . only 12.3% and 9% of students from pre - exam and post - exam groups , respectively , wrote reasons which they perceived to be responsible for their feelings . in the majority of instances ,
the examinations , course burden , and hectic schedule was regarded as the reason for their depression , anxiety , or stress . the main results of this study suggest that among medical students at alfaisal university , there is a high baseline level of depression , anxiety , and stress , which tends to remain much higher if a major examination is near . smoking and female sex appears to predict higher levels of depression , anxiety , or stress in the absence of an impending examination . the students perceive the course burden and hectic schedule as the most important reasons underlying their high dass-21 scores . to the best of our knowledge ,
this study was the largest in the kingdom where both saudi and expatriate students were included . further , we studied traits of depression , anxiety , and stress while most of the others had reported fewer parameters . we also studied the dass-21 scores at two different time - points to see the variation among medical students . we found that students would be highly stressed , anxious , and depressed if an examination was approaching . it appears that method and assessment grading systems affects student well - being more than any other component of the curriculum . it is possible that this setup is contributing to their high level of depression , anxiety , or stress . it is quite possible that the high prevalence of depression , anxiety , or stress could lead to burnout among them and thereby affect their performance , progress , or personal life in the long - term , thus requiring a follow up study . like many other countries , in saudi arabia ,
transition from a high school or college to a professional career is a big jump . it is no wonder 1st year medical students feel stress due to greater work load.23 similarly , the commencement of a hospital or practical phase of their career has its own demands . higher levels of depression have been reported during internship and residency due to non - supportive and highly challenging environments as compared to other undergraduate years.23,24 in the current study , although there was high stress among 1st year students , it peaked in year 4 , where the clerkship phase starts and students rotate through various disciplines of hospital as their major method of training . thus , this agrees with other similar studies elsewhere.23,24 mental health has been studied in various parts of the world . the baseline levels of stress , anxiety , and depression could vary due to different social , economic , and personal factors , and thus , these factors should affect the results of such studies . however , studies from the middle east in general , and saudi arabia in particular , are a recent phenomenon . in a recent study15 conducted simultaneously in saudi arabia and egypt by a single team
, it was concluded that saudi students had lower anxiety and depression than their egyptian counterparts . however , the students were studied in their respective home countries . in our study of students in the same setting
, we found that saudi students were more anxious and stressed than their expatriate counterparts . a number of studies done in various state - run medical colleges in the kingdom have reported high prevalence of depression , anxiety , or stress , where the students are primarily saudi nationals.1518 the results are compared in table 7 . those students who were admitted to medicine after a year - long upp had higher anxiety levels . thus , it could be inferred , albeit indirectly , that among those who had better preadmission qualifying grades , enabling a direct admission to the medical college ( without upp ) , there were lower anxiety levels . out of the direct admissions ,
whether this is the reason for higher dass-21 scores among saudi students , needs further research . some previous studies have concluded that female students are more likely to experience depression2325 and female physicians tend to have higher suicide rates than their male colleagues.8 in our study , the logistic regression ( table 4 ) suggested that females tends to have a significantly higher dass-21 score only in the post - exam group . it could be possible that an overall higher dass-21 score masked this phenomenon in the pre - exam group . although the levels of depression , anxiety , and stress were highest among those who lived alone , it did not reach statistical significance . it is observed that the medical students suffering from depression feel guilty about sharing their emotional state with others , or are reluctant to receive treatment.26,27 a recent review suggested that ignoring one s psychological problem could lead to serious consequences.28 unfortunately , medical students experience suicidal thoughts and ideas because of depression , anxiety , and other reasons.29 further , burnout and chronic stress have also been found to correlate strongly with professional misconduct or reduced altruistic values among physicians.30 this is a serious consequence not only for the health professionals but also for society . at least a few of the students comments hinted that they tend to remain content and patient in accordance with religious teachings . secondly , it gauges the prevalence of depression , anxiety , or stress among medical students in all 5 years at alfaisal university , which is a leading institution in the kingdom of saudi arabia . thirdly , it compares simultaneously the dass-21 scores in relation to sex , nationality , smoking status , and other possible underlying reasons in this culture . fourthly , the sample size is large and adequately represents the students in all 5 years from basic sciences to clerkship phases . firstly , the data on depression , anxiety , or stress among the study cohort before the commencement of medical education is not available . secondly , the prevalence of depression , anxiety , or stress in the general population is also not available . fourthly , the majority of students did not report their status for smoking and recreational drug use ; nor did they provide any descriptive comments about their perceived underlying reasons for their depression , anxiety , or stress . finally , the anonymity of students made it impossible to check for any change of individual dass-21 scores . first , the unnecessary course burden and busy schedule could be rectified by devising a justified list of observable specific course objectives in all courses . we found that students would be highly stressed , anxious , and depressed if an examination was approaching . it appears that method and assessment grading systems affects student well - being more than any other component of the curriculum . it is possible that this setup is contributing to their high level of depression , anxiety , or stress . it is quite possible that the high prevalence of depression , anxiety , or stress could lead to burnout among them and thereby affect their performance , progress , or personal life in the long - term , thus requiring a follow up study . like many other countries , in saudi arabia , the students are admitted to medical college after completing 12 years of school education . it is no wonder 1st year medical students feel stress due to greater work load.23 similarly , the commencement of a hospital or practical phase of their career has its own demands . higher levels of depression have been reported during internship and residency due to non - supportive and highly challenging environments as compared to other undergraduate years.23,24 in the current study , although there was high stress among 1st year students , it peaked in year 4 , where the clerkship phase starts and students rotate through various disciplines of hospital as their major method of training . the baseline levels of stress , anxiety , and depression could vary due to different social , economic , and personal factors , and thus , these factors should affect the results of such studies . however , studies from the middle east in general , and saudi arabia in particular , are a recent phenomenon . in a recent study15 conducted simultaneously in saudi arabia and egypt by a single team
, it was concluded that saudi students had lower anxiety and depression than their egyptian counterparts . however , the students were studied in their respective home countries . in our study of students in the same setting
, we found that saudi students were more anxious and stressed than their expatriate counterparts . a number of studies done in various state - run medical colleges in the kingdom have reported high prevalence of depression , anxiety , or stress , where the students are primarily saudi nationals.1518 the results are compared in table 7 . those students who were admitted to medicine after a year - long upp had higher anxiety levels . thus , it could be inferred , albeit indirectly , that among those who had better preadmission qualifying grades , enabling a direct admission to the medical college ( without upp ) , there were lower anxiety levels . out of the direct admissions ,
whether this is the reason for higher dass-21 scores among saudi students , needs further research . some previous studies have concluded that female students are more likely to experience depression2325 and female physicians tend to have higher suicide rates than their male colleagues.8 in our study , the logistic regression ( table 4 ) suggested that females tends to have a significantly higher dass-21 score only in the post - exam group . it could be possible that an overall higher dass-21 score masked this phenomenon in the pre - exam group . although the levels of depression , anxiety , and stress were highest among those who lived alone , it did not reach statistical significance . it is observed that the medical students suffering from depression feel guilty about sharing their emotional state with others , or are reluctant to receive treatment.26,27 a recent review suggested that ignoring one s psychological problem could lead to serious consequences.28 unfortunately , medical students experience suicidal thoughts and ideas because of depression , anxiety , and other reasons.29 further , burnout and chronic stress have also been found to correlate strongly with professional misconduct or reduced altruistic values among physicians.30 this is a serious consequence not only for the health professionals but also for society . at least a few of the students comments hinted that they tend to remain content and patient in accordance with religious teachings . secondly , it gauges the prevalence of depression , anxiety , or stress among medical students in all 5 years at alfaisal university , which is a leading institution in the kingdom of saudi arabia . thirdly , it compares simultaneously the dass-21 scores in relation to sex , nationality , smoking status , and other possible underlying reasons in this culture . fourthly , the sample size is large and adequately represents the students in all 5 years from basic sciences to clerkship phases . firstly , the data on depression , anxiety , or stress among the study cohort before the commencement of medical education is not available . secondly , the prevalence of depression , anxiety , or stress in the general population is also not available . fourthly , the majority of students did not report their status for smoking and recreational drug use ; nor did they provide any descriptive comments about their perceived underlying reasons for their depression , anxiety , or stress . finally , the anonymity of students made it impossible to check for any change of individual dass-21 scores . first , the unnecessary course burden and busy schedule could be rectified by devising a justified list of observable specific course objectives in all courses . thus , we conclude that the medical students had high levels of traits of depression , anxiety , and stress . anticipated examinations , attending upp , smoking , and female sex were associated with higher levels of depression , anxiety , or stress among them . students suggested that study burden and busy schedule were major reasons for their high scores . | [
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] |
the school of medicine and dentistry curriculum has early clinical exposure , and students have completed adult and pediatric ambulatory clerkship requirements by the end of year 2 .
the curriculum provides 82 hours of msk content early in the undergraduate curriculum , exclusive of physical diagnosis . of this ,
cadaveric extremity dissection during the first year constitutes approximately 30 hours . in year 2 , the basic science of msk medicine and rheumatology is covered in over 36 hours .
there are two 8-hour modules that focus on the orthopedic clinical aspects of common msk disorders . in the first year
there is a module composed of 2 hours of lecture with 6 hours of small group problem - based learning ( pbl ) material covering basic upper and lower extremity msk disorders .
the second year curriculum also has 2 hours of lecture and 6 hours of small group pbl material covering orthopedic emergencies , pediatric orthopedics , and low back pain .
the school of medicine and dentistry is one of many schools that do not require msk medicine instruction during the clinically heavy final 2 years of the medical school curriculum .
five hundred and sixty - eight medical students from the school of medicine and dentistry participated in the study .
participants included 304 second year students from the classes of 2009 , 2010 , and 2011 , and 264 fourth year students from the classes of 2008 , 2009 , and 2010 .
the class of 2009 and 2010 took the examination twice , once as second year and once as fourth year students . between may 2007 and february 2010
, participants were administered the national board of medical examiners ( nbme ) msk subject examination at the end of their second and fourth years of medical school .
the nbme msk assessment tool is a web - based multiple - choice examination developed in 2006 in conjunction with the bone and joint decade .
test materials were drawn from secure nbme item pools , reviewed , and approved by an external volunteer interdisciplinary msk task force composed of basic scientists and clinicians .
the msk subject examination is similar in format to the nbme subject examinations available in core clinical science disciplines , including medicine and pediatrics .
subject examinations are intended to complement other sources of information about the educational progression of medical students , and they are primarily designed for use as final examination after courses and clerkships .
the nmbe msk subject examination comprises 75 items , with 14 basic science and 61 clinical items plus a brief tutorial ; examinees are allotted 1 hour 55 min for completion .
the kr20 reliability of the examination in the initial reference group of senior students from three participating schools was 0.86 ; reliabilities greater than 0.85 are considered acceptable for low stakes examinations .
mean scores and sd correspond closely to performance on other nbme discipline based shelf examinations .
the school of medicine and dentistry participated in pilot testing and validation of the examination .
the examination was administered to students during a translational science course which included msk topics but did not review prior msk coursework .
the exam was required but students who missed the exam due to illness or out of town obligations did not have to make up the examination .
being a part of curricular evaluation , the score was not part of a course grade and there was no special preparation .
low stakes examination , yet provided a unique opportunity for self - assessment of msk medicine knowledge . participants clinical confidence in msk medicine and internal medicine / pediatrics was measured simultaneously using a 10-item self - assessment tool to quantitate self - reported confidence in evaluating and treating select clinical scenarios .
this clinical confidence tool is similar to those used in prior msk curricular evaluation studies and is not validated ( 9 , 10 ) .
there were two questions that asked students to rate their confidence in evaluating and treating common msk disorders .
students were then asked to rate their confidence in evaluating four msk clinical scenarios and four internal medicine / pediatrics scenarios .
the msk clinical scenarios were ankle pain , knee pain , shoulder pain , and wrist pain . the internal medicine / pediatrics scenarios were chest pain , delirium , dyspnea , and febrile infant .
the mean msk medicine and internal medicine / pediatrics clinical confidence scores were computed for each participant in the second and fourth years of medical school .
we then performed one - way analysis of variance ( anova ) to assess for overall differences in mean knowledge and confidence scores between classes .
analyses were conducted for both the second ( 20092011 ) and fourth ( 20082010 ) year classes .
we then excluded all paired data ( dependent scores ) and performed an independent samples t - test to compare second and fourth year scores . to compare participants second and fourth year knowledge and confidence scores over time , we performed paired sample t - tests on dependent scores for the classes of 2009 and 2010 .
paired analyses were also conducted to compare knowledge and confidence scores within second and fourth year classes .
relationships between msk knowledge and msk and internal medicine / pediatrics clinical confidence were then assessed by pearson correlations . in reporting results ,
the research subjects review board at the university of rochester school of medicine and dentistry approved this curricular evaluation study .
the school of medicine and dentistry curriculum has early clinical exposure , and students have completed adult and pediatric ambulatory clerkship requirements by the end of year 2 .
the curriculum provides 82 hours of msk content early in the undergraduate curriculum , exclusive of physical diagnosis . of this ,
cadaveric extremity dissection during the first year constitutes approximately 30 hours . in year 2 , the basic science of msk medicine and rheumatology is covered in over 36 hours .
there are two 8-hour modules that focus on the orthopedic clinical aspects of common msk disorders . in the first year
there is a module composed of 2 hours of lecture with 6 hours of small group problem - based learning ( pbl ) material covering basic upper and lower extremity msk disorders .
the second year curriculum also has 2 hours of lecture and 6 hours of small group pbl material covering orthopedic emergencies , pediatric orthopedics , and low back pain .
the school of medicine and dentistry is one of many schools that do not require msk medicine instruction during the clinically heavy final 2 years of the medical school curriculum .
five hundred and sixty - eight medical students from the school of medicine and dentistry participated in the study .
participants included 304 second year students from the classes of 2009 , 2010 , and 2011 , and 264 fourth year students from the classes of 2008 , 2009 , and 2010 .
the class of 2009 and 2010 took the examination twice , once as second year and once as fourth year students .
five hundred and sixty - eight medical students from the school of medicine and dentistry participated in the study .
participants included 304 second year students from the classes of 2009 , 2010 , and 2011 , and 264 fourth year students from the classes of 2008 , 2009 , and 2010 .
the class of 2009 and 2010 took the examination twice , once as second year and once as fourth year students .
between may 2007 and february 2010 , participants were administered the national board of medical examiners ( nbme ) msk subject examination at the end of their second and fourth years of medical school . the nbme msk assessment tool is a web - based multiple - choice examination developed in 2006 in conjunction with the bone and joint decade .
test materials were drawn from secure nbme item pools , reviewed , and approved by an external volunteer interdisciplinary msk task force composed of basic scientists and clinicians .
the msk subject examination is similar in format to the nbme subject examinations available in core clinical science disciplines , including medicine and pediatrics .
subject examinations are intended to complement other sources of information about the educational progression of medical students , and they are primarily designed for use as final examination after courses and clerkships .
the nmbe msk subject examination comprises 75 items , with 14 basic science and 61 clinical items plus a brief tutorial ; examinees are allotted 1 hour 55 min for completion .
the kr20 reliability of the examination in the initial reference group of senior students from three participating schools was 0.86 ; reliabilities greater than 0.85 are considered acceptable for low stakes examinations .
mean scores and sd correspond closely to performance on other nbme discipline based shelf examinations .
the school of medicine and dentistry participated in pilot testing and validation of the examination .
the examination was administered to students during a translational science course which included msk topics but did not review prior msk coursework . the exam was required but students who missed the exam due to illness or out of town obligations did not have to make up the examination .
being a part of curricular evaluation , the score was not part of a course grade and there was no special preparation .
low stakes examination , yet provided a unique opportunity for self - assessment of msk medicine knowledge . participants clinical confidence in msk medicine and internal medicine /
pediatrics was measured simultaneously using a 10-item self - assessment tool to quantitate self - reported confidence in evaluating and treating select clinical scenarios .
this clinical confidence tool is similar to those used in prior msk curricular evaluation studies and is not validated ( 9 , 10 ) .
there were two questions that asked students to rate their confidence in evaluating and treating common msk disorders .
students were then asked to rate their confidence in evaluating four msk clinical scenarios and four internal medicine / pediatrics scenarios .
the msk clinical scenarios were ankle pain , knee pain , shoulder pain , and wrist pain . the internal medicine / pediatrics scenarios were chest pain , delirium , dyspnea , and febrile infant .
the mean msk medicine and internal medicine / pediatrics clinical confidence scores were computed for each participant in the second and fourth years of medical school .
between may 2007 and february 2010 , participants were administered the national board of medical examiners ( nbme ) msk subject examination at the end of their second and fourth years of medical school . the nbme msk assessment tool is a web - based multiple - choice examination developed in 2006 in conjunction with the bone and joint decade .
test materials were drawn from secure nbme item pools , reviewed , and approved by an external volunteer interdisciplinary msk task force composed of basic scientists and clinicians .
the msk subject examination is similar in format to the nbme subject examinations available in core clinical science disciplines , including medicine and pediatrics .
subject examinations are intended to complement other sources of information about the educational progression of medical students , and they are primarily designed for use as final examination after courses and clerkships .
the nmbe msk subject examination comprises 75 items , with 14 basic science and 61 clinical items plus a brief tutorial ; examinees are allotted 1 hour 55 min for completion .
the kr20 reliability of the examination in the initial reference group of senior students from three participating schools was 0.86 ; reliabilities greater than 0.85 are considered acceptable for low stakes examinations .
mean scores and sd correspond closely to performance on other nbme discipline based shelf examinations .
the school of medicine and dentistry participated in pilot testing and validation of the examination .
the examination was administered to students during a translational science course which included msk topics but did not review prior msk coursework . the exam was required but students who missed the exam due to illness or out of town obligations did not have to make up the examination .
being a part of curricular evaluation , the score was not part of a course grade and there was no special preparation .
low stakes examination , yet provided a unique opportunity for self - assessment of msk medicine knowledge .
participants clinical confidence in msk medicine and internal medicine / pediatrics was measured simultaneously using a 10-item self - assessment tool to quantitate self - reported confidence in evaluating and treating select clinical scenarios .
this clinical confidence tool is similar to those used in prior msk curricular evaluation studies and is not validated ( 9 , 10 ) .
there were two questions that asked students to rate their confidence in evaluating and treating common msk disorders .
students were then asked to rate their confidence in evaluating four msk clinical scenarios and four internal medicine / pediatrics scenarios .
the msk clinical scenarios were ankle pain , knee pain , shoulder pain , and wrist pain . the internal medicine / pediatrics scenarios were chest pain , delirium , dyspnea , and febrile infant .
the mean msk medicine and internal medicine / pediatrics clinical confidence scores were computed for each participant in the second and fourth years of medical school .
we then performed one - way analysis of variance ( anova ) to assess for overall differences in mean knowledge and confidence scores between classes .
analyses were conducted for both the second ( 20092011 ) and fourth ( 20082010 ) year classes .
we then excluded all paired data ( dependent scores ) and performed an independent samples t - test to compare second and fourth year scores . to compare participants second and fourth year knowledge and confidence scores over time , we performed paired sample t - tests on dependent scores for the classes of 2009 and 2010 .
paired analyses were also conducted to compare knowledge and confidence scores within second and fourth year classes .
relationships between msk knowledge and msk and internal medicine / pediatrics clinical confidence were then assessed by pearson correlations . in reporting results ,
analyses were performed using sas version 9.1 ( sas inc . , cary , nc , usa ) .
the research subjects review board at the university of rochester school of medicine and dentistry approved this curricular evaluation study .
a total of 568 medical students completed the nbme msk subject examination between may 2007 and february 2010 .
overall participation rate was 98% , with table 1 presenting the details of participation by class year and by training level .
participation rates in nbme musculoskeletal ( msk ) knowledge assessment by class note : a total of 304 second year medical students and 264 fourth year medical students completed the nbme msk knowledge assessment .
the mean score for the nbme msk subject examination for all second year medical students was 59.210.6 .
the mean score for the nbme msk subject examination for all fourth year medical students was 69.79.6 . to compare second year and fourth year medical students
, we removed all paired data ( dependent scores ) and found a statistically significant increase in nbme scores ( p<0.0001 . )
table 2 shows nbme msk knowledge assessment scores for each class . there was a statistically significant difference between year 2 and year 4 scores for all classes .
nbme musculoskeletal knowledge assessment scores [ mean ( sd ) ] for years 2 and 4 by medical student class to compare second year and fourth year medical students , we removed all paired data ( dependent scores ) and found a statistically significant increase in nbme scores ( p<0.0001 . ) . for students who took the examination twice , longitudinal analyses over time showed statistically significant changes ( p<0.0001 ) . for students who took the examination twice , longitudinal analyses revealed statistically significant differences from year 2 to year 4 .
mean scores increased by 10.7 points ( p<0.0001 ) and 11.0 points ( p<0.0001 ) for the classes of 2009 and 2010 , respectively .
table 3 shows the mean clinical confidence scores ( 5-point likert scale ) for each group of medical students that took the test .
for all students , clinical confidence in internal medicine / pediatrics was higher than msk medicine .
clinical confidence for internal medicine / pediatrics improved between year 2 and year 4 , but clinical confidence for msk medicine was not significantly changed between year 2 and year 4 .
musculoskeletal ( msk ) and internal medicine / pediatrics clinical confidence scores ( mean ( sd ) ) for years 2 and 4 by medical student class in year 2 , a statistically significant difference ( p=0.001 ) exists between msk and internal medicine / pediatrics confidence scores . in year 4 , a statistically significant difference ( p=0.001 ) exists between msk and internal medicine / pediatrics confidence scores . from year 2 to year 4 , a statistically significant increase in internal medicine / pediatrics clinical confidence scores ( p= < 0.0001 ) .
by contrast , clinical confidence scores in msk medicine did not significantly differ ( p=0.41 ) .
the mean second year clinical confidence score for scenarios in internal medicine / pediatrics was 3.60.6 .
a statistically significant difference ( p=0.001 ) exists between the year 2 msk and internal medicine / pediatrics confidence scores ( table 3 ) . the mean fourth year internal medicine / pediatrics clinical confidence score was 4.20.6 .
again , a statistically significant difference ( p=0.001 ) exists between the year 4 msk and internal medicine / pediatrics confidence scores ( table 3 ) .
longitudinal analyses revealed a statistically significant increase in internal medicine / pediatrics clinical confidence scores from year 2 to year 4 ( p= < 0.0001 ) .
by contrast , clinical confidence scores in msk medicine did not significantly differ from year 2 to year 4 ( p=0.41 ) .
1 depicts the percentage of second and fourth year medical students reporting they were somewhat confident or
students at the second year level reported higher confidence in treating internal medicine / pediatrics core conditions than msk core conditions .
there was no correlation between nbme msk knowledge exam scores and clinical confidence scores for either internal medicine / pediatrics scenarios ( r
= 0.06 , p=0.34 ) or msk medicine scenarios for second year medical students ( r
= 0.10 , p=0.08 ) .
similarly , there was no correlation between nbme msk knowledge exam scores and clinical confidence scores for internal medicine / pediatrics scenarios for fourth year medical students ( r
= 0.08 , p=0.2 ) .
there was a weak , positive correlation between nbme msk assessment scores and clinical confidence scores for msk medicine scenarios for fourth year medical students ( r
= 0.13 , p=0.04 ) .
a total of 568 medical students completed the nbme msk subject examination between may 2007 and february 2010 .
overall participation rate was 98% , with table 1 presenting the details of participation by class year and by training level .
participation rates in nbme musculoskeletal ( msk ) knowledge assessment by class note : a total of 304 second year medical students and 264 fourth year medical students completed the nbme msk knowledge assessment .
the mean score for the nbme msk subject examination for all second year medical students was 59.210.6 .
the mean score for the nbme msk subject examination for all fourth year medical students was 69.79.6 . to compare second year and fourth year medical students , we removed all paired data ( dependent scores ) and found a statistically significant increase in nbme scores ( p<0.0001 . )
table 2 shows nbme msk knowledge assessment scores for each class . there was a statistically significant difference between year 2 and year 4 scores for all classes .
nbme musculoskeletal knowledge assessment scores [ mean ( sd ) ] for years 2 and 4 by medical student class to compare second year and fourth year medical students , we removed all paired data ( dependent scores ) and found a statistically significant increase in nbme scores ( p<0.0001 . ) . for students who took the examination twice , longitudinal analyses over time showed statistically significant changes ( p<0.0001 ) . for students who took the examination twice , longitudinal analyses revealed statistically significant differences from year 2 to year 4 .
mean scores increased by 10.7 points ( p<0.0001 ) and 11.0 points ( p<0.0001 ) for the classes of 2009 and 2010 , respectively .
table 3 shows the mean clinical confidence scores ( 5-point likert scale ) for each group of medical students that took the test . for all students , clinical confidence in internal medicine /
clinical confidence for internal medicine / pediatrics improved between year 2 and year 4 , but clinical confidence for msk medicine was not significantly changed between year 2 and year 4 .
musculoskeletal ( msk ) and internal medicine / pediatrics clinical confidence scores ( mean ( sd ) ) for years 2 and 4 by medical student class in year 2 , a statistically significant difference ( p=0.001 ) exists between msk and internal medicine / pediatrics confidence scores . in year 4 , a statistically significant difference ( p=0.001 ) exists between msk and internal medicine / pediatrics confidence scores . from year 2 to year 4 , a statistically significant increase in internal medicine / pediatrics clinical confidence scores ( p= < 0.0001 ) .
by contrast , clinical confidence scores in msk medicine did not significantly differ ( p=0.41 ) .
the mean second year clinical confidence score for scenarios in internal medicine / pediatrics was 3.60.6 .
a statistically significant difference ( p=0.001 ) exists between the year 2 msk and internal medicine / pediatrics confidence scores ( table 3 ) . the mean fourth year internal medicine / pediatrics clinical confidence score was 4.20.6 .
again , a statistically significant difference ( p=0.001 ) exists between the year 4 msk and internal medicine / pediatrics confidence scores ( table 3 ) .
longitudinal analyses revealed a statistically significant increase in internal medicine / pediatrics clinical confidence scores from year 2 to year 4 ( p= < 0.0001 ) . by contrast , clinical confidence scores in msk medicine did not significantly differ from year 2 to year 4 ( p=0.41 ) .
1 depicts the percentage of second and fourth year medical students reporting they were somewhat confident or
students at the second year level reported higher confidence in treating internal medicine / pediatrics core conditions than msk core conditions .
there was no correlation between nbme msk knowledge exam scores and clinical confidence scores for either internal medicine / pediatrics scenarios ( r
= 0.06 , p=0.34 ) or msk medicine scenarios for second year medical students ( r
= 0.10 , p=0.08 ) .
similarly , there was no correlation between nbme msk knowledge exam scores and clinical confidence scores for internal medicine / pediatrics scenarios for fourth year medical students ( r
= 0.08 , p=0.2 ) .
there was a weak , positive correlation between nbme msk assessment scores and clinical confidence scores for msk medicine scenarios for fourth year medical students ( r
= 0.13 , p=0.04 ) .
over the past decade , concerns have been raised by the aamc regarding the adequacy of msk training ( 5 ) .
recent studies have reported on deficiencies in msk knowledge ( 6 , 8 , 10 ) .
these studies have lead to concerns regarding the adequacy of msk training and the subsequent ability of future physicians to adequately care for the ever - increasing burden of msk disorders .
historically , the use of msk assessment tools in evaluating and documenting msk knowledge have been limited by availability , design , and application . in 1998 ,
an msk assessment tool was developed and reported on by freedman and bernstein ( 8) . this assessment tool was referred to as a basic competency examination in msk medicine and consisted of 25 short answer questions .
there was some criticism regarding the source of validation , and it was broadened and evaluated by internal medicine program directors ( 6 ) .
the msk basic competency examination represented a significant advancement in evaluation and has been employed in recent studies ( 911 ) .
nevertheless , its content outline , brevity , and item format raise questions about the validity and generalizability of results as measures of overall msk knowledge .
for these reasons , we used a simplified tool to quantitate confidence only and relied on the validated msk exam to assess knowledge . the standardized msk medical knowledge assessment method used in the current study
the exam blueprint was approved by an interdisciplinary task force of surgical and medical msk experts , and the overall examination pilot tested to establish performance metrics .
individual test items were drawn from secure validated nbme test libraries and evaluate knowledge of disease processes and therapeutics as well as diagnostic and clinical reasoning . when comparing the current study 's results with previous msk curricular reports , there are similarities as well as important differences ( 6 , 8 , 10 ) .
previous reports noted low levels of clinical confidence in evaluating msk disorders as well as low levels of msk knowledge among medical students . in the current study , low levels of clinical confidence in evaluating msk medicine disorders were similarly observed .
while clinical confidence for core medicine / pediatric conditions was either high at second year or increased to a higher level by fourth year , msk medicine confidence remained much lower in both the second and fourth years .
these findings of low clinical confidence in msk medicine are similar to reports by day et al .
it should be noted that these authors reported on a sampling of students throughout training and did not specifically evaluate pre - clinical versus clinical time periods .
initially low levels of msk knowledge were observed during the second year , but by the fourth year a statistically significant increase in msk knowledge was noted . in our cohort ,
clinical experience resulted in an increase in msk knowledge as measured by the nbme msk subject exam .
in fact , by the end of the fourth year , the exam results noted a mean of 69.7% correct , and this number approaches the anticipated or targeted mean score of 70% correct for established core nbme subject exams and the usmle step exams .
the current study is the first to specifically evaluate pre - clinical ( second year ) versus end of training post - clinical ( fourth year ) performance and to observe a high level of msk knowledge among graduating medical students .
given the frequency of msk disorders encountered in general medical practice ( 1 , 2 ) , the current study 's finding that msk clinical self - confidence as determined by our tool does not change with an increase in msk knowledge among graduating medical students is concerning . while we acknowledge that measures of confidence should not be interpreted as determinants of competence , we are concerned that medical students who graduate with a lower level of msk clinical confidence may either refer msk problems to subspecialists prematurely or order unnecessary or expensive diagnostic .
these possibilities could contribute to the escalation of health care costs , both directly and indirectly .
first , the nbme msk subject examination used to measure an increase in knowledge is new , and reliability information is based on a relatively small cohort of students at a few institutions .
additionally , since only one form of the nbme msk subject examination currently exists , students in the classes of 2009 and 2010 took the same examination in their second and fourth years of medical school .
, students were not informed that they would be taking the same exam again in their final year .
to evaluate this potential bias further , we stratified the data and removed all paired data ( dependent scores ) or those students who take the exam twice ( table 2 ) . when excluding paired data ( dependant scores ) , a statistically significant difference ( p<0.0001 ) was noted for unpaired year 2 ( 58.2 sd 10.4 ) and year 4 ( 69.3 sd 9.6 ) students .
another limitation is that our confidence self - assessment tool is not validated although as indicated , it was based on a previously published tool ( 9 , 10 ) .
however , because our students completed this self - assessment tool for all clinical scenarios at the same time , we believe that comparisons of the scores for msk scenarios with internal medicine / pediatrics scenarios is appropriate .
a review of the literature suggests that this is the first study to report a high level of msk medicine knowledge among graduating medical students .
the study introduces the nbme msk subject examination as a valid and reliable interdisciplinary external multiple - choice msk assessment tool .
large numbers of participants and high participation rates for both second and fourth year students strengthen the findings of the study .
this minimizes bias that can be seen in curriculum evaluation studies where students who are either pleased or dissatisfied with the curriculum participate more frequently . despite increased levels of msk knowledge , low levels of msk clinical confidence
were noted in our study and this is similar to the low levels reported in other msk assessment studies ( 2 , 8 , 10 ) .
higher levels of clinical confidence in core internal medicine / pediatrics may be due , in part , to a larger volume of encounters in internal medicine and pediatric conditions with reinforcement of appropriate evaluation of these conditions during required clerkships during the third and fourth years . a number of studies have reported a disparity between the curricular time in medical school and the prevalence of msk disorders ( 2 , 13 , 14 ) .
similar to the majority of medical schools , the school of medicine and dentistry requires exposure to msk medicine and disorders only during years 1 and 2 .
no msk courses or electives are required in years 3 and 4 , and it is unclear what aspects of the clinical years are influencing and contributing to the increase in msk knowledge .
further research should help to determine the factors that lead to increased msk knowledge in the clinical years and identify what increases or decreases msk clinical confidence .
it would be interesting to evaluate whether clinical skills in msk medicine , specifically physical examination of the extremities and spine , paralleled changes in either knowledge or confidence .
the authors have not received any funding or benefits from industry or elsewhere to conduct this study . | purposedespite the prevalence of musculoskeletal ( msk ) disorders , the degree to which medical schools are providing students the knowledge and confidence to treat these problems is unclear .
this study evaluated msk knowledge in second and fourth year medical students using a newly developed written assessment tool and examined the maturation of clinical confidence in treating core msk disorders.methodsover a 3-year period , the national board of medical examiners ( nbme ) msk subject examination consisting of 75 items was administered to 568 second and fourth year students at a single institution .
students were also asked to rate their confidence in treating a selection of medicine / pediatric and msk clinical scenarios on a 5-point likert scale.resultsparticipation rate was 98% .
the nbme msk assessment score was 59.210.6 for all second year medical students and 69.79.6 for all fourth year medical students .
there was a significant increase in nbme scores between the second and fourth years ( p<0.0001 ) .
students were more confident in treating internal medicine / pediatric conditions than msk medicine conditions ( p=0.001 ) .
confidence in treating msk medicine conditions did not improve between the second and fourth years ( p=0.41).conclusionsto our knowledge , this is the first study to report increased msk medicine knowledge as measured by a standardized examination after completing medical school core clinical rotations . despite increased msk knowledge , low levels of msk clinical confidence among graduating students were noted .
further research is needed to determine the factors that influence msk knowledge and clinical confidence in medical students . | Methods
MSK instruction
Medical student groups
Measures
MSK knowledge
Clinical confidence
Statistical analysis
Results
Student participation
MSK knowledge
Clinical confidence
Correlation between knowledge and confidence
Discussion
Conflict of interest and funding | in year 2 , the basic science of msk medicine and rheumatology is covered in over 36 hours . the school of medicine and dentistry is one of many schools that do not require msk medicine instruction during the clinically heavy final 2 years of the medical school curriculum . participants included 304 second year students from the classes of 2009 , 2010 , and 2011 , and 264 fourth year students from the classes of 2008 , 2009 , and 2010 . the class of 2009 and 2010 took the examination twice , once as second year and once as fourth year students . between may 2007 and february 2010
, participants were administered the national board of medical examiners ( nbme ) msk subject examination at the end of their second and fourth years of medical school . the nbme msk assessment tool is a web - based multiple - choice examination developed in 2006 in conjunction with the bone and joint decade . the msk subject examination is similar in format to the nbme subject examinations available in core clinical science disciplines , including medicine and pediatrics . subject examinations are intended to complement other sources of information about the educational progression of medical students , and they are primarily designed for use as final examination after courses and clerkships . the nmbe msk subject examination comprises 75 items , with 14 basic science and 61 clinical items plus a brief tutorial ; examinees are allotted 1 hour 55 min for completion . low stakes examination , yet provided a unique opportunity for self - assessment of msk medicine knowledge . participants clinical confidence in msk medicine and internal medicine / pediatrics was measured simultaneously using a 10-item self - assessment tool to quantitate self - reported confidence in evaluating and treating select clinical scenarios . there were two questions that asked students to rate their confidence in evaluating and treating common msk disorders . students were then asked to rate their confidence in evaluating four msk clinical scenarios and four internal medicine / pediatrics scenarios . the mean msk medicine and internal medicine / pediatrics clinical confidence scores were computed for each participant in the second and fourth years of medical school . we then performed one - way analysis of variance ( anova ) to assess for overall differences in mean knowledge and confidence scores between classes . we then excluded all paired data ( dependent scores ) and performed an independent samples t - test to compare second and fourth year scores . to compare participants second and fourth year knowledge and confidence scores over time , we performed paired sample t - tests on dependent scores for the classes of 2009 and 2010 . paired analyses were also conducted to compare knowledge and confidence scores within second and fourth year classes . relationships between msk knowledge and msk and internal medicine / pediatrics clinical confidence were then assessed by pearson correlations . in year 2 , the basic science of msk medicine and rheumatology is covered in over 36 hours . the school of medicine and dentistry is one of many schools that do not require msk medicine instruction during the clinically heavy final 2 years of the medical school curriculum . participants included 304 second year students from the classes of 2009 , 2010 , and 2011 , and 264 fourth year students from the classes of 2008 , 2009 , and 2010 . the class of 2009 and 2010 took the examination twice , once as second year and once as fourth year students . participants included 304 second year students from the classes of 2009 , 2010 , and 2011 , and 264 fourth year students from the classes of 2008 , 2009 , and 2010 . between may 2007 and february 2010 , participants were administered the national board of medical examiners ( nbme ) msk subject examination at the end of their second and fourth years of medical school . the nbme msk assessment tool is a web - based multiple - choice examination developed in 2006 in conjunction with the bone and joint decade . the msk subject examination is similar in format to the nbme subject examinations available in core clinical science disciplines , including medicine and pediatrics . subject examinations are intended to complement other sources of information about the educational progression of medical students , and they are primarily designed for use as final examination after courses and clerkships . the nmbe msk subject examination comprises 75 items , with 14 basic science and 61 clinical items plus a brief tutorial ; examinees are allotted 1 hour 55 min for completion . the examination was administered to students during a translational science course which included msk topics but did not review prior msk coursework . being a part of curricular evaluation , the score was not part of a course grade and there was no special preparation . low stakes examination , yet provided a unique opportunity for self - assessment of msk medicine knowledge . participants clinical confidence in msk medicine and internal medicine /
pediatrics was measured simultaneously using a 10-item self - assessment tool to quantitate self - reported confidence in evaluating and treating select clinical scenarios . there were two questions that asked students to rate their confidence in evaluating and treating common msk disorders . students were then asked to rate their confidence in evaluating four msk clinical scenarios and four internal medicine / pediatrics scenarios . the mean msk medicine and internal medicine / pediatrics clinical confidence scores were computed for each participant in the second and fourth years of medical school . between may 2007 and february 2010 , participants were administered the national board of medical examiners ( nbme ) msk subject examination at the end of their second and fourth years of medical school . the nbme msk assessment tool is a web - based multiple - choice examination developed in 2006 in conjunction with the bone and joint decade . the msk subject examination is similar in format to the nbme subject examinations available in core clinical science disciplines , including medicine and pediatrics . subject examinations are intended to complement other sources of information about the educational progression of medical students , and they are primarily designed for use as final examination after courses and clerkships . the nmbe msk subject examination comprises 75 items , with 14 basic science and 61 clinical items plus a brief tutorial ; examinees are allotted 1 hour 55 min for completion . the examination was administered to students during a translational science course which included msk topics but did not review prior msk coursework . being a part of curricular evaluation , the score was not part of a course grade and there was no special preparation . low stakes examination , yet provided a unique opportunity for self - assessment of msk medicine knowledge . participants clinical confidence in msk medicine and internal medicine / pediatrics was measured simultaneously using a 10-item self - assessment tool to quantitate self - reported confidence in evaluating and treating select clinical scenarios . there were two questions that asked students to rate their confidence in evaluating and treating common msk disorders . students were then asked to rate their confidence in evaluating four msk clinical scenarios and four internal medicine / pediatrics scenarios . the mean msk medicine and internal medicine / pediatrics clinical confidence scores were computed for each participant in the second and fourth years of medical school . we then performed one - way analysis of variance ( anova ) to assess for overall differences in mean knowledge and confidence scores between classes . we then excluded all paired data ( dependent scores ) and performed an independent samples t - test to compare second and fourth year scores . to compare participants second and fourth year knowledge and confidence scores over time , we performed paired sample t - tests on dependent scores for the classes of 2009 and 2010 . paired analyses were also conducted to compare knowledge and confidence scores within second and fourth year classes . relationships between msk knowledge and msk and internal medicine / pediatrics clinical confidence were then assessed by pearson correlations . a total of 568 medical students completed the nbme msk subject examination between may 2007 and february 2010 . participation rates in nbme musculoskeletal ( msk ) knowledge assessment by class note : a total of 304 second year medical students and 264 fourth year medical students completed the nbme msk knowledge assessment . the mean score for the nbme msk subject examination for all second year medical students was 59.210.6 . the mean score for the nbme msk subject examination for all fourth year medical students was 69.79.6 . to compare second year and fourth year medical students
, we removed all paired data ( dependent scores ) and found a statistically significant increase in nbme scores ( p<0.0001 . ) there was a statistically significant difference between year 2 and year 4 scores for all classes . nbme musculoskeletal knowledge assessment scores [ mean ( sd ) ] for years 2 and 4 by medical student class to compare second year and fourth year medical students , we removed all paired data ( dependent scores ) and found a statistically significant increase in nbme scores ( p<0.0001 . ) for students who took the examination twice , longitudinal analyses over time showed statistically significant changes ( p<0.0001 ) . mean scores increased by 10.7 points ( p<0.0001 ) and 11.0 points ( p<0.0001 ) for the classes of 2009 and 2010 , respectively . table 3 shows the mean clinical confidence scores ( 5-point likert scale ) for each group of medical students that took the test . for all students , clinical confidence in internal medicine / pediatrics was higher than msk medicine . clinical confidence for internal medicine / pediatrics improved between year 2 and year 4 , but clinical confidence for msk medicine was not significantly changed between year 2 and year 4 . musculoskeletal ( msk ) and internal medicine / pediatrics clinical confidence scores ( mean ( sd ) ) for years 2 and 4 by medical student class in year 2 , a statistically significant difference ( p=0.001 ) exists between msk and internal medicine / pediatrics confidence scores . in year 4 , a statistically significant difference ( p=0.001 ) exists between msk and internal medicine / pediatrics confidence scores . from year 2 to year 4 , a statistically significant increase in internal medicine / pediatrics clinical confidence scores ( p= < 0.0001 ) . by contrast , clinical confidence scores in msk medicine did not significantly differ ( p=0.41 ) . the mean second year clinical confidence score for scenarios in internal medicine / pediatrics was 3.60.6 . a statistically significant difference ( p=0.001 ) exists between the year 2 msk and internal medicine / pediatrics confidence scores ( table 3 ) . the mean fourth year internal medicine / pediatrics clinical confidence score was 4.20.6 . again , a statistically significant difference ( p=0.001 ) exists between the year 4 msk and internal medicine / pediatrics confidence scores ( table 3 ) . longitudinal analyses revealed a statistically significant increase in internal medicine / pediatrics clinical confidence scores from year 2 to year 4 ( p= < 0.0001 ) . by contrast , clinical confidence scores in msk medicine did not significantly differ from year 2 to year 4 ( p=0.41 ) . 1 depicts the percentage of second and fourth year medical students reporting they were somewhat confident or
students at the second year level reported higher confidence in treating internal medicine / pediatrics core conditions than msk core conditions . there was no correlation between nbme msk knowledge exam scores and clinical confidence scores for either internal medicine / pediatrics scenarios ( r
= 0.06 , p=0.34 ) or msk medicine scenarios for second year medical students ( r
= 0.10 , p=0.08 ) . similarly , there was no correlation between nbme msk knowledge exam scores and clinical confidence scores for internal medicine / pediatrics scenarios for fourth year medical students ( r
= 0.08 , p=0.2 ) . there was a weak , positive correlation between nbme msk assessment scores and clinical confidence scores for msk medicine scenarios for fourth year medical students ( r
= 0.13 , p=0.04 ) . a total of 568 medical students completed the nbme msk subject examination between may 2007 and february 2010 . participation rates in nbme musculoskeletal ( msk ) knowledge assessment by class note : a total of 304 second year medical students and 264 fourth year medical students completed the nbme msk knowledge assessment . the mean score for the nbme msk subject examination for all second year medical students was 59.210.6 . the mean score for the nbme msk subject examination for all fourth year medical students was 69.79.6 . to compare second year and fourth year medical students , we removed all paired data ( dependent scores ) and found a statistically significant increase in nbme scores ( p<0.0001 . ) there was a statistically significant difference between year 2 and year 4 scores for all classes . nbme musculoskeletal knowledge assessment scores [ mean ( sd ) ] for years 2 and 4 by medical student class to compare second year and fourth year medical students , we removed all paired data ( dependent scores ) and found a statistically significant increase in nbme scores ( p<0.0001 . ) for students who took the examination twice , longitudinal analyses over time showed statistically significant changes ( p<0.0001 ) . mean scores increased by 10.7 points ( p<0.0001 ) and 11.0 points ( p<0.0001 ) for the classes of 2009 and 2010 , respectively . table 3 shows the mean clinical confidence scores ( 5-point likert scale ) for each group of medical students that took the test . for all students , clinical confidence in internal medicine /
clinical confidence for internal medicine / pediatrics improved between year 2 and year 4 , but clinical confidence for msk medicine was not significantly changed between year 2 and year 4 . musculoskeletal ( msk ) and internal medicine / pediatrics clinical confidence scores ( mean ( sd ) ) for years 2 and 4 by medical student class in year 2 , a statistically significant difference ( p=0.001 ) exists between msk and internal medicine / pediatrics confidence scores . in year 4 , a statistically significant difference ( p=0.001 ) exists between msk and internal medicine / pediatrics confidence scores . from year 2 to year 4 , a statistically significant increase in internal medicine / pediatrics clinical confidence scores ( p= < 0.0001 ) . by contrast , clinical confidence scores in msk medicine did not significantly differ ( p=0.41 ) . the mean second year clinical confidence score for scenarios in internal medicine / pediatrics was 3.60.6 . a statistically significant difference ( p=0.001 ) exists between the year 2 msk and internal medicine / pediatrics confidence scores ( table 3 ) . the mean fourth year internal medicine / pediatrics clinical confidence score was 4.20.6 . again , a statistically significant difference ( p=0.001 ) exists between the year 4 msk and internal medicine / pediatrics confidence scores ( table 3 ) . longitudinal analyses revealed a statistically significant increase in internal medicine / pediatrics clinical confidence scores from year 2 to year 4 ( p= < 0.0001 ) . by contrast , clinical confidence scores in msk medicine did not significantly differ from year 2 to year 4 ( p=0.41 ) . 1 depicts the percentage of second and fourth year medical students reporting they were somewhat confident or
students at the second year level reported higher confidence in treating internal medicine / pediatrics core conditions than msk core conditions . there was no correlation between nbme msk knowledge exam scores and clinical confidence scores for either internal medicine / pediatrics scenarios ( r
= 0.06 , p=0.34 ) or msk medicine scenarios for second year medical students ( r
= 0.10 , p=0.08 ) . similarly , there was no correlation between nbme msk knowledge exam scores and clinical confidence scores for internal medicine / pediatrics scenarios for fourth year medical students ( r
= 0.08 , p=0.2 ) . there was a weak , positive correlation between nbme msk assessment scores and clinical confidence scores for msk medicine scenarios for fourth year medical students ( r
= 0.13 , p=0.04 ) . historically , the use of msk assessment tools in evaluating and documenting msk knowledge have been limited by availability , design , and application . previous reports noted low levels of clinical confidence in evaluating msk disorders as well as low levels of msk knowledge among medical students . in the current study , low levels of clinical confidence in evaluating msk medicine disorders were similarly observed . while clinical confidence for core medicine / pediatric conditions was either high at second year or increased to a higher level by fourth year , msk medicine confidence remained much lower in both the second and fourth years . these findings of low clinical confidence in msk medicine are similar to reports by day et al . initially low levels of msk knowledge were observed during the second year , but by the fourth year a statistically significant increase in msk knowledge was noted . in our cohort ,
clinical experience resulted in an increase in msk knowledge as measured by the nbme msk subject exam . the current study is the first to specifically evaluate pre - clinical ( second year ) versus end of training post - clinical ( fourth year ) performance and to observe a high level of msk knowledge among graduating medical students . given the frequency of msk disorders encountered in general medical practice ( 1 , 2 ) , the current study 's finding that msk clinical self - confidence as determined by our tool does not change with an increase in msk knowledge among graduating medical students is concerning . while we acknowledge that measures of confidence should not be interpreted as determinants of competence , we are concerned that medical students who graduate with a lower level of msk clinical confidence may either refer msk problems to subspecialists prematurely or order unnecessary or expensive diagnostic . first , the nbme msk subject examination used to measure an increase in knowledge is new , and reliability information is based on a relatively small cohort of students at a few institutions . additionally , since only one form of the nbme msk subject examination currently exists , students in the classes of 2009 and 2010 took the same examination in their second and fourth years of medical school . when excluding paired data ( dependant scores ) , a statistically significant difference ( p<0.0001 ) was noted for unpaired year 2 ( 58.2 sd 10.4 ) and year 4 ( 69.3 sd 9.6 ) students . however , because our students completed this self - assessment tool for all clinical scenarios at the same time , we believe that comparisons of the scores for msk scenarios with internal medicine / pediatrics scenarios is appropriate . a review of the literature suggests that this is the first study to report a high level of msk medicine knowledge among graduating medical students . the study introduces the nbme msk subject examination as a valid and reliable interdisciplinary external multiple - choice msk assessment tool . large numbers of participants and high participation rates for both second and fourth year students strengthen the findings of the study . despite increased levels of msk knowledge , low levels of msk clinical confidence
were noted in our study and this is similar to the low levels reported in other msk assessment studies ( 2 , 8 , 10 ) . higher levels of clinical confidence in core internal medicine / pediatrics may be due , in part , to a larger volume of encounters in internal medicine and pediatric conditions with reinforcement of appropriate evaluation of these conditions during required clerkships during the third and fourth years . a number of studies have reported a disparity between the curricular time in medical school and the prevalence of msk disorders ( 2 , 13 , 14 ) . similar to the majority of medical schools , the school of medicine and dentistry requires exposure to msk medicine and disorders only during years 1 and 2 . no msk courses or electives are required in years 3 and 4 , and it is unclear what aspects of the clinical years are influencing and contributing to the increase in msk knowledge . further research should help to determine the factors that lead to increased msk knowledge in the clinical years and identify what increases or decreases msk clinical confidence . | [
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because of the similarity in fdx structure to the structures of lpr [ 1 , 2 ] , it was expected that they would share the same target .
we found that fdx inhibited both rnap isolated from c. difficile by the method of pich and bahl and rnap from escherichia coli in a radiolabeled uridine triphosphate incorporation assay .
although the e. coli rnap enzyme is less sensitive than the c. difficile rnap , both enzymes are effectively inhibited by fdx .
mechanistic work was performed with the enzyme from e. coli , because this organism is more tractable for mutagenesis and recombinant protein purification . during the initiation phase of the transcription cycle ( figure 1a ) ,
the core rnap ( a complex of an -dimer , , , and subunits ) combines with a promoter - specificity factor to locate and bind to a promoter region of the dna , then separates , or melts , the double - stranded dna to form an open promoter complex ( rpo ) in which the melted region ( transcription bubble ) extends from positions 12 to + 2 ( relative to the transcription start site ) .
the incoming nucleotide ( nucleoside triphosphate ) substrate pairs with the template dna in the enzyme 's active site ( + 1 ) , and rna synthesis commences . when rnap adds 1 nucleotide to a growing rna chain , it moves 1 step forward on the dna template , repeating this cycle hundreds or thousands of times during the elongation phase until it reaches a terminator .
figure 1.a , transcription initiation pathway . core rna polymerase ( rnap ; gray oval , with active site depicted as a circle ) binds to a promoter specificity initiation factor ( 1 of many ) that directs the resulting holo rnap to a subset of promoters . in escherichia coli ,
the primary factor consists of separate domains ( numbered 1 4 ) ; domains 2 and 4 recognize the 10 and 35 elements of housekeeping
promoters to form a closed promoter complex , rpc , in which the double - stranded dna is loosely bound on the surface of rnap . in rpi1 , the first intermediate along the pathway , dna strand separation initiates around the 11 position ( relative to the transcription start site ) . melting propagates toward the active site ( as shown in rpi2 , but additional complexes also may exist ) . in the final ,
transcription - competent open promoter complex ( rpo ; boxed ) , the transcription bubble encompasses the + 1 position .
fidaxomicin ( fdx ; at 50 m ) was added to the in vitro transcription reaction with the e. coli rnap at different points .
the fraction of synthesized rna was measured ( as percentage of transcription in the absence of the antibiotic ) .
inhibition of the reaction was observed when fdx was added before steps 1 and 2 , but not 3 or 4 , after formation of the rpo .
abbreviations : [ p]gtp , -radiolabeled gtp ; apu , adenylyl ( 3-5 ) uridine ; apupcpg , tetranucleotide reaction product ; ctp , cytidine triphosphate ; fdx , fidaxomicin ; gtp , guanidine triphosphate ; rpo , open complex stabilized by the addition of a dinucleotide primer apu . a , transcription initiation pathway . core rna polymerase ( rnap ; gray oval , with active site depicted as a circle ) binds to a promoter specificity initiation factor ( 1 of many ) that directs the resulting holo rnap to a subset of promoters . in escherichia coli ,
the primary factor consists of separate domains ( numbered 1 4 ) ; domains 2 and 4 recognize the 10 and 35 elements of housekeeping promoters to form a closed promoter complex , rpc , in which the double - stranded dna is loosely bound on the surface of rnap . in rpi1 , the first intermediate along the pathway , dna strand separation initiates around the 11 position ( relative to the transcription start site ) . melting propagates toward the active site ( as shown in rpi2 , but additional complexes also may exist ) . in the final ,
transcription - competent open promoter complex ( rpo ; boxed ) , the transcription bubble encompasses the + 1 position .
fidaxomicin ( fdx ; at 50 m ) was added to the in vitro transcription reaction with the e. coli rnap at different points .
the fraction of synthesized rna was measured ( as percentage of transcription in the absence of the antibiotic ) .
inhibition of the reaction was observed when fdx was added before steps 1 and 2 , but not 3 or 4 , after formation of the rpo .
abbreviations : [ p]gtp , -radiolabeled gtp ; apu , adenylyl ( 3-5 ) uridine ; apupcpg , tetranucleotide reaction product ; ctp , cytidine triphosphate ; fdx , fidaxomicin ; gtp , guanidine triphosphate ; rpo , open complex stabilized by the addition of a dinucleotide primer apu .
order - of - addition experiments are commonly used to pinpoint a step along the pathway at which an antibiotic acts . in these assays ,
the antibiotic is added to a free rnap or to transcription complexes poised at different checkpoints . when the enzyme bypasses the step sensitive to the inhibitor , it becomes resistant to its action .
antibiotics that inhibit rna chain elongation block transcription when added at any step during transcription ; this group includes streptolydigin [ 14 , 15 ] , tagetitoxin , microcin j25 [ 17 , 18 ] , and cbrs ( a group of synthetic rna polymerase inhibitors ) , which are rifamycin - quinolone hybrids . rifampicin and sorangicin block extension of short transcripts and are no longer able to act when the nascent rna grows longer than 4 nt .
lpr and myxopyronins ( myxs ) , such as desmethylmyxopyronin ( dmyx ) [ 22 , 23 ] , inhibit rnap only if added before formation of the rpo .
we found that fdx also inhibits transcription only if added before the stable rpo has been formed ( figure 1b ) .
importantly , a common point of action does not prove the same mechanism . during both initiation and elongation ,
transcription complexes exist in many interconverting states that are differentially sensitive to both cellular regulators and antibiotics .
these states have been particularly well characterized in the course of initiation when several promoter complexes form sequentially , culminating in rpo .
in addition to the similar behavior of fdx , lpr , and dmyx in order - of - addition experiments , the patterns of mutations that confer resistance to fdx , lpr , and dmyx overlap , suggesting a similar site of binding / action on the rnap .
fdx and lpr do not inhibit thermus rnaps , the only bacterial enzymes for which detailed structural information currently is available .
however , structures of dmyx in complex with thermus thermophilus rnap revealed that the antibiotic binds to and drastically alters the conformation of a subunit element called switch-2 .
first , the switch-2 forms a hinge that connects the 2 pincers of the rnap crab - claw - like clamp ; opening of these pincers is thought to be required to load the dna into the rnap during initiation .
second , the switch-2 interacts with the template dna strand and determines its position .
, dmyx restricts movements of the switch-2 , blocking the clamp opening . in another , dmyx - stabilized refolding of the switch-2 sterically blocks the path of the template dna strand near the rnap active site .
analysis of dmyx - resistant mutants in vivo and in vitro validated the antibiotic - binding site [ 22 , 23 ] . footprinting
analysis revealed that dmyx does not prevent dna binding to e. coli rnap or nucleation of dna melting by the subunit .
however , dmyx blocks the propagation of dna melting toward the active site , stabilizing a partially melted intermediate , the existence of which has been postulated for many years . on the basis of the complex structure , we designed substitutions in the switch-2 that mimicked the dmyx - bound conformation even in the absence of the antibiotic ; these mutant enzymes were hypersensitive to dmyx and were locked in the same inactive promoter complex intermediate . of interest , dksa , a regulatory protein in e. coli ,
inhibits transcription through similar changes in rnap / dna interactions , and substitutions in the switch-2 alter the enzyme 's response to dksa .
switch-2 thus is a popular target for rnap inhibitors , and lpr - resistant substitutions therein initially suggested that lpr ( and based on structural similarities , also fdx ) may act similarly to dmyx .
for example , the same substitution , r337a , confers resistance both to antibiotics [ 21 , 22 ] and also to fdx .
however , a study by tupin et al revealed important differences between lpr and dmyx .
first , tupin et al demonstrated that lpr acts at a step preceding the one targeted by dmyx . in the presence of lpr , rnap bound to the promoter dna to form
second , this study suggested a key role for the subunit in lpr action : deletion of the hairpin loop that is positioned near the switch-2 conferred resistance to lpr .
thus , although the binding sites for lpr and dmyx may partially overlap , their mechanisms appear to be distinct .
in addition , it remained to be determined whether fdx acted similarly to lpr , particularly because these antibiotics target the highly dynamic switch-2 and their action could be strongly influenced by even subtle differences between the inhibitor scaffolds , the source of rnap , and the transcription complexes used for the analysis .
footprinting analyses were conducted with fdx to evaluate whether fdx behaved like lpr . similar to lpr and in contrast to dmyx , fdx inhibited dna melting in promoter complexes throughout the region surrounding the transcription start site ( figure 2 ) . however , our dnase i footprinting analysis revealed that fdx not only induced changes in the downstream dna interactions with the rnap ( as do dmyx and lpr ) , but also altered the dna conformation in the spacer region of the affected complexes ( unlike lpr ) .
also in contrast to lpr , fdx action was not altered by deletion of the hairpin loop .
one possibility is that they are attributable to the different promoters used for these analyses , pr in our work and placuv5 in tupin et al .
potassium permanganate ( kmno4 ) modifies single - stranded thymidine ( t ) residues , which subsequently are cleaved by piperidine ; positions of cleavage indicate the location of single - stranded dna regions .
a linear end - labeled pr promoter dna fragment was generated by polymerase chain reaction .
top , the 35 and 10 hexamers are indicated by black boxes , the start site ( + 1 ) is shown by a black dot .
reactivities of the 10 , 4 , 3 , and + 2 non - template strand t residues to kmno4 modification are summarized above the sequence ; black and white arrows indicate high and low reactivity , respectively .
bottom , a representative gel with the sensitive residues shown ; the middle portion of the gel was removed ( dashed line ) to conserve space .
results show that although myxopyronin blocks only dna melting near the start site ( at + 2 position ) , fdx reduces strand separation throughout the entire sensitive region with the wild - type rnap , but not with a mutant that carries an r337a substitution in the switch-2 region .
abbreviations : ab , antibiotic ; fdx , fidaxomicin ; myx , myxopyronin ; rnap , rna polymerase ; wt , wild - type .
potassium permanganate ( kmno4 ) modifies single - stranded thymidine ( t ) residues , which subsequently are cleaved by piperidine ; positions of cleavage indicate the location of single - stranded dna regions .
a linear end - labeled pr promoter dna fragment was generated by polymerase chain reaction .
top , the 35 and 10 hexamers are indicated by black boxes , the start site ( + 1 ) is shown by a black dot .
reactivities of the 10 , 4 , 3 , and + 2 non - template strand t residues to kmno4 modification are summarized above the sequence ; black and white arrows indicate high and low reactivity , respectively .
bottom , a representative gel with the sensitive residues shown ; the middle portion of the gel was removed ( dashed line ) to conserve space .
results show that although myxopyronin blocks only dna melting near the start site ( at + 2 position ) , fdx reduces strand separation throughout the entire sensitive region with the wild - type rnap , but not with a mutant that carries an r337a substitution in the switch-2 region .
abbreviations : ab , antibiotic ; fdx , fidaxomicin ; myx , myxopyronin ; rnap , rna polymerase ; wt , wild - type .
rnap presents a complex target for inhibition , and changes in the enzyme or antibiotic may confer substantial differences .
small changes in antibiotic structure may alter the contacts with the enzyme and consequently change the point for inhibition , as illustrated by the case of rifamycins .
substitutions in rnap ( naturally occurring in different bacterial enzymes ) bestow distinct kinetic properties and altered sensitivity to cellular effectors and antibiotics alike .
some of these changes impair the binding of a ligand to rnap , but others may act indirectly by altering the enzyme 's conformation and changing its response to the antibiotic .
for example , a subset of cbr - resistant mutations in rpoc conferred dependence on the inhibitor for growth of the mutant strains , indicating that their phenotypes can not be explained by the loss of cbr binding . thus , the phenotypes of any resistance mutations should be interpreted with caution because some of them could have indirect ( allosteric ) effects .
this may be particularly common for antibiotics that bind to the key sites on rnap , where substitutions would give rise to growth defects .
this complexity demands that when possible , structural analysis of the antibiotic / transcription complexes should be pursued in parallel with in - depth functional and genetic studies , using target rnaps from relevant bacterial species
. nevertheless , functional studies are useful for identifying differences in mechanism , particularly because they can provide information about the dynamics of enzyme inhibition rather than focusing on a static picture of binding .
the mechanistic studies presented here confirm that there are multiple steps that are targets for rnap inhibition by different antibiotics , and fdx inhibits a step distinct from elongation inhibitors , such as streptolydigin , and from the initiation inhibitors myx and the rifamycins .
the difference in mechanism compared with the rifamycins supports previous findings of lack of cross - resistance with this class of antibiotics and suggests that the rapid development of resistance observed with rifamycins does not indicate a rapid loss of susceptibility to fdx .
because of the similarity in fdx structure to the structures of lpr [ 1 , 2 ] , it was expected that they would share the same target .
we found that fdx inhibited both rnap isolated from c. difficile by the method of pich and bahl and rnap from escherichia coli in a radiolabeled uridine triphosphate incorporation assay .
although the e. coli rnap enzyme is less sensitive than the c. difficile rnap , both enzymes are effectively inhibited by fdx .
mechanistic work was performed with the enzyme from e. coli , because this organism is more tractable for mutagenesis and recombinant protein purification .
during the initiation phase of the transcription cycle ( figure 1a ) , the core rnap ( a complex of an -dimer , , , and subunits ) combines with a promoter - specificity factor to locate and bind to a promoter region of the dna , then separates , or melts , the double - stranded dna to form an open promoter complex ( rpo ) in which the melted region ( transcription bubble ) extends from positions 12 to + 2 ( relative to the transcription start site ) .
the incoming nucleotide ( nucleoside triphosphate ) substrate pairs with the template dna in the enzyme 's active site ( + 1 ) , and rna synthesis commences .
when rnap adds 1 nucleotide to a growing rna chain , it moves 1 step forward on the dna template , repeating this cycle hundreds or thousands of times during the elongation phase until it reaches a terminator .
figure 1.a , transcription initiation pathway . core rna polymerase ( rnap ; gray oval , with active site depicted as a circle ) binds to a promoter specificity initiation factor ( 1 of many ) that directs the resulting holo rnap to a subset of promoters . in escherichia coli ,
the primary factor consists of separate domains ( numbered 1 4 ) ; domains 2 and 4 recognize the 10 and 35 elements of housekeeping
promoters to form a closed promoter complex , rpc , in which the double - stranded dna is loosely bound on the surface of rnap . in rpi1 ,
the first intermediate along the pathway , dna strand separation initiates around the 11 position ( relative to the transcription start site ) . melting propagates toward the active site ( as shown in rpi2 , but additional complexes also may exist ) . in the final ,
transcription - competent open promoter complex ( rpo ; boxed ) , the transcription bubble encompasses the + 1 position .
. fidaxomicin ( fdx ; at 50 m ) was added to the in vitro transcription reaction with the e. coli rnap at different points .
the fraction of synthesized rna was measured ( as percentage of transcription in the absence of the antibiotic ) .
inhibition of the reaction was observed when fdx was added before steps 1 and 2 , but not 3 or 4 , after formation of the rpo .
abbreviations : [ p]gtp , -radiolabeled gtp ; apu , adenylyl ( 3-5 ) uridine ; apupcpg , tetranucleotide reaction product ; ctp , cytidine triphosphate ; fdx , fidaxomicin ; gtp , guanidine triphosphate ; rpo , open complex stabilized by the addition of a dinucleotide primer apu . a , transcription initiation pathway . core rna polymerase ( rnap ; gray oval , with active site depicted as a circle ) binds to a promoter specificity initiation factor ( 1 of many ) that directs the resulting holo rnap to a subset of promoters . in escherichia coli ,
the primary factor consists of separate domains ( numbered 1 4 ) ; domains 2 and 4 recognize the 10 and 35 elements of housekeeping
promoters to form a closed promoter complex , rpc , in which the double - stranded dna is loosely bound on the surface of rnap .
in rpi1 , the first intermediate along the pathway , dna strand separation initiates around the 11 position ( relative to the transcription start site ) . melting propagates toward the active site ( as shown in rpi2 , but additional complexes also may exist ) . in the final ,
transcription - competent open promoter complex ( rpo ; boxed ) , the transcription bubble encompasses the + 1 position .
fidaxomicin ( fdx ; at 50 m ) was added to the in vitro transcription reaction with the e. coli rnap at different points .
the fraction of synthesized rna was measured ( as percentage of transcription in the absence of the antibiotic ) .
inhibition of the reaction was observed when fdx was added before steps 1 and 2 , but not 3 or 4 , after formation of the rpo .
abbreviations : [ p]gtp , -radiolabeled gtp ; apu , adenylyl ( 3-5 ) uridine ; apupcpg , tetranucleotide reaction product ; ctp , cytidine triphosphate ; fdx , fidaxomicin ; gtp , guanidine triphosphate ; rpo , open complex stabilized by the addition of a dinucleotide primer apu .
order - of - addition experiments are commonly used to pinpoint a step along the pathway at which an antibiotic acts . in these assays ,
the antibiotic is added to a free rnap or to transcription complexes poised at different checkpoints .
when the enzyme bypasses the step sensitive to the inhibitor , it becomes resistant to its action .
antibiotics that inhibit rna chain elongation block transcription when added at any step during transcription ; this group includes streptolydigin [ 14 , 15 ] , tagetitoxin , microcin j25 [ 17 , 18 ] , and cbrs ( a group of synthetic rna polymerase inhibitors ) , which are rifamycin - quinolone hybrids . rifampicin and sorangicin block extension of short transcripts and are no longer able to act when the nascent rna grows longer than 4 nt .
lpr and myxopyronins ( myxs ) , such as desmethylmyxopyronin ( dmyx ) [ 22 , 23 ] , inhibit rnap only if added before formation of the rpo .
we found that fdx also inhibits transcription only if added before the stable rpo has been formed ( figure 1b ) .
importantly , a common point of action does not prove the same mechanism . during both initiation and elongation ,
transcription complexes exist in many interconverting states that are differentially sensitive to both cellular regulators and antibiotics .
these states have been particularly well characterized in the course of initiation when several promoter complexes form sequentially , culminating in rpo .
in addition to the similar behavior of fdx , lpr , and dmyx in order - of - addition experiments , the patterns of mutations that confer resistance to fdx , lpr , and dmyx overlap , suggesting a similar site of binding / action on the rnap . fdx and lpr
do not inhibit thermus rnaps , the only bacterial enzymes for which detailed structural information currently is available .
however , structures of dmyx in complex with thermus thermophilus rnap revealed that the antibiotic binds to and drastically alters the conformation of a subunit element called switch-2 .
first , the switch-2 forms a hinge that connects the 2 pincers of the rnap crab - claw - like clamp ; opening of these pincers is thought to be required to load the dna into the rnap during initiation .
second , the switch-2 interacts with the template dna strand and determines its position .
, dmyx restricts movements of the switch-2 , blocking the clamp opening . in another , dmyx - stabilized refolding of the switch-2 sterically blocks the path of the template dna strand near the rnap active site .
analysis of dmyx - resistant mutants in vivo and in vitro validated the antibiotic - binding site [ 22 , 23 ] .
footprinting analysis revealed that dmyx does not prevent dna binding to e. coli rnap or nucleation of dna melting by the subunit .
however , dmyx blocks the propagation of dna melting toward the active site , stabilizing a partially melted intermediate , the existence of which has been postulated for many years . on the basis of the complex structure , we designed substitutions in the switch-2 that mimicked the dmyx - bound conformation even in the absence of the antibiotic ; these mutant enzymes were hypersensitive to dmyx and were locked in the same inactive promoter complex intermediate
. of interest , dksa , a regulatory protein in e. coli , inhibits transcription through similar changes in rnap / dna interactions , and substitutions in the switch-2 alter the enzyme 's response to dksa .
switch-2 thus is a popular target for rnap inhibitors , and lpr - resistant substitutions therein initially suggested that lpr ( and based on structural similarities , also fdx ) may act similarly to dmyx .
for example , the same substitution , r337a , confers resistance both to antibiotics [ 21 , 22 ] and also to fdx .
however , a study by tupin et al revealed important differences between lpr and dmyx .
first , tupin et al demonstrated that lpr acts at a step preceding the one targeted by dmyx . in the presence of lpr , rnap bound to the promoter dna to form an unstable complex , but no strand separation could be detected .
second , this study suggested a key role for the subunit in lpr action : deletion of the hairpin loop that is positioned near the switch-2 conferred resistance to lpr .
thus , although the binding sites for lpr and dmyx may partially overlap , their mechanisms appear to be distinct .
in addition , it remained to be determined whether fdx acted similarly to lpr , particularly because these antibiotics target the highly dynamic switch-2 and their action could be strongly influenced by even subtle differences between the inhibitor scaffolds , the source of rnap , and the transcription complexes used for the analysis .
similar to lpr and in contrast to dmyx , fdx inhibited dna melting in promoter complexes throughout the region surrounding the transcription start site ( figure 2 ) .
however , our dnase i footprinting analysis revealed that fdx not only induced changes in the downstream dna interactions with the rnap ( as do dmyx and lpr ) , but also altered the dna conformation in the spacer region of the affected complexes ( unlike lpr ) . also in contrast to lpr , fdx action was not altered by deletion of the hairpin loop .
one possibility is that they are attributable to the different promoters used for these analyses , pr in our work and placuv5 in tupin et al .
potassium permanganate ( kmno4 ) modifies single - stranded thymidine ( t ) residues , which subsequently are cleaved by piperidine ; positions of cleavage indicate the location of single - stranded dna regions .
a linear end - labeled pr promoter dna fragment was generated by polymerase chain reaction .
top , the 35 and 10 hexamers are indicated by black boxes , the start site ( + 1 ) is shown by a black dot .
reactivities of the 10 , 4 , 3 , and + 2 non - template strand t residues to kmno4 modification are summarized above the sequence ; black and white arrows indicate high and low reactivity , respectively .
bottom , a representative gel with the sensitive residues shown ; the middle portion of the gel was removed ( dashed line ) to conserve space .
results show that although myxopyronin blocks only dna melting near the start site ( at + 2 position ) , fdx reduces strand separation throughout the entire sensitive region with the wild - type rnap , but not with a mutant that carries an r337a substitution in the switch-2 region .
abbreviations : ab , antibiotic ; fdx , fidaxomicin ; myx , myxopyronin ; rnap , rna polymerase ; wt , wild - type .
potassium permanganate ( kmno4 ) modifies single - stranded thymidine ( t ) residues , which subsequently are cleaved by piperidine ; positions of cleavage indicate the location of single - stranded dna regions .
a linear end - labeled pr promoter dna fragment was generated by polymerase chain reaction .
top , the 35 and 10 hexamers are indicated by black boxes , the start site ( + 1 ) is shown by a black dot .
reactivities of the 10 , 4 , 3 , and + 2 non - template strand t residues to kmno4 modification are summarized above the sequence ; black and white arrows indicate high and low reactivity , respectively .
bottom , a representative gel with the sensitive residues shown ; the middle portion of the gel was removed ( dashed line ) to conserve space .
results show that although myxopyronin blocks only dna melting near the start site ( at + 2 position ) , fdx reduces strand separation throughout the entire sensitive region with the wild - type rnap , but not with a mutant that carries an r337a substitution in the switch-2 region .
abbreviations : ab , antibiotic ; fdx , fidaxomicin ; myx , myxopyronin ; rnap , rna polymerase ; wt , wild - type .
rnap presents a complex target for inhibition , and changes in the enzyme or antibiotic may confer substantial differences .
small changes in antibiotic structure may alter the contacts with the enzyme and consequently change the point for inhibition , as illustrated by the case of rifamycins .
substitutions in rnap ( naturally occurring in different bacterial enzymes ) bestow distinct kinetic properties and altered sensitivity to cellular effectors and antibiotics alike .
some of these changes impair the binding of a ligand to rnap , but others may act indirectly by altering the enzyme 's conformation and changing its response to the antibiotic .
for example , a subset of cbr - resistant mutations in rpoc conferred dependence on the inhibitor for growth of the mutant strains , indicating that their phenotypes can not be explained by the loss of cbr binding .
thus , the phenotypes of any resistance mutations should be interpreted with caution because some of them could have indirect ( allosteric ) effects .
this may be particularly common for antibiotics that bind to the key sites on rnap , where substitutions would give rise to growth defects .
this complexity demands that when possible , structural analysis of the antibiotic / transcription complexes should be pursued in parallel with in - depth functional and genetic studies , using target rnaps from relevant bacterial species .
nevertheless , functional studies are useful for identifying differences in mechanism , particularly because they can provide information about the dynamics of enzyme inhibition rather than focusing on a static picture of binding .
the mechanistic studies presented here confirm that there are multiple steps that are targets for rnap inhibition by different antibiotics , and fdx inhibits a step distinct from elongation inhibitors , such as streptolydigin , and from the initiation inhibitors myx and the rifamycins .
the difference in mechanism compared with the rifamycins supports previous findings of lack of cross - resistance with this class of antibiotics and suggests that the rapid development of resistance observed with rifamycins does not indicate a rapid loss of susceptibility to fdx . | fidaxomicin was recently approved for the treatment of clostridium difficile infection .
it inhibits transcription by bacterial rna polymerase .
because transcription is a multistep process , experiments were conducted in which fidaxomicin was added at different stages of transcriptional initiation to identify the blocked step .
dna footprinting experiments were also conducted to further elucidate the stage inhibited .
fidaxomicin blocks initiation only if added before the formation of the open promoter complex , in which the template dna strands have separated but rna synthesis has not yet begun .
binding of fidaxomicin precludes the initial separation of dna strands that is prerequisite to rna synthesis .
these studies show that it has a mechanism distinct from that of elongation inhibitors , such as streptolydigin , and from the transcription initiation inhibitors myxopyronin and the rifamycins . | RESULTS AND DISCUSSION
Establishment of RNAP as a Target of FDX
RNAP Mechanism
Order-of-Addition Experiments
MYX as an Initial Model and Switch-2 Region as a Target
Footprinting Analysis to Compare MYX, LPR, and FDX | because of the similarity in fdx structure to the structures of lpr [ 1 , 2 ] , it was expected that they would share the same target . during the initiation phase of the transcription cycle ( figure 1a ) ,
the core rnap ( a complex of an -dimer , , , and subunits ) combines with a promoter - specificity factor to locate and bind to a promoter region of the dna , then separates , or melts , the double - stranded dna to form an open promoter complex ( rpo ) in which the melted region ( transcription bubble ) extends from positions 12 to + 2 ( relative to the transcription start site ) . the incoming nucleotide ( nucleoside triphosphate ) substrate pairs with the template dna in the enzyme 's active site ( + 1 ) , and rna synthesis commences . figure 1.a , transcription initiation pathway . core rna polymerase ( rnap ; gray oval , with active site depicted as a circle ) binds to a promoter specificity initiation factor ( 1 of many ) that directs the resulting holo rnap to a subset of promoters . in escherichia coli ,
the primary factor consists of separate domains ( numbered 1 4 ) ; domains 2 and 4 recognize the 10 and 35 elements of housekeeping
promoters to form a closed promoter complex , rpc , in which the double - stranded dna is loosely bound on the surface of rnap . in the final ,
transcription - competent open promoter complex ( rpo ; boxed ) , the transcription bubble encompasses the + 1 position . fidaxomicin ( fdx ; at 50 m ) was added to the in vitro transcription reaction with the e. coli rnap at different points . the fraction of synthesized rna was measured ( as percentage of transcription in the absence of the antibiotic ) . inhibition of the reaction was observed when fdx was added before steps 1 and 2 , but not 3 or 4 , after formation of the rpo . a , transcription initiation pathway . core rna polymerase ( rnap ; gray oval , with active site depicted as a circle ) binds to a promoter specificity initiation factor ( 1 of many ) that directs the resulting holo rnap to a subset of promoters . in escherichia coli ,
the primary factor consists of separate domains ( numbered 1 4 ) ; domains 2 and 4 recognize the 10 and 35 elements of housekeeping promoters to form a closed promoter complex , rpc , in which the double - stranded dna is loosely bound on the surface of rnap . in rpi1 , the first intermediate along the pathway , dna strand separation initiates around the 11 position ( relative to the transcription start site ) . in the final ,
transcription - competent open promoter complex ( rpo ; boxed ) , the transcription bubble encompasses the + 1 position . fidaxomicin ( fdx ; at 50 m ) was added to the in vitro transcription reaction with the e. coli rnap at different points . the fraction of synthesized rna was measured ( as percentage of transcription in the absence of the antibiotic ) . inhibition of the reaction was observed when fdx was added before steps 1 and 2 , but not 3 or 4 , after formation of the rpo . in these assays ,
the antibiotic is added to a free rnap or to transcription complexes poised at different checkpoints . antibiotics that inhibit rna chain elongation block transcription when added at any step during transcription ; this group includes streptolydigin [ 14 , 15 ] , tagetitoxin , microcin j25 [ 17 , 18 ] , and cbrs ( a group of synthetic rna polymerase inhibitors ) , which are rifamycin - quinolone hybrids . lpr and myxopyronins ( myxs ) , such as desmethylmyxopyronin ( dmyx ) [ 22 , 23 ] , inhibit rnap only if added before formation of the rpo . we found that fdx also inhibits transcription only if added before the stable rpo has been formed ( figure 1b ) . in addition to the similar behavior of fdx , lpr , and dmyx in order - of - addition experiments , the patterns of mutations that confer resistance to fdx , lpr , and dmyx overlap , suggesting a similar site of binding / action on the rnap . first , the switch-2 forms a hinge that connects the 2 pincers of the rnap crab - claw - like clamp ; opening of these pincers is thought to be required to load the dna into the rnap during initiation . second , the switch-2 interacts with the template dna strand and determines its position . , dmyx restricts movements of the switch-2 , blocking the clamp opening . in another , dmyx - stabilized refolding of the switch-2 sterically blocks the path of the template dna strand near the rnap active site . footprinting
analysis revealed that dmyx does not prevent dna binding to e. coli rnap or nucleation of dna melting by the subunit . however , dmyx blocks the propagation of dna melting toward the active site , stabilizing a partially melted intermediate , the existence of which has been postulated for many years . on the basis of the complex structure , we designed substitutions in the switch-2 that mimicked the dmyx - bound conformation even in the absence of the antibiotic ; these mutant enzymes were hypersensitive to dmyx and were locked in the same inactive promoter complex intermediate . of interest , dksa , a regulatory protein in e. coli ,
inhibits transcription through similar changes in rnap / dna interactions , and substitutions in the switch-2 alter the enzyme 's response to dksa . switch-2 thus is a popular target for rnap inhibitors , and lpr - resistant substitutions therein initially suggested that lpr ( and based on structural similarities , also fdx ) may act similarly to dmyx . in the presence of lpr , rnap bound to the promoter dna to form
second , this study suggested a key role for the subunit in lpr action : deletion of the hairpin loop that is positioned near the switch-2 conferred resistance to lpr . in addition , it remained to be determined whether fdx acted similarly to lpr , particularly because these antibiotics target the highly dynamic switch-2 and their action could be strongly influenced by even subtle differences between the inhibitor scaffolds , the source of rnap , and the transcription complexes used for the analysis . footprinting analyses were conducted with fdx to evaluate whether fdx behaved like lpr . similar to lpr and in contrast to dmyx , fdx inhibited dna melting in promoter complexes throughout the region surrounding the transcription start site ( figure 2 ) . however , our dnase i footprinting analysis revealed that fdx not only induced changes in the downstream dna interactions with the rnap ( as do dmyx and lpr ) , but also altered the dna conformation in the spacer region of the affected complexes ( unlike lpr ) . also in contrast to lpr , fdx action was not altered by deletion of the hairpin loop . reactivities of the 10 , 4 , 3 , and + 2 non - template strand t residues to kmno4 modification are summarized above the sequence ; black and white arrows indicate high and low reactivity , respectively . bottom , a representative gel with the sensitive residues shown ; the middle portion of the gel was removed ( dashed line ) to conserve space . results show that although myxopyronin blocks only dna melting near the start site ( at + 2 position ) , fdx reduces strand separation throughout the entire sensitive region with the wild - type rnap , but not with a mutant that carries an r337a substitution in the switch-2 region . abbreviations : ab , antibiotic ; fdx , fidaxomicin ; myx , myxopyronin ; rnap , rna polymerase ; wt , wild - type . reactivities of the 10 , 4 , 3 , and + 2 non - template strand t residues to kmno4 modification are summarized above the sequence ; black and white arrows indicate high and low reactivity , respectively . bottom , a representative gel with the sensitive residues shown ; the middle portion of the gel was removed ( dashed line ) to conserve space . results show that although myxopyronin blocks only dna melting near the start site ( at + 2 position ) , fdx reduces strand separation throughout the entire sensitive region with the wild - type rnap , but not with a mutant that carries an r337a substitution in the switch-2 region . abbreviations : ab , antibiotic ; fdx , fidaxomicin ; myx , myxopyronin ; rnap , rna polymerase ; wt , wild - type . rnap presents a complex target for inhibition , and changes in the enzyme or antibiotic may confer substantial differences . some of these changes impair the binding of a ligand to rnap , but others may act indirectly by altering the enzyme 's conformation and changing its response to the antibiotic . this complexity demands that when possible , structural analysis of the antibiotic / transcription complexes should be pursued in parallel with in - depth functional and genetic studies , using target rnaps from relevant bacterial species
. the mechanistic studies presented here confirm that there are multiple steps that are targets for rnap inhibition by different antibiotics , and fdx inhibits a step distinct from elongation inhibitors , such as streptolydigin , and from the initiation inhibitors myx and the rifamycins . the difference in mechanism compared with the rifamycins supports previous findings of lack of cross - resistance with this class of antibiotics and suggests that the rapid development of resistance observed with rifamycins does not indicate a rapid loss of susceptibility to fdx . because of the similarity in fdx structure to the structures of lpr [ 1 , 2 ] , it was expected that they would share the same target . during the initiation phase of the transcription cycle ( figure 1a ) , the core rnap ( a complex of an -dimer , , , and subunits ) combines with a promoter - specificity factor to locate and bind to a promoter region of the dna , then separates , or melts , the double - stranded dna to form an open promoter complex ( rpo ) in which the melted region ( transcription bubble ) extends from positions 12 to + 2 ( relative to the transcription start site ) . the incoming nucleotide ( nucleoside triphosphate ) substrate pairs with the template dna in the enzyme 's active site ( + 1 ) , and rna synthesis commences . figure 1.a , transcription initiation pathway . core rna polymerase ( rnap ; gray oval , with active site depicted as a circle ) binds to a promoter specificity initiation factor ( 1 of many ) that directs the resulting holo rnap to a subset of promoters . in escherichia coli ,
the primary factor consists of separate domains ( numbered 1 4 ) ; domains 2 and 4 recognize the 10 and 35 elements of housekeeping
promoters to form a closed promoter complex , rpc , in which the double - stranded dna is loosely bound on the surface of rnap . in rpi1 ,
the first intermediate along the pathway , dna strand separation initiates around the 11 position ( relative to the transcription start site ) . in the final ,
transcription - competent open promoter complex ( rpo ; boxed ) , the transcription bubble encompasses the + 1 position . fidaxomicin ( fdx ; at 50 m ) was added to the in vitro transcription reaction with the e. coli rnap at different points . the fraction of synthesized rna was measured ( as percentage of transcription in the absence of the antibiotic ) . inhibition of the reaction was observed when fdx was added before steps 1 and 2 , but not 3 or 4 , after formation of the rpo . a , transcription initiation pathway . core rna polymerase ( rnap ; gray oval , with active site depicted as a circle ) binds to a promoter specificity initiation factor ( 1 of many ) that directs the resulting holo rnap to a subset of promoters . in escherichia coli ,
the primary factor consists of separate domains ( numbered 1 4 ) ; domains 2 and 4 recognize the 10 and 35 elements of housekeeping
promoters to form a closed promoter complex , rpc , in which the double - stranded dna is loosely bound on the surface of rnap . in the final ,
transcription - competent open promoter complex ( rpo ; boxed ) , the transcription bubble encompasses the + 1 position . fidaxomicin ( fdx ; at 50 m ) was added to the in vitro transcription reaction with the e. coli rnap at different points . inhibition of the reaction was observed when fdx was added before steps 1 and 2 , but not 3 or 4 , after formation of the rpo . in these assays ,
the antibiotic is added to a free rnap or to transcription complexes poised at different checkpoints . antibiotics that inhibit rna chain elongation block transcription when added at any step during transcription ; this group includes streptolydigin [ 14 , 15 ] , tagetitoxin , microcin j25 [ 17 , 18 ] , and cbrs ( a group of synthetic rna polymerase inhibitors ) , which are rifamycin - quinolone hybrids . lpr and myxopyronins ( myxs ) , such as desmethylmyxopyronin ( dmyx ) [ 22 , 23 ] , inhibit rnap only if added before formation of the rpo . we found that fdx also inhibits transcription only if added before the stable rpo has been formed ( figure 1b ) . in addition to the similar behavior of fdx , lpr , and dmyx in order - of - addition experiments , the patterns of mutations that confer resistance to fdx , lpr , and dmyx overlap , suggesting a similar site of binding / action on the rnap . first , the switch-2 forms a hinge that connects the 2 pincers of the rnap crab - claw - like clamp ; opening of these pincers is thought to be required to load the dna into the rnap during initiation . second , the switch-2 interacts with the template dna strand and determines its position . , dmyx restricts movements of the switch-2 , blocking the clamp opening . in another , dmyx - stabilized refolding of the switch-2 sterically blocks the path of the template dna strand near the rnap active site . however , dmyx blocks the propagation of dna melting toward the active site , stabilizing a partially melted intermediate , the existence of which has been postulated for many years . on the basis of the complex structure , we designed substitutions in the switch-2 that mimicked the dmyx - bound conformation even in the absence of the antibiotic ; these mutant enzymes were hypersensitive to dmyx and were locked in the same inactive promoter complex intermediate
. of interest , dksa , a regulatory protein in e. coli , inhibits transcription through similar changes in rnap / dna interactions , and substitutions in the switch-2 alter the enzyme 's response to dksa . switch-2 thus is a popular target for rnap inhibitors , and lpr - resistant substitutions therein initially suggested that lpr ( and based on structural similarities , also fdx ) may act similarly to dmyx . in the presence of lpr , rnap bound to the promoter dna to form an unstable complex , but no strand separation could be detected . second , this study suggested a key role for the subunit in lpr action : deletion of the hairpin loop that is positioned near the switch-2 conferred resistance to lpr . in addition , it remained to be determined whether fdx acted similarly to lpr , particularly because these antibiotics target the highly dynamic switch-2 and their action could be strongly influenced by even subtle differences between the inhibitor scaffolds , the source of rnap , and the transcription complexes used for the analysis . similar to lpr and in contrast to dmyx , fdx inhibited dna melting in promoter complexes throughout the region surrounding the transcription start site ( figure 2 ) . however , our dnase i footprinting analysis revealed that fdx not only induced changes in the downstream dna interactions with the rnap ( as do dmyx and lpr ) , but also altered the dna conformation in the spacer region of the affected complexes ( unlike lpr ) . also in contrast to lpr , fdx action was not altered by deletion of the hairpin loop . reactivities of the 10 , 4 , 3 , and + 2 non - template strand t residues to kmno4 modification are summarized above the sequence ; black and white arrows indicate high and low reactivity , respectively . bottom , a representative gel with the sensitive residues shown ; the middle portion of the gel was removed ( dashed line ) to conserve space . abbreviations : ab , antibiotic ; fdx , fidaxomicin ; myx , myxopyronin ; rnap , rna polymerase ; wt , wild - type . reactivities of the 10 , 4 , 3 , and + 2 non - template strand t residues to kmno4 modification are summarized above the sequence ; black and white arrows indicate high and low reactivity , respectively . bottom , a representative gel with the sensitive residues shown ; the middle portion of the gel was removed ( dashed line ) to conserve space . results show that although myxopyronin blocks only dna melting near the start site ( at + 2 position ) , fdx reduces strand separation throughout the entire sensitive region with the wild - type rnap , but not with a mutant that carries an r337a substitution in the switch-2 region . abbreviations : ab , antibiotic ; fdx , fidaxomicin ; myx , myxopyronin ; rnap , rna polymerase ; wt , wild - type . rnap presents a complex target for inhibition , and changes in the enzyme or antibiotic may confer substantial differences . some of these changes impair the binding of a ligand to rnap , but others may act indirectly by altering the enzyme 's conformation and changing its response to the antibiotic . for example , a subset of cbr - resistant mutations in rpoc conferred dependence on the inhibitor for growth of the mutant strains , indicating that their phenotypes can not be explained by the loss of cbr binding . this complexity demands that when possible , structural analysis of the antibiotic / transcription complexes should be pursued in parallel with in - depth functional and genetic studies , using target rnaps from relevant bacterial species . the mechanistic studies presented here confirm that there are multiple steps that are targets for rnap inhibition by different antibiotics , and fdx inhibits a step distinct from elongation inhibitors , such as streptolydigin , and from the initiation inhibitors myx and the rifamycins . the difference in mechanism compared with the rifamycins supports previous findings of lack of cross - resistance with this class of antibiotics and suggests that the rapid development of resistance observed with rifamycins does not indicate a rapid loss of susceptibility to fdx . | [
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the p2y receptors ( p2yrs )
are g protein - coupled receptors ( gpcrs )
that are activated by extracellular nucleotides .
the widely expressed utp - activated
( p2y2r and p2y4r ) and udp - activated ( p2y6r ) receptors belong to the subfamily of p2y1-like
receptors that promote inositol lipid signaling through gq - mediated activation of phospholipase c- ( plc- ) .
few
selective agonists and antagonists are
available for pharmacological differentiation of these uracil nucleotide - regulated
receptors , and p2y2r , p2y4r , and p2y6r exhibit much overlap in affinities for pyrimidine nucleotides .
this study was initiated to synthesize nucleoside 5-triphosphate
derivatives as potent agonist probes of the p2y4r , including
chain derivatization for incorporation of bulky reporter groups .
the
message for this p2yr subtype is expressed at high levels in intestine ,
pituitary , and brain , with lower levels observed in adipose tissue ,
prostate , spleen , lymphocytes , skeletal muscle , and lung .
the p2y4r has been shown to regulate cardiovascular development , and activation of this signaling protein results
in intestinal chloride secretion , inhibition
of contraction of the mouse ileum longitudinal muscle , and k secretion in the luminal membrane of the distal colon .
thus , ligands of the p2y4r are of potential interest
for pharmacotherapies directed at intestinal function , angiogenesis ,
cardiac remodeling , postischemic revascularization , cns function ,
and inflammation .
uridine-5-triphosphate
( utp , 1 ) is the endogenous
p2y4r agonist ( ec50 = 80 nm ) , while
adenosine-5-triphosphate ( atp ) antagonizes the human homologue
of this receptor and activates the rat homologue .
activity relationship ( sar ) studies of
the p2y4r have been performed through preparation of modified
analogues in conjunction with molecular modeling .
two analogues of
utp , n-(3-phenylpropyloxy)cytidine-5-triphosphate , 2 , and 2-azido-2-deoxy - utp , 3 , and one 5-monophosphate derivative , iso - cmp 4 , have demonstrated
( chart 1 ) low to moderate selectivity for this
subtype , but highly potent and selective p2y4r agonists
have not been identified to date .
the 4-benzyloxyiminopyrimidine group was identified as a structurally
permissive site for synthesis of functionalized congeners , leading to high affinity molecular probes for
the closely related udp - activated p2y6r .
because of the structural similarity of the p2y4r and p2y6r , we focused on the same site for derivatization
of analogues of 1 that included an elongated alkyl chain ,
i.e. , a n - phenylpropyloxy group , which
we hypothesized would be more suitable for conferring p2y4r selectivity .
one goal of these studies
was to generate an analogue(s ) with tolerance for steric bulk when
bound to the receptor , potentially allowing attachment of a fluorophore
or other reporter group to serve as a tracer for the receptor on live
cells .
aec50 values for
activation of plc via the human p2y4r are : 2 , 23 nm ; 2-azido-2-deoxy - utp 3 , 1100
nm ; iso - cmp 4 , 4980 nm .
ec50 values for
activation of plc via the human p2y4r are : 2 , 23 nm ; 2-azido-2-deoxy - utp 3 , 1100
nm ; iso - cmp 4 , 4980 nm .
we considered a 4-arylalkyloxyimino
group in the pyrimidine nucleobase
as an approach to expand the range of potent agonists of p2y4r and related p2yrs . in summary , we have modified the 4-(3-phenylpropyloxy)imino
group of 2 with various ring substitutions and chain
elongations and evaluated the new nucleotide derivatives in functional
assays of the hp2y4r , p2y6r , and p2y2r .
because dinucleoside tetraphosphates are also known to activate
the p2y4r , we included several
derivatives in the dinucleotide series to test their compatibility
with 4-alkyloxyimino modifications .
novel nucleotide derivatives 7 , 1028 , and 3031 ( table 1 ) were prepared
and purified to homogeneity .
two
series of nucleotide derivatives were chosen : 5-triphosphates
related to utp and dinucleotides related to up4u 29 , which have relatively high potency at both p2y2r and p2y4r .
the synthetic routes to these groups of structurally
modified pyrimidine nucleotides include : utp analogues containing
a n - alkoxy group ( scheme 1 ) , modified up4u dinucleotide analogues ( scheme 2 ) , and utp analogues containing alexa fluor 488
and bodipy 630/650 fluorescent dyes ( scheme 3 ) .
reagents and conditions : ( a )
cytidine , pyridine , 110 c ; ( b ) proton sponge , pocl3 , po(ome)3 , tributylammonium pyrophosphate , dmf ; ( c ) 1
n naoh , thf , 50 c ; ( d ) pybop , diea , but-4-yn-1-amine . reagents : ( a ) dic , mgcl2 , dmf . reagents and conditions : ( a )
azide ( e.g. , 124 , 1 equiv ) and alkyne ( 1.4 equiv ) precursors , buoh / water , tbta , sodium ascorbate , cupric sulfate .
functional assays were conducted
with 1321n1 astrocytoma cells expressing recombinant hp2y2 , p2y4 , or p2y6rs .
the synthesis of n - alkoxy cytidines 1026 from cytidine was performed using
corresponding alkoxyamine hydrochlorides .
various alkoxy groups including
phenyl , pyridyl , thienyl , naphthyl , and alkyl ester derivatives were
chosen .
these alkoxyamine derivatives were synthesized from the corresponding
3-arylpropyl bromides using gabriel synthesis or a variation thereof
using n , n-di - tert - butoxycarbonylhydroxylamine as the source of the hydroxylamine moiety .
the 3-alkylpropyl bromides 5662 and 67 were synthesized from the corresponding benzyl
bromides by homologation in four steps ( scheme 4a ) .
the 3-arylpropyl bromides 73 , 76 , and 78 were synthesized
from the corresponding alcohols and converted to the alkyoxyimino
derivatives ( scheme 4b ) .
( 3-thien-3-yl)propyl bromide 71 was synthesized
from the corresponding ,-unsaturated carboxylic acid
( scheme s1 , supporting information ) .
the
resulting n - alkoxy - cytidines were phosphorylated
by standard methods to give the desired n - alkoxy - cytidine 5-triphosphates 1026 ( scheme 1 ) . in each case , the unprotected nucleoside was first treated with
phosphorus oxychloride and the reaction mixture was treated immediately
with bis(tri - n - butylammonium ) pyrophosphate to produce
the 5-triphosphate .
the synthesis of dinucleoside tetraphosphates ,
including an asymmetrically substituted analogue 30 ,
was performed using n , n-diisopropylcarbodiimide
( dic ) as a coupling reagent for condensing a 5-monophosphate
and 5-triphosphate in the presence of magnesium chloride ( scheme 2 ) , which dramatically increased the reactivity compared
to carbodiimide alone .
the fluorescent
alexa fluor 488 5-triphosphate 27 and bodipy
5-triphosphate 28 derivatives were synthesized
from alkyne nucleotide precursor 26 and an azide derivative
( e.g. , a bodipy 630/650 azide 124 for product 28 , scheme s2 , supporting information ) using
copper - catalyzed [ 2 + 3 ] cycloaddition click reactions with azides
( scheme 3 ) . reagents and conditions : ( a )
( a ) diethyl malonate , nah , thf ; ( b ) nacl , dmso ; ( c ) dibal - h ; ( d ) cbr4 , pph3 ; ( e ) et3n , dmf , 100c ; ( f )
nh2nh2h2o ; ( g ) 1 n hcl .
n - alkoxyamine intermediates were
prepared by analogous methods ( scheme s1 , supporting
information ) : ( a ) k2co3 , ch3cn , reflux ; ( b ) cbr4 , pph3 ; ( c ) et3n , dmf , 100 c ; ( d ) nh2nh2h2o ; ( e ) 10% pd / c , h2 , meoh ; ( f ) lialh4 , thf ; ( g ) br2 , pph3 , 2,6-lutidine .
( b ) ( a ) hbr in acetic acid , 50 c ; ( b )
dbu , ch3cn , 50 c ; ( c ) 1 n hcl , ch2cl2 .
the newly synthesized analogues
were tested for potency against
the p2y2r , p2y4r , and p2y6r .
similar n-3-phenylalkyloxy derivatives
were shown to be only weakly active at the p2y14r ; thus , this subtype was not part of the initial
screen .
the potencies of nine known reference compounds ( 2 , 56 , 89 , and 29 ) were included for comparison .
standard assays of potency for activation of plc were performed
as described in 1321n1 human astrocytoma
cell lines stably expressing either the p2y2r , p2y4r , or p2y6r
( 1321n1-p2yr cells ) . among substituted
phenyl groups , 3-f 12 , 4-i 16 , and 4-meo 19 provided relatively high potency
at the three p2y2 , p2y4 , and p2y6rs .
thus , we decided to elongate functionalized congeners based on p - substituted ethers related to 19 .
other aryl
moieties that appeared to enhance potency were thien-3-yl 22 and naphth-2-yl 23 , indicating a tolerance of steric
bulk in this region .
the optimal chain length at the p2y4r was reached , with the n - propyl analogue 2 not substituted with an ether in the chain ( cf .
in contrast , the longer n - butyl homologue 10 displayed increased potency at the p2y2r and
p2y6r .
high potency at the p2y4r was achieved
with a terminal alkyne at the n - linked
chain for tethering sterically bulky groups by click chemistry ( 26 , ec50 = 40 nm , figure 1 ) . however , potency at p2y2r and p2y6r also
was largely retained in this ether- and amide - linked alkyne , with
ec50 values of 109 and 183 nm , respectively .
comparison
of potencies of alkyne 26 and fluorescent
analogues 27 and 28 in activation of plc
via the human p2y2r ( a ) , p2y4r ( b ) , and p2y6r ( c ) in stably transfected 1321n1 human astrocytoma cells . the fluorescent dye
, alexa fluor
488 , is a suitable fluorophore
for flow cytometry ( fcm ) binding assays and live cell imaging .
this fluorophore was incorporated in conjugate 27 ,
but only an intermediate potency was observed with this molecule at
the three p2yrs .
curiously , in comparison to the precursor
molecule 26 , the bodipy conjugate exhibited enhanced
potency at the p2y2r ( ec50 = 66 nm ) and p2y6r ( ec50 = 23 nm ) but slightly decreased potency
at the p2y4r .
thus , 28 is a high affinity
pan - agonist fluorescent probe with nearly equivalent potency at the
hp2y2r , hp2y4r , and hp2y6r .
a preliminary feasibility study was carried out to determine if 28 could serve as a fluorescent p2yr probe .
these experiments
were with p2y6r - expressing 1321n1 cells because 28 is most potent at this p2yr subtype ( figure 2 ) .
fluorescent binding of 28 was compared by fcm , using
several known p2y6r ligands to compete for cell labeling .
incubation of cells with 100 nm 28 for 30 min at 37 c
in the presence of the ecto - nucleotidase inhibitor ,-methylene - adenosine
5-diphosphate 125 ( 100 m ) resulted in significant cell labeling ( figure 2 ) .
the level of cell - associated fluorescence was not reduced by coincubation
of 28 with 10 m n - methyl-2-deoxyadenosine 3,5-bisphosphate ( 117 , mrs2179 ) , a p2y1r - selective antagonist that is inactive
at p2y2 , p2y4 , and p2y6rs .
however , significant decreases in mean fluorescence
intensity were observed after preincubation with p2y6r
ligands , including the p2y6r - selective noncompetitive antagonist n , n-1,4-butanediyl - bis[n-(3-isothiocyanatophenyl)]thiourea ( 118 , mrs2578 , 10 m ) and the competitive antagonist suramin ( 119 , 10 m ) .
although 119 is a nonselective p2x and p2y antagonist , we included it in this
assay because it has been shown to effectively block the p2y6r .
the potent p2y6r - selective
dinucleotide agonist p-(uridine 5-)-p-(n - methoxycytidine
5-)triphosphate ( 120 , mrs2957 , 10 m ) produced the largest decrease in cell - associated
fluorescence .
fluorescence microscopy comparing binding to 1321n1-p2y6r cells in the presence and absence of 118 ( figure 3 ) indicated that 1 m 28 specifically
labeled cells expressing the receptor .
fluorescence ligand binding
experiments using fcm in stably transfected
1321n1-p2y6r cells with 28 after preincubation
at 37 c with known p2y6r agonist or p2y6r antagonists .
each column shows the brightness of each compound
using 28 set at 100% and after correcting the mfi values
for autofluorescence .
binding of 28 at 20 and 30 min to the
p2y6r was significantly blocked after preincubation with
selective antagonist 118 , selective agonist 120 , and nonselective antagonist 119 .
no significant difference
in mfi was observed for p2y1r antagonist 117 . * , p < 0.05 , when compared to cells treated
with only 28 . average of mfi after 20 min incubation
of 28 : 107.0 30.2 ; after 30 min incubation of 28 : 222.8 72.1 .
fluorescent micrographs using a keyence fluorescent microscope
( bz-9000 ) of p2y6r - expressing 1321n1 astrocytoma cells
exposed to the fluorescent agonist 28 ( 1 m , 30
min incubation at 37 c ) .
( b ) incubation with 1 m 14 at 37 c
for 30 min in medium .
( c ) incubation with 1 m 14 at 37 c for 30 min in cells pretreated with noncompetitive
antagonist 118 ( 10 m ) .
we have identified a functionalization approach
that provides versatile
nucleotide analogues for further derivatization of high potency p2y2r , p2y4r , and p2y6r agonists .
the initial
approach was based on a p2y4r - selective agonist , and many
of the new derivatives tend toward selectivity at this subtype .
however ,
upon elongation of a functionalized chain at the n position , significant potency also was observed at the
related p2y2r and p2y6r subtypes .
the derivatization strategy was aimed
to clarify where to modify
the pyrimidine nucleotides to preserve or increase potency at any
or all of the relevant p2yrs to allow the attachment of sterically
large fluorophores and other reporter
groups .
questions to be answered were : if the pseudo - oxime chain of 2 proved to be a suitable attachment point , how long should
the alkyl linker ( i.e. , propyl in 2 ) be for optimal potency
( cf .
8 , 9 , 11 , 25 ) ? were ring - substitutions ( cf .
1219 ) or replacement of the terminal phenyl moiety with heteroaryl or
other group ( cf . 7 , 2024 ) desirable for enhancing potency ?
1219 ) or other aryl group was best suited for high potency ?
thus , model compounds of various intermediate substructures were prepared
and tested before the final target fluorescent conjugates were designed .
briefly , we chose the p - position for extension of
an alkyl ether chain on the 4-(3-phenylpropyloxy)imino group of 2 , which was click - linked through
a terminal alkyne , i.e. , 26 , to an azide - bearing fluorophore .
other possible approaches could have been through chain elongation
at the 2 , 3 , or 5 positions of a uracil ring .
modifications of the distal phenyl ring of a 3-phenylpropyloxyimino
group , shown previously to be optimal for p2y4r potency ,
allowed a bulky fluorescent reporter group to be introduced , but the
p2y4r selectivity was compromised .
nevertheless , these
molecules also provide high affinity leads for further modified agonists
that could exhibit higher selectivity at one of these receptors .
possible
directions for structural modifications of pyrimidine nucleotides
conducive to p2y4r selectivity might include ribose and
nucleobase modifications such as appear in compounds 3 and 4 .
this discovery provides an optimal new
route for generation of
fluorescent probes for facile pooled quantification of these three
p2yrs in living cells . in cells expressing a single p2yr subtype ,
such as our astrocytoma cells
, there is no ambiguity . following the
development of standard assay conditions , the potent fluorescent ligand 28
could potentially be used for high - throughput screening
of drug libraries with p2y2r- , p2y4r- , or p2y6r - expressing cells .
radioligands are not available for any
of these receptors . our previously reported fluorescent nucleotide
p2yr probe ,
a conjugate of alexa fluor 488 , tethered from a cytosine
nucleobase in the diphosphate series , was selective for the p2y6r .
the current work adds a second
viable fluorescent probes for studying the p2y6r . although
we did not directly examine its binding to p2y2r- and p2y4r - expressing cells , compound 28 adds the potential
capability of fluorescent characterization of these subtypes for which
no high affinity assay probes are available .
assay development for
routine application of this fluorescent probe presents a separate
experimental challenge that will be addressed in future work .
the high potency of 28 at the hp2y2r , hp2y4r , and hp2y6r suggests the existence of energetically
favorable interactions with groups on the conformationally flexible
extracellular regions of p2y2r and p2y6r .
the
modeling of these extracellular regions is difficult because of their
flexibility and the lack of a close structural template .
further characterization
of the location of the fluorescent label within the cells as well
as delineation of the saturability , reversibility , kinetics , and other
measurements will be required before this probe molecule can be used
routinely for fcm labeling of cells expressing p2yrs .
a series of n-(3-arylpropyloxy ) derivatives
of uridine-5-triphosphate were synthesized and found to display
high potency as agonists of the initial target g protein - coupled hp2y4r and also hp2y2r and hp2y6r .
the phenyl
group could be substituted with heterocyclic rings or a naphthyl ring
with retention of p2yr affinity , indicating a broad tolerance for
steric bulk in this distal region of the nucleotide .
thus , an extended n-(3-arylpropyloxy ) group accessed a structurally
permissive region on various gq - coupled p2yrs and was modulated
by distal structural changes to alter selectivity .
this freedom of
substitution can be utilized for the design of pan - agonist affinity
probes of the uracil nucleotide - activated , gq - coupled p2y2/4/6 subset of receptors .
h nmr spectra were obtained
with a varian mercury 400 spectrometers using d2o , meod ,
cdcl3 , or dmso - d6 as a solvent .
p nmr spectra were recorded at room temperature ( rt ) on varian
mercury 400 ( 162.10 mhz ) spectrometers ; orthophosphoric acid ( 85% )
was used as an external standard . in several cases ,
the signal of
the terminal phosphate moiety was not visible due to high dilution .
high - resolution mass measurements were performed using a micromass / waters
lct premier electrospray time of flight ( tof ) mass spectrometer coupled
with a waters hplc system , unless noted .
all other reagents were of analytical
grade and were purchased from sigma - aldrich ( st .
purification of the nucleotide analogues for biological testing was
carried out on ( diethylamino)ethyl ( deae)-a25 sephadex columns with
a linear gradient ( 0.010.5 m ) of 0.5 m ammonium bicarbonate
as the mobile phase .
then these nucleotides were additionally purified
by hplc with a luna 5 rp - c18(2 ) semipreparative column ( 250
mm 10.0 mm ; phenomenex , torrance , ca ) and using the following
conditions : flow rate of 2 ml / min ; 10 mm triethylammonium acetate
( teaa)-ch3cn from 100:0 to 95:5 ( system b ) ( or up to 99:1
to 50:50 ( system c ) ) in 30 min ( and isolated in the triethylammonium
salt form ) .
all other compounds ( cytidine derivatives ) were purified
from a silica column chromatography ( chcl3:meoh - gradient
from 100:0 to 70:30 ) .
packard 1100 hplc equipped with
a zorbax sb - aq 5 m analytical column ( 50 mm 4.6 mm ;
agilent technologies inc . ,
mobile phase : linear gradient
solvent system ; 5 mm tbap ( tetrabutylammonium dihydrogenphosphate)-ch3cn from 80:20 to 40:60 in 13 min ; the flow rate was 0.5 ml / min
( system a ) .
peaks were detected by uv absorption with a diode array
detector at 230 , 254 , and 280 nm .
all nucleotide derivatives tested
for biological activity showed > 98% purity by hplc analysis ( detection
at 254 nm ) .
a solution of the nucleoside 97112 , 114 ( 73.0 mol ) , and 1,8-bis(dimethylamino)naphthalene
( proton sponge , 24.0 mg , 0.11 mmol ) in trimethyl phosphate ( 0.4 ml )
was stirred for 10 min at 0 c .
then ,
phosphorus oxychloride ( 13.0 l , 0.13 mmol ) was added dropwise ,
and the reaction mixture was stirred for 2 h at 0 c . a solution
of tributylammonium pyrophosphate ( 0.80 ml , 0.44 mmol ) and tributylamine
( 69.0 l , 0.29 mmol ) in n
, n - dimethylformamide ( dmf , 1 ml ) was added , and stirring was continued
at 0 c for additional 10 min . then 0.2 m triethylammonium bicarbonate
solution ( 1.5 ml ) was added , and the clear solution was stirred at
rt for 1 h. after removal of solvents , the residue was purified by
sephadex - deae a-25 resin and preparative hplc .
compound 26 ( 7.0 mg , 6.9 mol , 10% ) was obtained as a white solid using n-(3-((4-but-3-yn-1-aminocarbonylmethyleneoxy)phenyl)propyloxy)cytidine 116 ( 35.5 mg , 70.1 mol ) .
h nmr ( d2o ) 7.277.19 ( m , 3h ) , 6.94 ( d , j =
8.6 , 2h ) , 5.94 ( d , j = 6.4 hz , 1h ) , 5.77 ( d , j = 8.2 , 1h ) , 4.60 ( s , 2h ) , 4.444.40 ( m , 1h ) , 4.36
( t , j = 5.9 , 1h ) , 4.264.14 ( m , 3h ) , 4.06
( t , j = 6.0 , 2h ) , 3.44 ( t , j = 6.6 ,
2h ) , 2.752.63 ( m , 2h ) , 2.44 ( dt , j = 6.6 , j = 2.7 , 2h ) , 2.33 ( t , j = 2.6 , 1h ) , 2.071.91
( m , 2h ) . hrms - ei found : 741.0974 ( m h ) .
c24h32n4o17p3 requires 741.0970 ; analytical hplc ( system a : 6.54 min ) . to a mixture of alexa
fluor 488 azide ( alexa fluor 488 5-carboxamido-(6-azidohexanyl ) , bis(triethylammonium
salt ) ) , 5-isomer , 0.5 mg , 0.8 mol , 1 equiv ) , and alkyne 26 ( 0.8 mg , 0.8 mol , 1.4 equiv ) in a 0.20 ml of ( 1:1 )
mixture of buoh and water , were added tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl ] amine ( tbta , 0.1 mg ) and freshly
prepared 1 m aqueous sodium ascorbate solution ( 1.6 mol , 1.6
l , 2 equiv ) followed by 7.5% aqueous copper sulfate pentahydrate
solution ( 4.0 l , 1.2 mol , 1.5 equiv ) .
the reaction mixture
was stirred overnight at rt , solvent was evaporated , and the residue
was purified by semipreparative hplc as described above to obtain 27 ( 1.64 mg , 34% yield ) as a yellow / orange solid . hrms - ei
found : 1399.2106 ( m h ) .
c51h58n10o27s2p3 requires 1399.2104 . to a mixture of bodipy
630/650 azide ( 124 , supporting information , 1.0 mg , 1.0 mol , 1 equiv ) and alkyne 26 ( 2.0
mg , 1.8 mol , 1.2 equiv ) in a 200 l of ( 1:1 ) mixture
of buoh and water , and 20 l of dmf , were added
tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl ] amine
( tbta , 0.05 mg ) and freshly prepared 1 m aqueous sodium ascorbate
solution ( 1.1 l , 1.6 mol , 1 equiv ) followed by 7.5%
aqueous copper sulfate pentahydrate solution ( 2.5 l , 0.8 mol ,
0.8 equiv ) .
the bright - blue color reaction mixture was stirred overnight
at rt , solvent was evaporated , and the residue was purified by semipreparative
hplc as described above to obtain desired triphosphate 28 as blue - colored solids . hrms - ei m / z ( m + es ms ) for 28 found , 1388.3648 ( m + h ) ; calcd for c64h68n11o20bf2p3s , 1388.3631 ; analytical
hplc ( system a : 7.91 min ) . fluorescence absorption and
emission spectra
indicated max values at 634 and 650 nm , respectively ( supporting information ) , as measured in solution
in a cuvette using a spectramax m5 reader ( molecular devices , sunnyvale ,
ca ) .
uridine 5-monophosphate
disodium salt ( 4.4 mg , 11.0 mol , 2 equiv ) and n-(3-phenylpropoxy)cytidine-5-triphosphate triethylammonium
salt 2 ( 4.5 mg , 5.4 mol , 1 equiv ) were converted
to the tributylammonium salts by treatment with ion - exchange resin
( dowex 50wx2 - 200 ( h ) ) and tributylamine .
after removal of the water ,
the obtained uridine monophosphate dibutylammonium salt and the tributylammonium
salt of n - phenylpropoxycytidine-5-triphosphate 2 ( 4.5 mg , 5.5 mol , 1 equiv ) were dried under high
vacuum for 1 h in separate vials .
dic ( 1 l , 5.5 mol ,
1 equiv ) was added to a solution of n-(3-phenylpropoxy)cytidine-5-triphosphate tributylammonium
salt 2 in dmf ( 200 l ) . after stirring the reaction
mixture at rt for 3 h , a solution of the uridine 5-monophosphate
tributylammonium salt ( 11.0 mol ) and mgcl2 ( 1.0
mg , 11.0 mol , 2 equiv ) in dmf ( 100 l ) were added .
after removal of the
solvent , the mgcl2 was removed by treatment with ion - exchange
resin ( dowex 50wx2 - 200 ( h ) ) and ammonia bicarbonate or tri - n - butylamine , and the residue was purified by a semipreparative
hplc purification using system c. compound 30 ( 1.0 mg ,
0.8 mol , 16% ) was obtained as a white solid . h
nmr ( d2o ) 7.93 ( d , j = 7.9 hz ,
1h ) , 7.357.30 ( m , j = 8.3 hz , 4h ) , 7.257.21
( m , 2h ) , 6.025.91 ( m , 2h ) , 5.78 ( d , j = 7.9
hz , 1h ) , 4.424.33 ( m , 4h ) , 4.304.16 ( m , 6h ) , 4.05
( t , j = 4.8 hz , 2h ) , 2.74 ( t , j =
8.3 hz , 2h ) , 2.051.79 ( m , 2h ) .
p nmr ( d2o ) 11.51 ( br ) , 23.22 ( br ) .
compound 31 ( 0.5 mg , 0.3 mol , 10% ) was obtained as a white solid using n-(3-phenylpropoxy)cytidine-5-triphosphate
triethylammonium salt ( 3.0 mg , 3.0 mol ) and n-(3-phenylpropoxy)cytidine-5-monophosphate diethylammonium
salt ( 3.0 mg , 6.1 mol ) from the previous procedure .
h nmr ( d2o ) 7.457.10 ( m , 12h ) , 5.955.87
( m , 2h ) , 5.795.63 ( m , 2h ) , 4.394.29 ( m , 4h ) , 4.274.16
( m , 6h ) , 4.064.00 ( m , 4h ) , 2.802.65 ( m , 4h ) , 2.051.93
( m , 4h ) .
synthesis of compounds 4096 is
described in the supporting information . a suspension of cytidine ( 360 mg , 1.5 mmol )
and 4-phenylbutoxyamine hydrochloride ( 87 , 617 mg , 3.0
mmol , 2 equiv ) in pyridine ( 4 ml ) was stirred at 100 c overnight .
the reaction mixture was evaporated , and the residue was evaporated
twice with toluene , triturated with chloroform , and filtered . the
filtrate was evaporated , and the residue was purified by flash chromatography
( chloroform methanol , gradient of 310% ) to afford n-(4-phenylbutyloxy)cytidine ( 97 ) ( 0.430 g , 1.10 mmol , 73% ) .
h nmr ( 400 mhz , meod ) : 7.52
( d , j1 = 8.20 hz , 1h ) , 7.207.17
( m , 2h ) , 7.157.05 ( m , 3h ) , 5.83 ( d , j1 = 4.84 hz , 1h ) , 5.63 ( d , j1 =
8.12 hz , 1h ) , 4.13 ( t , j1 = 5.20 hz , 1h ) ,
4.09 ( m , 1h ) , 4.003.92 ( m , 3h ) , 3.78 ( dd , j1 = 12.16 hz , j1 = 2.72 hz ,
1h ) , 3.68 ( dd , j1 = 12.20 hz , j2 = 3.16 hz , 1h ) , 2.60 ( t , j1 = 6.88 hz , 2h ) , 1.701.60 ( m , 4h ) .
c nmr ( 400
mhz , meod ) : 149.2 , 147.6 , 142.0 , 137.1 , 135.0 , 128.0 , 127.9 , 125.4 ,
95.0 , 88.9 , 84.8 , 74.4 , 73.7 , 69.9 , 60.9 , 35.1 , 27.7 , 27.3 .
m / z ( m + esi ms ) found , 392.1813 ; calcd
for c19h26n3o6 , 392.1816 .
compound 115 ( 45 mg , 88.5
mol , 65% ) was obtained as a white solid from cytidine ( 29 mg ,
0.12 mmol ) and n-(3-(4-tert - butyloxy - carbonylmethoxy)phenyl)propyloxyamine hydrochloride ( 91 ) ( 79 mg , 0.25 mmol ) using the above procedure .
h nmr ( 400 mhz , meod ) : 7.16 ( d , j1 =
8.24 hz , 1h ) , 7.06 ( d , j1 = 8.36 hz , 2h ) ,
6.76 ( d , j1 = 8.52 hz , 1h ) , 5.82 ( d , j1 = 5.20 hz , 1h ) , 5.55 ( d , j1 = 8.24 hz , 1h ) , 4.46 ( s , 2h ) , 4.134.10 ( m , 2h ) ,
3.943.91 ( m , 3h ) , 3.76 ( d , j1 =
12.08 hz , 1h ) , 3.66 ( dd , j1 = 12.12 hz , j2 = 3.00 hz , 1h ) , 2.58 ( t , j1 = 7.08 hz , 2h ) , 2.011.86(m , 2h ) , 1.42(s , 9h ) .
c nmr ( 400 mhz , meod ) : 169.0 , 156.2 , 150.1 , 144.7 , 134.7 ,
131.4 , 129.1 , 114.2 , 97.6 , 88.4 , 84.6 , 82.0 , 73.4 , 72.8 , 70.3 , 65.4 ,
61.3 , 30.8 , 30.4 , 27.0 .
m / z ( m +
esi ms ) found , 508.2294 ; calcd for c24h34n3o9 , 508.2290 .
n-(3-((4-tert - butyloxy - carbonylmethoxy)phenyl)propyloxy)cytidine 115 ( 44 mg , 0.12 mmol ) was dissolved in 1.0 ml of thf and
treated with 2.0 ml of 1 n naoh .
the reaction mixture was stirred
at 50 c for 2 h. the mixture was cooled to rt and neutralized
by adding 1n hcl .
after removal of the solvent , n-(3-((4-methoxycarbonate)phenyl)propyloxy)cytidine ( 31
mg , 68 mol , 56% ) was isolated after column chromatography .
h nmr ( 400 mhz , meod ) : 7.17 ( d , j1 = 8.24 hz , 1h ) , 7.09 ( d , j1 = 8.44 hz ,
2h ) , 6.81 ( d , j1 = 8.64 hz , 1h ) , 5.82
( d , j1 = 5.44 hz , 1h ) , 5.55 ( d , j1 = 8.24 hz , 1h ) , 4.57 ( s , 2h ) , 4.12 ( t , j1 = 5.40 hz , 1h ) , 4.08 ( m , 1h ) , 3.953.90
( m , 3h ) , 3.74 ( dd , j1 = 12.08 hz , j1 = 2.88 hz , 1h ) , 3.66 ( dd , j1 = 12.16 hz , j2 = 3.40 hz ,
1h ) , 2.60 ( t , j1 = 7.40 hz , 2h ) , 1.951.86
( m , 2h ) .
c nmr ( 400 mhz , meod ) : 156.2 , 150.1 , 144.7 ,
134.7 , 131.3 , 129.0 , 114.1 , 97.4 , 88.3 , 84.7 , 73.3 , 72.7 , 70.3 , 64.6 ,
61.3 , 30.8 , 30.4 , 27.0 .
m / z ( m +
esi ms ) found , 442.1665 ; calcd for c20h26n3o9 , 452.1664 .
the above isolated acid ( 50
mg , 0.1 mmol ) , diea ( 0.128 ml , 0.93 mmol , 8.5 equiv ) , and 6.0 ml of
dmf were added to round - bottom flask . then 4-amino-1-butyne ( 13.5
l , 0.16 mmol , 1.5 equiv ) and 1.0 ml of dmf were added to the
above mixture , which was then stirred for 10 min at rt .
pybop ( 57
mg , 0.11 mmol , 1.1 equiv ) was added , and the mixture was stirred overnight
.
solvent was removed , and 116 ( 45.1 mg , 89.9 mol ,
80% ) was isolated following column chromatography .
h nmr
( 400 mhz , meod ) : 7.17 ( d , j1 = 8.28 hz ,
1h ) , 7.11 ( d , j1 = 8.56 hz , 2h ) , 6.86
( d , j1 = 8.64 hz , 2h ) , 5.82 ( d , j1 = 5.44 hz , 1h ) 5.53 ( d , j1 = 8.24 hz , 1h ) , 4.43 ( s , 2h ) , 4.154.00 ( m , 2h ) ,
3.95 ( t , j1 = 6.36 hz , 2h ) , 3.91 ( q , j1 = 3.32 hz , 1h ) , 3.75 ( dd , j1 = 12.16 hz , j1 = 2.88 hz ,
1h ) , 3.66 ( dd , j1 = 12.12 hz , j2 = 3.36 hz , 1h ) , 3.38 ( t , j1 = 7.08 hz , 2h ) , 2.62 ( t , j1 = 7.40 hz , 2h ) , 2.37 ( td , j1 = 7.08
hz , j1 = 2.64 hz , 2h ) , 2.25 ( t , j1 = 2.64 hz , lh ) , 2.011.85 ( m , 2h ) .
c nmr ( 400 mhz , meod ) : 170.11 , 155.98 , 150.03 , 144.68 , 135.27 ,
131.37 , 12 129.5 , 123.9 , 114.7 , 114.5 , 112.1 , 111.9 , 97.4 , 88.3 , 84.7 ,
73.3 , 72.5 , 70.3 , 61.3 , 31.4 , 30.1 .
m / z ( m + h esi ms ) found , 503.2139 ; calcd for c24h31n4o8 , 503.2136 .
stable cell lines
for study of the human ( h ) p2y2 , p2y4 , and p2y6rs were produced by retroviral expression of the individual
receptors in 1321n1 human astrocytoma cells , which do not natively
express p2yrs .
agonist - stimulated [ h]inositol phosphate accumulation was quantified in cells
plated at 20000 cells / well on 96-well plates two days prior to assay .
the inositol lipid pool of cells was radiolabeled 16 h prior to the
assay by incubation with 100 l of serum - free inositol - free
dulbecco s modified eagle s medium , containing 1.0 ci
of [ h]myo - inositol .
test
drugs were added in 25 l of 100 mm hepes ( n-(2-hydroxyethyl)-piperazine - n-2-ethanesulfonic
acid ) , ph 7.3 , in hbss containing 50 mm licl .
incubations were for
30 min at 37 c and were terminated by aspiration of the drug - containing
medium and addition of 90 l of ice - cold 50 mm formic acid .
the samples were neutralized with 30 l of 150 mm nh4oh and applied to dowex ag1-x8 anion exchange columns .
total inositol
phosphates were eluted , and radioactivity was measured using a liquid
scintillation counter .
agonist potencies ( ec50 values )
were determined from concentration response curves by nonlinear
regression analysis using the graphpad software package prism ( graphpad ,
san diego , ca ) .
each concentration of drug was tested in triplicate
assays , and concentration effect curves for each test drug were repeated
in at least three separate experiments with freshly diluted molecule .
the results are presented as mean sem from multiple experiments
or in the case of concentration effect curves from a single experiment
carried out with triplicate assays that were representative of results
from multiple experiments .
1321n1 human
astrocytoma cells
overexpressing the p2y6r were grown in dmem with 5% fbs ,
50 u / ml penicillin / streptomycin , and 2 mm l - glutamine .
cells
were grown in 6-well plates ( approximately 3 10 cells / well ) and incubated at 37 c and 5% co2 for
24 h. at 80% confluency , the medium was replaced with fresh preheated
medium , in addition to ,-methylene - adenosine 5-diphosphate
( 125 , amp - cp , 100 m ) and a known agonist or antagonist
of the p2y6r .
after a 30 min incubation , 28 was added at different time intervals and the decrease in fluorescence
intensity was measured by fcm .
binding of 28 to the p2y6r in overexpressing 1321n1 astrocytoma cells was blocked by
using known p2y6r ligands , such as antagonists 118 and 119 or the agonist 120 ( all 10 m ) .
ecto - nucleotidase inhibitor 125 ( 100 m ) was added
to prevent the metabolism of 5-di and triphosphate derivatives
to the 5-monophosphates .
after a 30 min preincubation
with the appropriate agonist or antagonist and 125 ( 100
m ) , 28 ( 100 nm ) was added at different time intervals
from 20 to 30 min . at the end of incubation ,
the medium was removed ,
the cells were washed three times with ice - cold dpbs , and 1 ml of
0.2% edta was added to each well to detach the cells from the plate .
the cell
suspensions were transferred to 5 ml of polystyrene round - bottom bd
falcon tubes ( bd , franklin lakes , nj ) and centrifuged for 5 min at
23 c and 400 g . after the supernatant was discarded ,
the cells were washed with 3 ml of dbps and centrifuged again at 23
c and 400 g for 5 min .
after centrifugation ,
the supernatant was discarded and the cells suspended in 0.5 ml of
dpbs for analysis by fcm .
the intensity of fluorescence
emission
of each sample was measured by using fcm .
cell suspensions were vortexed
briefly before analysis on a becton and dickinson facscalibur flow
cytometer ( bd , franklin lakes , nj ) .
measured fluorescent
intensities ( mfis ) were obtained in the fl-4 channel in log mode .
data were collected
and analyzed using cell quest pro software ( bd , franklin lakes , nj ) .
p2y6r - expressing
astrocytoma cells were grown on glass coverslips placed in a 6-well
plate .
when the cells reached 80% confluence , the medium was replaced
with fresh medium , p2y6r antagonist 118 ( 10
m ) was added to the cells , and incubation was continued for
30 min at 37 c .
then 16 was added to achieve a
final concentration of 1 m , and incubation continued for 30
min at 37 c . at the end of the incubation ,
the cells were washed
with ice - cold pbs and mounted on a glass slide .
data analysis was performed with the
prism 5 ( graphpad , san diego ca ) software .
the mean autofluorescence
of 1321n1 astrocytoma cells was measured in the absence of the fluorescent
ligand .
the mean fluorescence intensity in the presence of fluorescent
ligand was corrected by subtracting the autofluorescence . | extended n4-(3-arylpropyl)oxy derivatives
of uridine-5-triphosphate were synthesized and potently stimulated
phospholipase c stimulation in astrocytoma cells expressing g protein - coupled
human ( h ) p2y receptors ( p2yrs ) activated by utp ( p2y2/4r ) or udp ( p2y6r ) .
the potent p2y4r - selective n4-(3-phenylpropyl)oxy agonist was phenyl ring - substituted
or replaced with terminal heterocyclic or naphthyl rings with retention
of p2yr potency .
this broad tolerance for steric bulk in a distal
region was not observed for dinucleoside tetraphosphate agonists with
both nucleobases substituted . the potent n4-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 ( ec50 : p2y2r , 47 nm ; p2y4r , 23 nm ) was functionalized
for chain extension using click tethering of fluorophores as prosthetic
groups .
the bodipy 630/650 conjugate 28 ( mrs4162 ) exhibited
ec50 values of 70 , 66 , and 23 nm at the hp2y2/4/6rs , respectively , and specifically labeled cells expressing the p2y6r .
thus , an extended n4-(3-arylpropyl)oxy
group accessed a structurally permissive region on three gq - coupled p2yrs , and potency and selectivity were modulated by distal
structural changes .
this freedom of substitution was utilized to design
of a pan - agonist fluorescent probe of a subset of uracil nucleotide - activated
hp2yrs . | Introduction
Results
Discussion
Conclusions
Experimental
Section | the p2y receptors ( p2yrs )
are g protein - coupled receptors ( gpcrs )
that are activated by extracellular nucleotides . the widely expressed utp - activated
( p2y2r and p2y4r ) and udp - activated ( p2y6r ) receptors belong to the subfamily of p2y1-like
receptors that promote inositol lipid signaling through gq - mediated activation of phospholipase c- ( plc- ) . few
selective agonists and antagonists are
available for pharmacological differentiation of these uracil nucleotide - regulated
receptors , and p2y2r , p2y4r , and p2y6r exhibit much overlap in affinities for pyrimidine nucleotides . this study was initiated to synthesize nucleoside 5-triphosphate
derivatives as potent agonist probes of the p2y4r , including
chain derivatization for incorporation of bulky reporter groups . thus , ligands of the p2y4r are of potential interest
for pharmacotherapies directed at intestinal function , angiogenesis ,
cardiac remodeling , postischemic revascularization , cns function ,
and inflammation . uridine-5-triphosphate
( utp , 1 ) is the endogenous
p2y4r agonist ( ec50 = 80 nm ) , while
adenosine-5-triphosphate ( atp ) antagonizes the human homologue
of this receptor and activates the rat homologue . the 4-benzyloxyiminopyrimidine group was identified as a structurally
permissive site for synthesis of functionalized congeners , leading to high affinity molecular probes for
the closely related udp - activated p2y6r . one goal of these studies
was to generate an analogue(s ) with tolerance for steric bulk when
bound to the receptor , potentially allowing attachment of a fluorophore
or other reporter group to serve as a tracer for the receptor on live
cells . aec50 values for
activation of plc via the human p2y4r are : 2 , 23 nm ; 2-azido-2-deoxy - utp 3 , 1100
nm ; iso - cmp 4 , 4980 nm . ec50 values for
activation of plc via the human p2y4r are : 2 , 23 nm ; 2-azido-2-deoxy - utp 3 , 1100
nm ; iso - cmp 4 , 4980 nm . the synthetic routes to these groups of structurally
modified pyrimidine nucleotides include : utp analogues containing
a n - alkoxy group ( scheme 1 ) , modified up4u dinucleotide analogues ( scheme 2 ) , and utp analogues containing alexa fluor 488
and bodipy 630/650 fluorescent dyes ( scheme 3 ) . functional assays were conducted
with 1321n1 astrocytoma cells expressing recombinant hp2y2 , p2y4 , or p2y6rs . these alkoxyamine derivatives were synthesized from the corresponding
3-arylpropyl bromides using gabriel synthesis or a variation thereof
using n , n-di - tert - butoxycarbonylhydroxylamine as the source of the hydroxylamine moiety . the 3-arylpropyl bromides 73 , 76 , and 78 were synthesized
from the corresponding alcohols and converted to the alkyoxyimino
derivatives ( scheme 4b ) . the fluorescent
alexa fluor 488 5-triphosphate 27 and bodipy
5-triphosphate 28 derivatives were synthesized
from alkyne nucleotide precursor 26 and an azide derivative
( e.g. the newly synthesized analogues
were tested for potency against
the p2y2r , p2y4r , and p2y6r . similar n-3-phenylalkyloxy derivatives
were shown to be only weakly active at the p2y14r ; thus , this subtype was not part of the initial
screen . the potencies of nine known reference compounds ( 2 , 56 , 89 , and 29 ) were included for comparison . standard assays of potency for activation of plc were performed
as described in 1321n1 human astrocytoma
cell lines stably expressing either the p2y2r , p2y4r , or p2y6r
( 1321n1-p2yr cells ) . among substituted
phenyl groups , 3-f 12 , 4-i 16 , and 4-meo 19 provided relatively high potency
at the three p2y2 , p2y4 , and p2y6rs . thus , we decided to elongate functionalized congeners based on p - substituted ethers related to 19 . other aryl
moieties that appeared to enhance potency were thien-3-yl 22 and naphth-2-yl 23 , indicating a tolerance of steric
bulk in this region . high potency at the p2y4r was achieved
with a terminal alkyne at the n - linked
chain for tethering sterically bulky groups by click chemistry ( 26 , ec50 = 40 nm , figure 1 ) . however , potency at p2y2r and p2y6r also
was largely retained in this ether- and amide - linked alkyne , with
ec50 values of 109 and 183 nm , respectively . comparison
of potencies of alkyne 26 and fluorescent
analogues 27 and 28 in activation of plc
via the human p2y2r ( a ) , p2y4r ( b ) , and p2y6r ( c ) in stably transfected 1321n1 human astrocytoma cells . curiously , in comparison to the precursor
molecule 26 , the bodipy conjugate exhibited enhanced
potency at the p2y2r ( ec50 = 66 nm ) and p2y6r ( ec50 = 23 nm ) but slightly decreased potency
at the p2y4r . thus , 28 is a high affinity
pan - agonist fluorescent probe with nearly equivalent potency at the
hp2y2r , hp2y4r , and hp2y6r . the level of cell - associated fluorescence was not reduced by coincubation
of 28 with 10 m n - methyl-2-deoxyadenosine 3,5-bisphosphate ( 117 , mrs2179 ) , a p2y1r - selective antagonist that is inactive
at p2y2 , p2y4 , and p2y6rs . however , significant decreases in mean fluorescence
intensity were observed after preincubation with p2y6r
ligands , including the p2y6r - selective noncompetitive antagonist n , n-1,4-butanediyl - bis[n-(3-isothiocyanatophenyl)]thiourea ( 118 , mrs2578 , 10 m ) and the competitive antagonist suramin ( 119 , 10 m ) . the potent p2y6r - selective
dinucleotide agonist p-(uridine 5-)-p-(n - methoxycytidine
5-)triphosphate ( 120 , mrs2957 , 10 m ) produced the largest decrease in cell - associated
fluorescence . fluorescence microscopy comparing binding to 1321n1-p2y6r cells in the presence and absence of 118 ( figure 3 ) indicated that 1 m 28 specifically
labeled cells expressing the receptor . binding of 28 at 20 and 30 min to the
p2y6r was significantly blocked after preincubation with
selective antagonist 118 , selective agonist 120 , and nonselective antagonist 119 . fluorescent micrographs using a keyence fluorescent microscope
( bz-9000 ) of p2y6r - expressing 1321n1 astrocytoma cells
exposed to the fluorescent agonist 28 ( 1 m , 30
min incubation at 37 c ) . we have identified a functionalization approach
that provides versatile
nucleotide analogues for further derivatization of high potency p2y2r , p2y4r , and p2y6r agonists . the initial
approach was based on a p2y4r - selective agonist , and many
of the new derivatives tend toward selectivity at this subtype . however ,
upon elongation of a functionalized chain at the n position , significant potency also was observed at the
related p2y2r and p2y6r subtypes . other possible approaches could have been through chain elongation
at the 2 , 3 , or 5 positions of a uracil ring . modifications of the distal phenyl ring of a 3-phenylpropyloxyimino
group , shown previously to be optimal for p2y4r potency ,
allowed a bulky fluorescent reporter group to be introduced , but the
p2y4r selectivity was compromised . in cells expressing a single p2yr subtype ,
such as our astrocytoma cells
, there is no ambiguity . following the
development of standard assay conditions , the potent fluorescent ligand 28
could potentially be used for high - throughput screening
of drug libraries with p2y2r- , p2y4r- , or p2y6r - expressing cells . the high potency of 28 at the hp2y2r , hp2y4r , and hp2y6r suggests the existence of energetically
favorable interactions with groups on the conformationally flexible
extracellular regions of p2y2r and p2y6r . further characterization
of the location of the fluorescent label within the cells as well
as delineation of the saturability , reversibility , kinetics , and other
measurements will be required before this probe molecule can be used
routinely for fcm labeling of cells expressing p2yrs . a series of n-(3-arylpropyloxy ) derivatives
of uridine-5-triphosphate were synthesized and found to display
high potency as agonists of the initial target g protein - coupled hp2y4r and also hp2y2r and hp2y6r . the phenyl
group could be substituted with heterocyclic rings or a naphthyl ring
with retention of p2yr affinity , indicating a broad tolerance for
steric bulk in this distal region of the nucleotide . thus , an extended n-(3-arylpropyloxy ) group accessed a structurally
permissive region on various gq - coupled p2yrs and was modulated
by distal structural changes to alter selectivity . this freedom of
substitution can be utilized for the design of pan - agonist affinity
probes of the uracil nucleotide - activated , gq - coupled p2y2/4/6 subset of receptors . all nucleotide derivatives tested
for biological activity showed > 98% purity by hplc analysis ( detection
at 254 nm ) . a solution of the nucleoside 97112 , 114 ( 73.0 mol ) , and 1,8-bis(dimethylamino)naphthalene
( proton sponge , 24.0 mg , 0.11 mmol ) in trimethyl phosphate ( 0.4 ml )
was stirred for 10 min at 0 c . then ,
phosphorus oxychloride ( 13.0 l , 0.13 mmol ) was added dropwise ,
and the reaction mixture was stirred for 2 h at 0 c . a solution
of tributylammonium pyrophosphate ( 0.80 ml , 0.44 mmol ) and tributylamine
( 69.0 l , 0.29 mmol ) in n
, n - dimethylformamide ( dmf , 1 ml ) was added , and stirring was continued
at 0 c for additional 10 min . then 0.2 m triethylammonium bicarbonate
solution ( 1.5 ml ) was added , and the clear solution was stirred at
rt for 1 h. after removal of solvents , the residue was purified by
sephadex - deae a-25 resin and preparative hplc . to a mixture of alexa
fluor 488 azide ( alexa fluor 488 5-carboxamido-(6-azidohexanyl ) , bis(triethylammonium
salt ) ) , 5-isomer , 0.5 mg , 0.8 mol , 1 equiv ) , and alkyne 26 ( 0.8 mg , 0.8 mol , 1.4 equiv ) in a 0.20 ml of ( 1:1 )
mixture of buoh and water , were added tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl ] amine ( tbta , 0.1 mg ) and freshly
prepared 1 m aqueous sodium ascorbate solution ( 1.6 mol , 1.6
l , 2 equiv ) followed by 7.5% aqueous copper sulfate pentahydrate
solution ( 4.0 l , 1.2 mol , 1.5 equiv ) . to a mixture of bodipy
630/650 azide ( 124 , supporting information , 1.0 mg , 1.0 mol , 1 equiv ) and alkyne 26 ( 2.0
mg , 1.8 mol , 1.2 equiv ) in a 200 l of ( 1:1 ) mixture
of buoh and water , and 20 l of dmf , were added
tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl ] amine
( tbta , 0.05 mg ) and freshly prepared 1 m aqueous sodium ascorbate
solution ( 1.1 l , 1.6 mol , 1 equiv ) followed by 7.5%
aqueous copper sulfate pentahydrate solution ( 2.5 l , 0.8 mol ,
0.8 equiv ) . fluorescence absorption and
emission spectra
indicated max values at 634 and 650 nm , respectively ( supporting information ) , as measured in solution
in a cuvette using a spectramax m5 reader ( molecular devices , sunnyvale ,
ca ) . uridine 5-monophosphate
disodium salt ( 4.4 mg , 11.0 mol , 2 equiv ) and n-(3-phenylpropoxy)cytidine-5-triphosphate triethylammonium
salt 2 ( 4.5 mg , 5.4 mol , 1 equiv ) were converted
to the tributylammonium salts by treatment with ion - exchange resin
( dowex 50wx2 - 200 ( h ) ) and tributylamine . after removal of the
solvent , the mgcl2 was removed by treatment with ion - exchange
resin ( dowex 50wx2 - 200 ( h ) ) and ammonia bicarbonate or tri - n - butylamine , and the residue was purified by a semipreparative
hplc purification using system c. compound 30 ( 1.0 mg ,
0.8 mol , 16% ) was obtained as a white solid . after removal of the solvent , n-(3-((4-methoxycarbonate)phenyl)propyloxy)cytidine ( 31
mg , 68 mol , 56% ) was isolated after column chromatography . pybop ( 57
mg , 0.11 mmol , 1.1 equiv ) was added , and the mixture was stirred overnight
. solvent was removed , and 116 ( 45.1 mg , 89.9 mol ,
80% ) was isolated following column chromatography . stable cell lines
for study of the human ( h ) p2y2 , p2y4 , and p2y6rs were produced by retroviral expression of the individual
receptors in 1321n1 human astrocytoma cells , which do not natively
express p2yrs . agonist potencies ( ec50 values )
were determined from concentration response curves by nonlinear
regression analysis using the graphpad software package prism ( graphpad ,
san diego , ca ) . 1321n1 human
astrocytoma cells
overexpressing the p2y6r were grown in dmem with 5% fbs ,
50 u / ml penicillin / streptomycin , and 2 mm l - glutamine . cells
were grown in 6-well plates ( approximately 3 10 cells / well ) and incubated at 37 c and 5% co2 for
24 h. at 80% confluency , the medium was replaced with fresh preheated
medium , in addition to ,-methylene - adenosine 5-diphosphate
( 125 , amp - cp , 100 m ) and a known agonist or antagonist
of the p2y6r . binding of 28 to the p2y6r in overexpressing 1321n1 astrocytoma cells was blocked by
using known p2y6r ligands , such as antagonists 118 and 119 or the agonist 120 ( all 10 m ) . ecto - nucleotidase inhibitor 125 ( 100 m ) was added
to prevent the metabolism of 5-di and triphosphate derivatives
to the 5-monophosphates . after a 30 min preincubation
with the appropriate agonist or antagonist and 125 ( 100
m ) , 28 ( 100 nm ) was added at different time intervals
from 20 to 30 min . at the end of incubation ,
the medium was removed ,
the cells were washed three times with ice - cold dpbs , and 1 ml of
0.2% edta was added to each well to detach the cells from the plate . p2y6r - expressing
astrocytoma cells were grown on glass coverslips placed in a 6-well
plate . when the cells reached 80% confluence , the medium was replaced
with fresh medium , p2y6r antagonist 118 ( 10
m ) was added to the cells , and incubation was continued for
30 min at 37 c . | [
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the phenomenon of population aging has been observed throughout the world and brazil is one of the countries with the fastest rates of aging . in 2025 , brazil will be the 6th country in number of elderly people worldwide .
there was a decrease of acute processes that evolve to cure or death and an increase in chronic degenerative non - communicable diseases ( ncds ) , which nowadays are primarily responsible for disability and mortality in our population .
elderly has the highest prevalence of comorbidity , disability , and frailty of any age group . because of these characteristics the treatment of older adults with pain is complex and affected by age - related changes in pharmacokinetics and pharmacodynamics .
polypharmacy , defined as either the use of multiple medications or the use of unnecessary medications , is common in older people and increases the risk of adverse drug reactions , nonadherence , and increased cost . nowadays
most elderly people die after a period of deterioration caused by one or more chronic progressive illnesses and the suffering that characterizes the period before death can be related , at least in part , to the experience of unrelieved symptoms .
pain is among the most prevalent of these symptoms and management of chronic nonmalignant and malignant pain prevents disability and improves quality of life in elderly at the end of life . despite this , pain management is reported to be poor in elderly .
chronic pain encompasses a complex array of sensory - discriminatory , motivational - affective , and cognitive - evaluative components .
because of this complexity , both pharmacologic and nonpharmacologic approaches should be considered to treat pain .
given the large number of older persons with pain at the end of life and the few data about this issue , the objective of this article is to review the pharmacologic and nonpharmacologic approaches of the pain treatment in this population .
we searched the cochrane library , medline and lilacs from 1990 to 2011 and the references in retrieved manuscripts .
the two most commonly used scales for adults are a verbal descriptor scale ( i.e. which word best describes your pain ; none , mild moderate , severe or excruciating ? ) or a numerical scale ( i.e. on a scale from 0 to 10 , where 0 indicates no pain and 10 indicates the worst pain you can imagine , how would you rate your pain ? ) .
however , elderly patients often have significant cognitive , motor , visual , or auditory impairments that must be considered when evaluating the presence and intensity of symptoms , and the assessment method should be modified accordingly . some signs and symptoms
are suggestive of the presence of pain and can be used to assess non - communicative elderly and patients with cognitive impairment or delirium ( table 1 ) .
assessment of pain in the confused non - communicative elderly pain can be alleviated in elderly through the careful use of analgesic drags combined with nonpharmacologic strategies .
the nonpharmacological treatment of pain at the end of life has been little explored and yet little used .
lack of a scientific support for the use of these resources has hindered their implementation as effective tools for the treatment of pain .
however , these non invasive methods can be of great help to patients suffering from pain .
the association of nonpharmacologic resources with the pharmacological treatment can help reduce the use of analgesics minimizing the side effects of long term medication .
physical therapiesthe role of physiotherapists in palliative care is primarily defined by expert opinion , which is not backed up by good quality evidence .
however , an absence of research evidence does not mean that a service or treatment is ineffective , just that we do nt know for certain yet .
the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources.physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort .
therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . because persistent pain is commonly associated with prolonged physical inactivity , these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
psychological therapiesformal cognitive - behavioral therapies are helpful for many older adults with persistent pain .
oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death .
patients and families learn to utilize their preexisting coping abilities and to develop new ones for difficult situations , including uncontrolled pain[1315 ] .
delirium and cognitive impairment are obstacles to psychotherapy.complementary and alternative therapiesunrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy .
although there is little scientific evidence for the efficacy of most of these strategies for controlling persistent pain , it is important that clinicians not leave patients with a sense of hopelessness as a result of their efforts to discourage unapproved but benign therapies or to debunk healthcare quackery and fraud .
the effectiveness of homeopathy as a symptomatic treatment for acute and chronic pain remains unproven .
recent neuroscience research suggested that acupuncture induces clinical responses though the modulation of the nervous system .
a cochrane 's review concluded that touch therapies may have a modest effect in pain relief and more studies on healing touch and reiki in relieving pain are needed .
another cochrane 's review showed that listening to music reduces pain intensity levels and opioid requirements , but the magnitude of these benefits is small and , therefore , its clinical importance unclear .
table 2 shows the national center for complementary and alternative medicine ( nccam ) classification of available complementary and alternative medicine ( cam ) .
the role of physiotherapists in palliative care is primarily defined by expert opinion , which is not backed up by good quality evidence .
however , an absence of research evidence does not mean that a service or treatment is ineffective , just that we do nt know for certain yet .
the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources .
physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort .
therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . because persistent pain is commonly associated with prolonged physical inactivity , these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range .
cold / heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points .
electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients .
exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . because persistent pain is commonly associated with prolonged physical inactivity
, these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
psychological therapies formal cognitive - behavioral therapies are helpful for many older adults with persistent pain .
oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death .
patients and families learn to utilize their preexisting coping abilities and to develop new ones for difficult situations , including uncontrolled pain[1315 ] .
complementary and alternative therapies unrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy .
although there is little scientific evidence for the efficacy of most of these strategies for controlling persistent pain , it is important that clinicians not leave patients with a sense of hopelessness as a result of their efforts to discourage unapproved but benign therapies or to debunk healthcare quackery and fraud .
the effectiveness of homeopathy as a symptomatic treatment for acute and chronic pain remains unproven .
recent neuroscience research suggested that acupuncture induces clinical responses though the modulation of the nervous system .
a cochrane 's review concluded that touch therapies may have a modest effect in pain relief and more studies on healing touch and reiki in relieving pain are needed .
another cochrane 's review showed that listening to music reduces pain intensity levels and opioid requirements , but the magnitude of these benefits is small and , therefore , its clinical importance unclear .
table 2 shows the national center for complementary and alternative medicine ( nccam ) classification of available complementary and alternative medicine ( cam ) .
the two most commonly used scales for adults are a verbal descriptor scale ( i.e. which word best describes your pain ; none , mild moderate , severe or excruciating ? ) or a numerical scale ( i.e. on a scale from 0 to 10 , where 0 indicates no pain and 10 indicates the worst pain you can imagine , how would you rate your pain ? ) .
however , elderly patients often have significant cognitive , motor , visual , or auditory impairments that must be considered when evaluating the presence and intensity of symptoms , and the assessment method should be modified accordingly . some signs and symptoms
are suggestive of the presence of pain and can be used to assess non - communicative elderly and patients with cognitive impairment or delirium ( table 1 ) .
assessment of pain in the confused non - communicative elderly pain can be alleviated in elderly through the careful use of analgesic drags combined with nonpharmacologic strategies .
the nonpharmacological treatment of pain at the end of life has been little explored and yet little used .
lack of a scientific support for the use of these resources has hindered their implementation as effective tools for the treatment of pain .
however , these non invasive methods can be of great help to patients suffering from pain .
the association of nonpharmacologic resources with the pharmacological treatment can help reduce the use of analgesics minimizing the side effects of long term medication .
physical therapiesthe role of physiotherapists in palliative care is primarily defined by expert opinion , which is not backed up by good quality evidence .
however , an absence of research evidence does not mean that a service or treatment is ineffective , just that we do nt know for certain yet .
the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources.physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort .
therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . because persistent pain is commonly associated with prolonged physical inactivity , these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
psychological therapiesformal cognitive - behavioral therapies are helpful for many older adults with persistent pain .
oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death .
patients and families learn to utilize their preexisting coping abilities and to develop new ones for difficult situations , including uncontrolled pain[1315 ] .
delirium and cognitive impairment are obstacles to psychotherapy.complementary and alternative therapiesunrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy .
although there is little scientific evidence for the efficacy of most of these strategies for controlling persistent pain , it is important that clinicians not leave patients with a sense of hopelessness as a result of their efforts to discourage unapproved but benign therapies or to debunk healthcare quackery and fraud .
the effectiveness of homeopathy as a symptomatic treatment for acute and chronic pain remains unproven .
recent neuroscience research suggested that acupuncture induces clinical responses though the modulation of the nervous system .
a cochrane 's review concluded that touch therapies may have a modest effect in pain relief and more studies on healing touch and reiki in relieving pain are needed .
another cochrane 's review showed that listening to music reduces pain intensity levels and opioid requirements , but the magnitude of these benefits is small and , therefore , its clinical importance unclear .
table 2 shows the national center for complementary and alternative medicine ( nccam ) classification of available complementary and alternative medicine ( cam ) .
the role of physiotherapists in palliative care is primarily defined by expert opinion , which is not backed up by good quality evidence .
however , an absence of research evidence does not mean that a service or treatment is ineffective , just that we do nt know for certain yet .
the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources .
physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort .
therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . because persistent pain is commonly associated with prolonged physical inactivity , these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range .
cold / heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points .
electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients .
exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain .
because persistent pain is commonly associated with prolonged physical inactivity , these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
psychological therapies formal cognitive - behavioral therapies are helpful for many older adults with persistent pain .
oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death .
patients and families learn to utilize their preexisting coping abilities and to develop new ones for difficult situations , including uncontrolled pain[1315 ] .
complementary and alternative therapies unrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy .
although there is little scientific evidence for the efficacy of most of these strategies for controlling persistent pain , it is important that clinicians not leave patients with a sense of hopelessness as a result of their efforts to discourage unapproved but benign therapies or to debunk healthcare quackery and fraud .
the effectiveness of homeopathy as a symptomatic treatment for acute and chronic pain remains unproven .
recent neuroscience research suggested that acupuncture induces clinical responses though the modulation of the nervous system .
a cochrane 's review concluded that touch therapies may have a modest effect in pain relief and more studies on healing touch and reiki in relieving pain are needed .
another cochrane 's review showed that listening to music reduces pain intensity levels and opioid requirements , but the magnitude of these benefits is small and , therefore , its clinical importance unclear .
table 2 shows the national center for complementary and alternative medicine ( nccam ) classification of available complementary and alternative medicine ( cam ) .
the two most commonly used scales for adults are a verbal descriptor scale ( i.e. which word best describes your pain ; none , mild moderate , severe or excruciating ? ) or a numerical scale ( i.e. on a scale from 0 to 10 , where 0 indicates no pain and 10 indicates the worst pain you can imagine , how would you rate your pain ? ) .
however , elderly patients often have significant cognitive , motor , visual , or auditory impairments that must be considered when evaluating the presence and intensity of symptoms , and the assessment method should be modified accordingly . some signs and symptoms
are suggestive of the presence of pain and can be used to assess non - communicative elderly and patients with cognitive impairment or delirium ( table 1 ) .
pain can be alleviated in elderly through the careful use of analgesic drags combined with nonpharmacologic strategies .
the nonpharmacological treatment of pain at the end of life has been little explored and yet little used .
lack of a scientific support for the use of these resources has hindered their implementation as effective tools for the treatment of pain .
however , these non invasive methods can be of great help to patients suffering from pain .
the association of nonpharmacologic resources with the pharmacological treatment can help reduce the use of analgesics minimizing the side effects of long term medication .
physical therapiesthe role of physiotherapists in palliative care is primarily defined by expert opinion , which is not backed up by good quality evidence .
however , an absence of research evidence does not mean that a service or treatment is ineffective , just that we do nt know for certain yet .
the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources.physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort .
therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . because persistent pain is commonly associated with prolonged physical inactivity , these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
psychological therapiesformal cognitive - behavioral therapies are helpful for many older adults with persistent pain .
oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death .
patients and families learn to utilize their preexisting coping abilities and to develop new ones for difficult situations , including uncontrolled pain[1315 ] .
delirium and cognitive impairment are obstacles to psychotherapy.complementary and alternative therapiesunrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy .
although there is little scientific evidence for the efficacy of most of these strategies for controlling persistent pain , it is important that clinicians not leave patients with a sense of hopelessness as a result of their efforts to discourage unapproved but benign therapies or to debunk healthcare quackery and fraud .
the effectiveness of homeopathy as a symptomatic treatment for acute and chronic pain remains unproven .
recent neuroscience research suggested that acupuncture induces clinical responses though the modulation of the nervous system .
a cochrane 's review concluded that touch therapies may have a modest effect in pain relief and more studies on healing touch and reiki in relieving pain are needed .
another cochrane 's review showed that listening to music reduces pain intensity levels and opioid requirements , but the magnitude of these benefits is small and , therefore , its clinical importance unclear .
table 2 shows the national center for complementary and alternative medicine ( nccam ) classification of available complementary and alternative medicine ( cam ) .
the role of physiotherapists in palliative care is primarily defined by expert opinion , which is not backed up by good quality evidence .
however , an absence of research evidence does not mean that a service or treatment is ineffective , just that we do nt know for certain yet .
the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources .
physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . because persistent pain is commonly associated with prolonged physical inactivity , these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range .
cold / heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points .
electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease .
therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients .
exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . because persistent pain is commonly associated with prolonged physical inactivity , these effects may be partly due to the reversal of the physiologic consequences of reconditioning .
psychological therapies formal cognitive - behavioral therapies are helpful for many older adults with persistent pain .
oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death .
patients and families learn to utilize their preexisting coping abilities and to develop new ones for difficult situations , including uncontrolled pain[1315 ] .
complementary and alternative therapies unrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy .
although there is little scientific evidence for the efficacy of most of these strategies for controlling persistent pain , it is important that clinicians not leave patients with a sense of hopelessness as a result of their efforts to discourage unapproved but benign therapies or to debunk healthcare quackery and fraud .
the effectiveness of homeopathy as a symptomatic treatment for acute and chronic pain remains unproven .
recent neuroscience research suggested that acupuncture induces clinical responses though the modulation of the nervous system .
a cochrane 's review concluded that touch therapies may have a modest effect in pain relief and more studies on healing touch and reiki in relieving pain are needed .
another cochrane 's review showed that listening to music reduces pain intensity levels and opioid requirements , but the magnitude of these benefits is small and , therefore , its clinical importance unclear .
table 2 shows the national center for complementary and alternative medicine ( nccam ) classification of available complementary and alternative medicine ( cam ) .
inferences about the type and mechanisms that may be responsible for the pain are helpful in the evaluation and the management of pain syndromes .
a)nociceptive pain : may be visceral or somatic and is most often derived from the stimulation of pain receptors .
nociceptive pain may arise from tissue inflammation , mechanical deformation , ongoing injury or destruction .
nociceptive mechanisms usually respond well to traditional approaches to pain management , including common analgesic medications and nonpharmacologic strategies.b)neuropathic pain : results from a pathophysiologic process that involves the peripheral or central nervous system .
examples include diabetic neuropathy , trigeminal neuralgia , post - herpetic neuralgia , poststroke central or thalamic pain , and postamputation phantom limb pain .
these pain syndromes do not respond as predictably as do nociceptive pain to conventional analgesic therapy .
however , they have been noted to respond to unconventional analgesic drugs , such as tricyclic antidepressants , anticonvulsants , or antiarrhythmic drugs .
nociceptive pain : may be visceral or somatic and is most often derived from the stimulation of pain receptors .
nociceptive pain may arise from tissue inflammation , mechanical deformation , ongoing injury or destruction .
nociceptive mechanisms usually respond well to traditional approaches to pain management , including common analgesic medications and nonpharmacologic strategies .
neuropathic pain : results from a pathophysiologic process that involves the peripheral or central nervous system .
examples include diabetic neuropathy , trigeminal neuralgia , post - herpetic neuralgia , poststroke central or thalamic pain , and postamputation phantom limb pain .
these pain syndromes do not respond as predictably as do nociceptive pain to conventional analgesic therapy .
however , they have been noted to respond to unconventional analgesic drugs , such as tricyclic antidepressants , anticonvulsants , or antiarrhythmic drugs .
the use of analgesics for the treatment of acute pain is the same as for non - palliative care patients .
according to world health organization treatment is escalated in an orderly manner from non - opioid to weak opioid to strong opioid - analgesic ladder(tables 3 and 4 ) .
world health organization ( who ) analgesic ladder adjuvant drugs . adapted from doyle d , 2008
the only international guidelines that are available are from the american geriatric society , the most recent being from 2002 which made a number of important recommendations ( table 5 ) .
however , the approach may require modification because of comorbidities or physiologic changes associated with aging .
polypharmacy , defined as either the use of multiple medications or the use of unnecessary medications , is common in older people and increases the risk of adverse drug reactions , nonadherence , and increased cost .
several drugs used to treat pain , such as opioids , tricyclic antidepressants , gabapentin , and pregabalin , are among those associated with sedation , dizziness , and falls , particularly in frail or vulnerable elderly patients .
drug absorption is generally unchanged , but studies of drug distribution reveal increased plasma concentration of water - soluble drugs and increased half - life of fat - soluble drugs in older adults . for all opioids half - life of the active drug and metabolites is increased in the elderly .
it is , therefore , recommended that doses be reduced , a longer time interval be used between doses , and creatinine clearance be monitored .
age - related changes in hepatic metabolism and renal excretion ( adapted from schmader et al )
a)non - opwids : the non - opioid analgesics include acetylsalicylic acid ( asa ) , other nsaids , and acetaminophen .
1)nsaids : the gastrointestinal harm induced by nsaids may be the most prevalent adverse event associated with this drug .
gastrointestinal adverse events are more likely to occur with long - term use and with higher doses of the nsaid .
advanced age is a risk factor for gastrointestinal complications and a proton pump inhibitor or misoprostol should be used during the treatment with the nsaids .
the use of cox-2 inhibitors may reduce the risk for gastrointestinal events ; however , this benefit is negated if the patient is using aspirin , even at low doses .
the patient populations most likely to be prescribed nsaids for pain relief are often the same populations with cardiovascular risk factors and it does not appear that the toxic effects can be avoided by restricting their use to short durations .
selection of therapy must weigh cardiovascular risk as well as benefit , other risks of therapy , and costs .
the cardiovascular risks first appeared to be a class effect of the cox-2 ( cycloxygenase-2 ) inhibitors .
naproxen and ibuprofen appear to be the safest with respect to cardiovascular risk , when specifically measuring cardiovascular events and mortality .
naproxen appears to convey a statistically significant protective outcome in terms of cardiovascular events however for those with cardiovascular disease or risk factors for ischemic heart disease the american heart association recommends acetaminophen , aspirin , tramadol , and opioid analgesics as firstline therapy .
although the rate of nsaid - induced nephrotoxicity has been estimated to be low ( 1 - 5% ) the nsaids , including cox-2 inhibitors , must be used cautiously or not at all in patients with predisposing renal conditions .
it is important to monitor blood pressure , weight , and serum creatinine and potassium concentrations when appropriate , use the lowest dose possible , and consider limiting dietary salt intake.2)acetaminophen : the adverse effects are liver and renal toxicity and caution is necessary in patients with liver damage .
both tramadol and codeine require dose adjustment for renal impairment.c)strong opioids : all opioids are considered effective in cancer and non - cancer pain management , but no well designed specific studies in the elderly patient are available .
higher opioid doses are often needed for neuropathic pain than for nociceptive pain . with all opioids ,
the adverse reactions and events can be limited by using lower starting doses , longer dose intervals , and slow titration ; however , constipation , nausea , and vomiting often require prophylaxis or therapy .
other common adverse reactions with opioids use include sedation , impaired judgment , impaired psychomotor function and respiratory depression.d)adjuvant drugs : the adjuvant drugs ( table 4 ) may be necessary for one of three reasons :
to treat the adverse effects of analgesics ( e.g. antiemetics and laxatives);to enhance pain relief ( e.g. a corticosteroid in nerve compression pain and a anticonvulsant in neuropathic pain);to treat concomitant psychological disturbances such as insomnia , anxiety and depression ( e.g. night sedatives , anxiolytics , antidepressants ) .
non - opwids : the non - opioid analgesics include acetylsalicylic acid ( asa ) , other nsaids , and acetaminophen .
1)nsaids : the gastrointestinal harm induced by nsaids may be the most prevalent adverse event associated with this drug . gastrointestinal adverse events are more likely to occur with long - term use and with higher doses of the nsaid .
advanced age is a risk factor for gastrointestinal complications and a proton pump inhibitor or misoprostol should be used during the treatment with the nsaids .
the use of cox-2 inhibitors may reduce the risk for gastrointestinal events ; however , this benefit is negated if the patient is using aspirin , even at low doses .
the patient populations most likely to be prescribed nsaids for pain relief are often the same populations with cardiovascular risk factors and it does not appear that the toxic effects can be avoided by restricting their use to short durations .
selection of therapy must weigh cardiovascular risk as well as benefit , other risks of therapy , and costs .
the cardiovascular risks first appeared to be a class effect of the cox-2 ( cycloxygenase-2 ) inhibitors .
naproxen and ibuprofen appear to be the safest with respect to cardiovascular risk , when specifically measuring cardiovascular events and mortality .
naproxen appears to convey a statistically significant protective outcome in terms of cardiovascular events however for those with cardiovascular disease or risk factors for ischemic heart disease the american heart association recommends acetaminophen , aspirin , tramadol , and opioid analgesics as firstline therapy .
although the rate of nsaid - induced nephrotoxicity has been estimated to be low ( 1 - 5% ) the nsaids , including cox-2 inhibitors , must be used cautiously or not at all in patients with predisposing renal conditions .
it is important to monitor blood pressure , weight , and serum creatinine and potassium concentrations when appropriate , use the lowest dose possible , and consider limiting dietary salt intake.2)acetaminophen : the adverse effects are liver and renal toxicity and caution is necessary in patients with liver damage .
nsaids : the gastrointestinal harm induced by nsaids may be the most prevalent adverse event associated with this drug .
gastrointestinal adverse events are more likely to occur with long - term use and with higher doses of the nsaid .
advanced age is a risk factor for gastrointestinal complications and a proton pump inhibitor or misoprostol should be used during the treatment with the nsaids .
the use of cox-2 inhibitors may reduce the risk for gastrointestinal events ; however , this benefit is negated if the patient is using aspirin , even at low doses .
the patient populations most likely to be prescribed nsaids for pain relief are often the same populations with cardiovascular risk factors and it does not appear that the toxic effects can be avoided by restricting their use to short durations .
selection of therapy must weigh cardiovascular risk as well as benefit , other risks of therapy , and costs .
the cardiovascular risks first appeared to be a class effect of the cox-2 ( cycloxygenase-2 ) inhibitors .
naproxen and ibuprofen appear to be the safest with respect to cardiovascular risk , when specifically measuring cardiovascular events and mortality .
naproxen appears to convey a statistically significant protective outcome in terms of cardiovascular events however for those with cardiovascular disease or risk factors for ischemic heart disease the american heart association recommends acetaminophen , aspirin , tramadol , and opioid analgesics as firstline therapy .
although the rate of nsaid - induced nephrotoxicity has been estimated to be low ( 1 - 5% ) the nsaids , including cox-2 inhibitors , must be used cautiously or not at all in patients with predisposing renal conditions .
it is important to monitor blood pressure , weight , and serum creatinine and potassium concentrations when appropriate , use the lowest dose possible , and consider limiting dietary salt intake .
acetaminophen : the adverse effects are liver and renal toxicity and caution is necessary in patients with liver damage .
strong opioids : all opioids are considered effective in cancer and non - cancer pain management , but no well designed specific studies in the elderly patient are available .
higher opioid doses are often needed for neuropathic pain than for nociceptive pain . with all opioids ,
the adverse reactions and events can be limited by using lower starting doses , longer dose intervals , and slow titration ; however , constipation , nausea , and vomiting often require prophylaxis or therapy .
other common adverse reactions with opioids use include sedation , impaired judgment , impaired psychomotor function and respiratory depression .
adjuvant drugs : the adjuvant drugs ( table 4 ) may be necessary for one of three reasons :
to treat the adverse effects of analgesics ( e.g. antiemetics and laxatives);to enhance pain relief ( e.g. a corticosteroid in nerve compression pain and a anticonvulsant in neuropathic pain);to treat concomitant psychological disturbances such as insomnia , anxiety and depression ( e.g. night sedatives , anxiolytics , antidepressants ) .
to treat the adverse effects of analgesics ( e.g. antiemetics and laxatives ) ; to enhance pain relief ( e.g. a corticosteroid in nerve compression pain and a anticonvulsant in neuropathic pain ) ; to treat concomitant psychological disturbances such as insomnia , anxiety and depression ( e.g. night sedatives , anxiolytics , antidepressants ) .
elderly is a population at risk for developing side effects of drugs acting on central nervous system .
dizziness , somnolence or sedation , and peripheral edema are reported common side effects of anticonvulsants .
dry mouth and somnolence or sedation are reported side effects of tricyclic antidepressants and benzodiazepines .
the exclusion of older adults with comorbidities and the underrepresentation of the old old
( people > 80 years ) are phenomena seen in clinical trials of medications for neuropathic pain .
table 7 shows drug classes , examples and adverse reactions of the pharmacological treatment of the neuropathic pain .
when other treatments fail to relieve suffering in the imminently dying patient , pst may be a valid palliative care option .
pst is defined as the use of specific sedative medications to relieve intolerable suffering from refractory symptoms by a reduction in patient consciousness , using appropriate drugs carefully titrated to the cessation of symptoms .
the initial dose of sedatives should usually be small enough to maintain the patients ability to communicate periodically . in the case of continuous and deep pst
, the disease should be irreversible and advanced , with death expected within hours to days .
both pharmacologic and nonpharmacologic approaches should be considered to treat pain in elderly at the end of life .
it is difficult to make specific recommendations about the long - term use of complementary and alternative therapies but it is important that clinicians not leave patients with a sense of hopelessness as a result of their efforts to discourage unapproved but benign therapies . the sequential use of analgesics drugs according the who ladder is a relatively inexpensive yet effective method for relieving pain .
all opioids are considered effective and are used in cancer and non - cancer pain management , but no well designed specific studies in the elderly patient are available . to relieve suffering in the imminently dying patient palliative sedation may be a valid palliative care option . | background : the treatment of older adults with pain is complex and affected by age - related changes in pharmacokinetics and pharmacodynamics .
chronic pain encompasses a complex array of sensory - discriminatory , motivational - affective , and cognitive - evaluative components .
because of this complexity , both pharmacologic and nonpharmacologic approaches should be considered to treat pain.aims:given the large number of older persons with pain at the end of life and the few data about this issue , the objective of this article is to review the treatment of pain in this population.patients and methods : we searched the cochrane library , medline and lilacs from 1990 to 2011 and the references in retrieved manuscripts .
the search terms were pain and elderly and end of life.results:there are evidences of undertreatment among elderly people .
the association of nonpharmacologic resources with the pharmacological treatment can help reduce the use of analgesics minimizing the side effects of long term medication .
pharmacological treatment is escalated in an orderly manner from non - opioid to weak opioid to strong opioid .
adjuvant drugs like anticonvulsants and antidepressants may be necessary.conclusions:the sequential use of analgesics drugs and opioids are considered effective and relatively inexpensive for relieving pain , but no well designed specific studies in the elderly patient are available .
there are not specific recommendations about the long - term use of complementary and alternative therapies and although their effectiveness remains unproven they should not be discouraged .
palliative sedation may be a valid palliative care option to relieve suffering in the imminently dying patient . | Introduction
Methods
None
Pain assessment in the elderly
Nonpharmacologic therapy of pain
Pharmacologic therapy of pain
Nociceptive pain
Elderly's particularities and cautions
Neuropathic pain
Palliative sedation
Conclusions | because of these characteristics the treatment of older adults with pain is complex and affected by age - related changes in pharmacokinetics and pharmacodynamics . polypharmacy , defined as either the use of multiple medications or the use of unnecessary medications , is common in older people and increases the risk of adverse drug reactions , nonadherence , and increased cost . pain is among the most prevalent of these symptoms and management of chronic nonmalignant and malignant pain prevents disability and improves quality of life in elderly at the end of life . chronic pain encompasses a complex array of sensory - discriminatory , motivational - affective , and cognitive - evaluative components . because of this complexity , both pharmacologic and nonpharmacologic approaches should be considered to treat pain . given the large number of older persons with pain at the end of life and the few data about this issue , the objective of this article is to review the pharmacologic and nonpharmacologic approaches of the pain treatment in this population . we searched the cochrane library , medline and lilacs from 1990 to 2011 and the references in retrieved manuscripts . however , elderly patients often have significant cognitive , motor , visual , or auditory impairments that must be considered when evaluating the presence and intensity of symptoms , and the assessment method should be modified accordingly . some signs and symptoms
are suggestive of the presence of pain and can be used to assess non - communicative elderly and patients with cognitive impairment or delirium ( table 1 ) . assessment of pain in the confused non - communicative elderly pain can be alleviated in elderly through the careful use of analgesic drags combined with nonpharmacologic strategies . the nonpharmacological treatment of pain at the end of life has been little explored and yet little used . lack of a scientific support for the use of these resources has hindered their implementation as effective tools for the treatment of pain . the association of nonpharmacologic resources with the pharmacological treatment can help reduce the use of analgesics minimizing the side effects of long term medication . the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources.physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . psychological therapiesformal cognitive - behavioral therapies are helpful for many older adults with persistent pain . oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death . delirium and cognitive impairment are obstacles to psychotherapy.complementary and alternative therapiesunrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range . cold / heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients . exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . psychological therapies formal cognitive - behavioral therapies are helpful for many older adults with persistent pain . oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death . complementary and alternative therapies unrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy . the effectiveness of homeopathy as a symptomatic treatment for acute and chronic pain remains unproven . however , elderly patients often have significant cognitive , motor , visual , or auditory impairments that must be considered when evaluating the presence and intensity of symptoms , and the assessment method should be modified accordingly . some signs and symptoms
are suggestive of the presence of pain and can be used to assess non - communicative elderly and patients with cognitive impairment or delirium ( table 1 ) . assessment of pain in the confused non - communicative elderly pain can be alleviated in elderly through the careful use of analgesic drags combined with nonpharmacologic strategies . the nonpharmacological treatment of pain at the end of life has been little explored and yet little used . lack of a scientific support for the use of these resources has hindered their implementation as effective tools for the treatment of pain . the association of nonpharmacologic resources with the pharmacological treatment can help reduce the use of analgesics minimizing the side effects of long term medication . the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources.physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . psychological therapiesformal cognitive - behavioral therapies are helpful for many older adults with persistent pain . oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death . delirium and cognitive impairment are obstacles to psychotherapy.complementary and alternative therapiesunrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients . exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . psychological therapies formal cognitive - behavioral therapies are helpful for many older adults with persistent pain . oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death . complementary and alternative therapies unrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy . however , elderly patients often have significant cognitive , motor , visual , or auditory impairments that must be considered when evaluating the presence and intensity of symptoms , and the assessment method should be modified accordingly . some signs and symptoms
are suggestive of the presence of pain and can be used to assess non - communicative elderly and patients with cognitive impairment or delirium ( table 1 ) . the nonpharmacological treatment of pain at the end of life has been little explored and yet little used . lack of a scientific support for the use of these resources has hindered their implementation as effective tools for the treatment of pain . the association of nonpharmacologic resources with the pharmacological treatment can help reduce the use of analgesics minimizing the side effects of long term medication . the maintenance or improvement of the functional component of quality of life and significant improvements in pain are noted in patients who received optimized levels of physiotherapy time and resources.physical therapy techniques used in pain control :
massage : touch is probably the oldest method of relieving pain and discomfort . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . psychological therapiesformal cognitive - behavioral therapies are helpful for many older adults with persistent pain . oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death . delirium and cognitive impairment are obstacles to psychotherapy.complementary and alternative therapiesunrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range.cold/heat : where local skin sensation is normal and tissue is intact , local applications of heat and ice may be used to reduce spasm , for acute inflammatory conditions , and over myofascial trigger points.electrophysical modalities : transcutaneous electrical nerve stimulation ( tens ) is the only electrical modality currently recommended for use in the presence of active neoplastic disease . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients.exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . therapeutic massage using stroking and gentle kneading may be used to reduce muscle spasm , relieve pain , aid relaxation , and maintenance of joint range . therapeutic ultrasound , interferential or pulsed shortwave diathermy relieves pain and muscle spasm and may be used over normal tissue and for non - cancer patients . exercise : systematic reviews of observational and randomized controlled clinical trials conclude that there is strong evidence that participation in regular physical activity reduces the pain and enhances the functional capacity of older adults with persistent pain . psychological therapies formal cognitive - behavioral therapies are helpful for many older adults with persistent pain . oriented therapy may provide patients and families with the support , knowledge , and skills they need to cope with cancer treatment , pain , and death . complementary and alternative therapies unrelieved persistent pain commonly causes patients to seek relief with alternative medicine , including homeopathy , acupuncture , healing touch , and music therapy . inferences about the type and mechanisms that may be responsible for the pain are helpful in the evaluation and the management of pain syndromes . the use of analgesics for the treatment of acute pain is the same as for non - palliative care patients . according to world health organization treatment is escalated in an orderly manner from non - opioid to weak opioid to strong opioid - analgesic ladder(tables 3 and 4 ) . adapted from doyle d , 2008
the only international guidelines that are available are from the american geriatric society , the most recent being from 2002 which made a number of important recommendations ( table 5 ) . polypharmacy , defined as either the use of multiple medications or the use of unnecessary medications , is common in older people and increases the risk of adverse drug reactions , nonadherence , and increased cost . several drugs used to treat pain , such as opioids , tricyclic antidepressants , gabapentin , and pregabalin , are among those associated with sedation , dizziness , and falls , particularly in frail or vulnerable elderly patients . drug absorption is generally unchanged , but studies of drug distribution reveal increased plasma concentration of water - soluble drugs and increased half - life of fat - soluble drugs in older adults . age - related changes in hepatic metabolism and renal excretion ( adapted from schmader et al )
a)non - opwids : the non - opioid analgesics include acetylsalicylic acid ( asa ) , other nsaids , and acetaminophen . gastrointestinal adverse events are more likely to occur with long - term use and with higher doses of the nsaid . advanced age is a risk factor for gastrointestinal complications and a proton pump inhibitor or misoprostol should be used during the treatment with the nsaids . the use of cox-2 inhibitors may reduce the risk for gastrointestinal events ; however , this benefit is negated if the patient is using aspirin , even at low doses . both tramadol and codeine require dose adjustment for renal impairment.c)strong opioids : all opioids are considered effective in cancer and non - cancer pain management , but no well designed specific studies in the elderly patient are available . other common adverse reactions with opioids use include sedation , impaired judgment , impaired psychomotor function and respiratory depression.d)adjuvant drugs : the adjuvant drugs ( table 4 ) may be necessary for one of three reasons :
to treat the adverse effects of analgesics ( e.g. non - opwids : the non - opioid analgesics include acetylsalicylic acid ( asa ) , other nsaids , and acetaminophen . gastrointestinal adverse events are more likely to occur with long - term use and with higher doses of the nsaid . advanced age is a risk factor for gastrointestinal complications and a proton pump inhibitor or misoprostol should be used during the treatment with the nsaids . the use of cox-2 inhibitors may reduce the risk for gastrointestinal events ; however , this benefit is negated if the patient is using aspirin , even at low doses . gastrointestinal adverse events are more likely to occur with long - term use and with higher doses of the nsaid . advanced age is a risk factor for gastrointestinal complications and a proton pump inhibitor or misoprostol should be used during the treatment with the nsaids . the use of cox-2 inhibitors may reduce the risk for gastrointestinal events ; however , this benefit is negated if the patient is using aspirin , even at low doses . strong opioids : all opioids are considered effective in cancer and non - cancer pain management , but no well designed specific studies in the elderly patient are available . adjuvant drugs : the adjuvant drugs ( table 4 ) may be necessary for one of three reasons :
to treat the adverse effects of analgesics ( e.g. to treat the adverse effects of analgesics ( e.g. dizziness , somnolence or sedation , and peripheral edema are reported common side effects of anticonvulsants . the exclusion of older adults with comorbidities and the underrepresentation of the old old
( people > 80 years ) are phenomena seen in clinical trials of medications for neuropathic pain . table 7 shows drug classes , examples and adverse reactions of the pharmacological treatment of the neuropathic pain . when other treatments fail to relieve suffering in the imminently dying patient , pst may be a valid palliative care option . pst is defined as the use of specific sedative medications to relieve intolerable suffering from refractory symptoms by a reduction in patient consciousness , using appropriate drugs carefully titrated to the cessation of symptoms . in the case of continuous and deep pst
, the disease should be irreversible and advanced , with death expected within hours to days . both pharmacologic and nonpharmacologic approaches should be considered to treat pain in elderly at the end of life . it is difficult to make specific recommendations about the long - term use of complementary and alternative therapies but it is important that clinicians not leave patients with a sense of hopelessness as a result of their efforts to discourage unapproved but benign therapies . the sequential use of analgesics drugs according the who ladder is a relatively inexpensive yet effective method for relieving pain . all opioids are considered effective and are used in cancer and non - cancer pain management , but no well designed specific studies in the elderly patient are available . to relieve suffering in the imminently dying patient palliative sedation may be a valid palliative care option . | [
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] |
radical nephrectomy , partial nephrectomy , and ablative modalities are well described [ 1 , 2 ] .
the open , laparoscopic , and percutaneous approaches are prevalent techniques [ 1 , 2 ] .
robotic partial nephrectomy is in a fledgling state , but the complexity and reconstructive nature of laparoscopic partial nephrectomy may theoretically be well suited to a robotic assisted approach . there have been a few reports of the procedure , with differing techniques [ 35 ] . as evidenced with robotic prostatectomy , there are several advantages of da vinci assisted laparoscopic surgery .
the robotic approach subjectively allows better visualization , improved control via multiple effector arms , and reduced surgeon fatigue .
the goal of this publication is to describe one method to perform a da vinci four - arm approach to partial nephrectomy using a parallelogram port placement .
this approach has been validated in tumors in all locations except directly posterior midpole lesions .
robotic partial nephrectomy will be outlined in several steps , with a similar format as a recently published communication regarding robotic prostatectomy . for the purposes of this article
, the instruments in the working arms may be interchanged depending on whether or the surgeon is right or left handed.step 1 : trocar placement with parallelogram arrangement and docking ( fig .
1).fig . 1four - arm approach to da vinci partial nephrectomy . 1 cephalad arm .
. assistant port is not visualized , but is usually placed between the camera port and the cephalad arm .
umbilicus is visualized step 1 : trocar placement with parallelogram arrangement and docking ( fig .
assistant port is not visualized , but is usually placed between the camera port and the cephalad arm .
umbilicus is visualized four - arm approach to da vinci partial nephrectomy . 1 cephalad arm .
assistant port is not visualized , but is usually placed between the camera port and the cephalad arm .
umbilicus is visualized three robotic trocars with cannulae.two 12 mm disposable trocars ( camera and assistant).handheld electrocautery ( 35 w).insufflation needle .
extensive ex vivo work was performed to create a trocar arrangement that could be used in most renal tumors .
several challenges were present , especially since the kidney is many times larger than the prostate , and thus , a larger range of motion is necessary .
the trocar arrangement was optimized for hilar dissection and retraction , with the acceptance of some minor collisions in the extreme upper and lower parts of the surgical field .
the camera and the cephalad arm are always paired on one side of the robot ( with the joint on the camera arm towards the cephalad arm ) , and the caudal and fourth arms are paired on the other side of the robot .
after the abdomen is inspected , the remaining robotic trocars are placed , as seen in fig . 1 .
the trocars are placed 812 cm apart depending on the size of the patient s torso .
the assistant s trocar is placed in between the camera and the cephalad port . for the purposes of this article
, the ports will be referred to as cephalad ( ce ) , lateral ( la ) , caudal ( ca ) , assistant ( as ) , and camera ( cam ) . in this communication
the ce and la arms are classified as the working arms . the caudal port is attached to the fourth arm . in older three - arm da vinci systems , the assistant could place a second trocar in lieu of the fourth arm .
docking is performed diagonally so that the center of the robot is in a line with the camera port and the renal hilum ( usually off the tip of the 11th rib ) .
the robot s position may be tested prior to the patient arriving in the room to ensure that the arms do not become contaminated by collisions with anesthesia monitors or overhead lights .
after insufflation and port placement , the robot is docked , and the primary surgeon engages the console .
the author prefers to attach insufflation tubing to the assistant s trocar . for obese patients ,
all ports should be shifted laterally.step 2 : reflection of the colon ( fig .
2colon is reflected over the anterior surface of the kidney step 2 : reflection of the colon ( fig .
2colon is reflected over the anterior surface of the kidney colon is reflected over the anterior surface of the kidney working arms ( ce and la ) : monopolar scissor ( 35 w ) and prograsp ( alternative : bipolar or plasmakinetic forceps).caudal arm ( ca ) : dual - blade atrial retractor.assistant : suction / irrigator.scope : 30 downward orientation .
working arms ( ce and la ) : monopolar scissor ( 35 w ) and prograsp ( alternative : bipolar or plasmakinetic forceps ) .
caudal arm ( ca ) : dual - blade atrial retractor . assistant : suction / irrigator .
reflection of the colon is a critical step , as it confers exposure for the remainder of the operation .
when beginning ones experience with robotic renal surgery , the surgeon should reflect the colon as much as possible to allow wide access to the retroperitoneum .
however , with experience , the surgeon may choose to reflect the colon in a more limited fashion .
the peritoneum should be incised and the colon reflected medially until gerota s fascia is visible along the medial length of the kidney . note that the left colon lies quite laterally in comparison to the right colon , and more mobilization may be needed on the left . under robotic visualization , the surgeon may see that the perinephric fat and gerota s fascia often possess a paler yellow color than the mesenteric fat .
the proper plane between the peritoneum and retroperitoneum is often avascular , so the colo - renal attachments may be swept downwards or cut with an athermal technique when the plane is reached .
the cephalad extent of the dissection should occur up to the liver edge or splenic edge and then laterally along the edge of the spleen or liver .
the mesenteric fat and the colo - renal attachments can be retracted by the assistant or with the fourth arm .
the surgeon can easily provide countertraction with the prograsp , and the dual - blade retractor may be used to push the colon medially . on the right ,
on the left , the ureter and the gonadal vein may be identified , but the aorta is often not directly seen , since it is surrounded by more adherent lymphatics and fat .
caution is advised near the iliac vessels and appendix when incising the peritoneum near the pelvis ; one usually does not need to dissect so caudally .
caution is also advised on dissecting too medially on the right side and hitting the duodenum .
on the left , it is critical to avoid the pancreas by performing the dissection directly on the perinephric fat.step 3 : lateral retraction of the lower pole of the kidney to put hilum on stretch ( fig .
3the kidney is retracted laterally to allow exposure and dissection of the renal hilum step 3 : lateral retraction of the lower pole of the kidney to put hilum on stretch ( fig .
3the kidney is retracted laterally to allow exposure and dissection of the renal hilum the kidney is retracted laterally to allow exposure and dissection of the renal hilum working arms ( ce and la ) : monopolar scissor and prograsp.fourth arm ( ca ) : dual - blade retractor.assistant : suction / irrigator . working arms ( ce and la ) : monopolar scissor and prograsp .
fourth arm ( ca ) : dual - blade retractor . assistant : suction / irrigator . lateral retraction of the kidney , originally popularized by kavoussi laparoscopically , suspends the kidney to allow the renal hilum to be on stretch .
open surgeons who perform flank incisions also lateralize the kidney when they lift the kidney up from the psoas muscle during dissection .
after reflection of the colon and identification of the ureter , the psoas muscle is identified medial to the ureter .
once a glimpse of the psoas muscle is identified , it is exploited with upward retraction of the kidney and perinephric fat .
the dual - blade atrial retractor is the ideal instrument to cradle the kidney from its medial surface .
the retractor is placed under the lower pole of the kidney and is used to elevate the kidney away from the great vessels .
as such , the renal hilum is stretched and working space is created between the kidney and the great vessels .
any remaining colo - renal attachments will be visualized as they will also be placed on stretch and they can be taken down sharply .
once the hilum is on stretch , there are several maneuvers to find the renal vein : one may trace the gonadal vein to the cava and then trace the cava to the renal vein ( or , on the left , trace the gonadal vein directly to the renal vein ) . with experience
this tracing becomes unnecessary , and one may actually visualize the large biphasic venous pulsation .
the console surgeon may use the scissors to gently push the fat off the anterior surface of the renal vein to gain exposure .
once the renal vein is identified , the artery is then dissected with the help of the assistant who can retract the vein cranially and caudally .
the artery is generally harder to identify than the vein ; however it is unnecessary to totally clean off the periarterial fat and lymphatics . on the left side
it may be difficult to identify the artery posterior to the renal vein , usually because the left gonadal vein obscures the underlying arterial vessel . to gain mobility around the renal vein , the surgeon may staple or clip the gonadal vein near the renal vein . on the right side ,
the artery is often easier to identify since the right renal vein is rarely tethered by other branches.step 4 : dissection of kidney off of the adrenal gland ( fig .
4the upper pole is mobilized from the adrenal attachments step 4 : dissection of kidney off of the adrenal gland ( fig .
4the upper pole is mobilized from the adrenal attachments the upper pole is mobilized from the adrenal attachments working arms ( ce and la ) : monopolar scissor and prograsp.fourth arm ( ca ) : dual - blade retractor or robotic clip applier.assistant : suction / irrigator . working arms ( ce and la ) : monopolar scissor and prograsp .
fourth arm ( ca ) : dual - blade retractor or robotic clip applier . assistant : suction / irrigator .
dissection of the kidney s upper pole from the adrenal gland is useful for tumors in the upper part of the kidney , in which the adrenal resection is not anticipated .
often , there are significant unnamed blood vessels traveling in between the adrenal and the kidney .
the area between the kidney parenchyma and adrenal gland can be divided with careful use of monopolar cautery .
the assistant can thin out the fat with blunt dissection and strands are divided by the console surgeon .
the prograsp is useful to give countertraction to the assistant s suction device to allow dissection of the area .
the fourth arm ( ca ) with the dual - blade retractor should be repositioned on the anterior surface of the kidney to retract the whole kidney caudally .
alternatively , the dual - blade retractor could be exchanged for a robotic clip applier .
if the upper pole renal tumor is very large , then the surgeon may elect to resect the adrenal gland en bloc with the mass .
thus , the surgeon will be dissecting along the lateral border of the vena cava or aorta .
in such cases , careful dissection is necessary to visualize the adrenal vein ( right ) or the aorta ( left side ) .
to gain additional exposure for adrenal resection , the colon may need additional medialization . on the right , the colon should be reflected such that the infrahepatic cava is visualized ; this involves incising the peritoneum along the edge of the liver .
on the left side , the surgeon may incise the lateral splenic attachments near the diaphragm and the splenorenal ligaments .
the spleen will then fall medially with the colon , gaining exposure to the medial aspect of the left adrenal gland .
the surgeon should generally not take down the splenocolic attachments , as several blood vessels travel in this area .
if collisions ensue or mobility is impaired , the scissors are switched to the cephalad arm and the fourth - arm instrument is removed .
on the right , superior retraction of the liver is often needed with an extra 5 mm port in the subxyphoid location.step 5 : remove fat around lesion to expose renal parenchyma ( fig .
5the parenchymal margins of the kidney are exposed by removing the fat around the lesion step 5 : remove fat around lesion to expose renal parenchyma ( fig .
5the parenchymal margins of the kidney are exposed by removing the fat around the lesion the parenchymal margins of the kidney are exposed by removing the fat around the lesion working arms ( ce and la ) : monopolar scissors and prograsp.fourth arm ( ca ) : dual - blade retractor.assistant : laparoscopic ultrasound probe , suction / irrigator , 10 mm spoon .
fourth arm ( ca ) : dual - blade retractor . assistant : laparoscopic ultrasound probe , suction / irrigator , 10 mm spoon .
the tumor is identified with intraoperative ultrasound . using cautery and retraction , the parenchyma is exposed circumferentially around the tumor .
exposure of the parenchyma is important , as this edge will be sewn later in the case .
the fourth arm can be used to give countertraction on the fat or the kidney .
if fat is totally detached , the assistant may extract the tissue with a 10 mm spoon .
technically , removal of adipose tissue around the tumor is not difficult except in patients with abundant adherent perinephric fat .
if the perinephric fat does not easily slide off the kidney , this step becomes very tedious and time consuming , with numerous entries into vessels in gerota s fascia . in the initial experience
, it would be prudent to avoid patients with abundant perinephric fat . during exposure of the renal parenchyma
all anticipated staff changes should occur during this step to maintain consistency of staff during tumor excision .
thrombogenic agents should be prepared so there are no distractions during clamping and tumor excision .
intraoperative ultrasound is used to define the margins of resection and the parenchyma may be scored to delineate the margins .
the deep margins should be mentally visualized with three - dimensional spatial orientation.step 6 : clamping the renal hilum ( fig .
6the renal hilum is clamped step 6 : clamping the renal hilum ( fig .
6the renal hilum is clamped the renal hilum is clamped working arms ( ce and la ) : monopolar scissors and prograsp.fourth arm ( ca ) : dual - blade retractor.assistant : laparoscopic bulldog applier and multiple bulldogs . working arms ( ce and la ) : monopolar scissors and prograsp .
fourth arm ( ca ) : dual - blade retractor . assistant : laparoscopic bulldog applier and multiple bulldogs .
some bulldogs are controlled by the console surgeon ; most are placed by the assistant .
mannitol ( 12.5 or 25 gm ) and furosemide ( 1020 mg ) may be given to the patient prior to vascular occlusion .
the hilum is exposed by lifting the lower pole of the kidney with the dual - blade retractor .
the console surgeon may use the prograsp to retract the vein slightly to expose the artery .
the artery is then clamped with a bulldog . clamping the vein is optional , but the author prefers to clamp the vein to ensure a bloodless field and to help identify problems during resection ( see step 6 , below ) .
theoretically , the kidney should only have one arterial vessel , but in practice , multiple arteries are not uncommon .
accessory upper pole arteries may be overlooked if the upper pole is not freed from the adrenal gland .
arterial branches may also be missed if the hilar dissection is performed too laterally , as the main artery may have branched .
several pearls may aid in this step : 1 . the author avoids the satinsky clamp , as robotic arms may hit the device and theoretically could avulse the hilum .
2 . if the assistant is inexperienced , the console surgeon may scrub into the field and clamp the hilum him / herself .
some bulldogs may be handled by the prograsp and may be manipulated by the console surgeon ; if this is desired , then rehearse ex vivo prior to the operation .
the or timer / stopwatch should be started so that the console surgeon is aware of cumulative clamp time . the circulating nurse or anesthesiologist should call out the clamp times every 10 min so the console surgeon does not have to glance at the clock.step 7 : tumor excision ( fig .
7the renal mass is excised the renal mass is excised working arms ( ce and la ) : monopolar scissors and prograspfourth arm ( ca ) : dual - blade retractor.assistant : suction / irrigator .
working arms ( ce and la ) : monopolar scissors and prograsp fourth arm ( ca ) : dual - blade retractor .
the author also uses tilepro technology to visualize computed tomography ( ct ) or magnetic resonance ( mr ) images in the surgical field .
oncological principles should be followed ; a negative margin is the paramount goal , and collecting system or hilar structures should be entered with impunity if needed .
the author does not use a ureteral catheter , as the robotic camera offers superb visualization in the bloodless field , and the collecting system is identifiable .
nevertheless , a ureteral catheter , with retrograde indigo carmine injection , may be employed if desired .
after excision of the tumor , the author sends frozen sections of the base of the nephrotomy .
bleeding from the nephrotomy while clamped is a major source of anxiety during resection , and troubleshooting must be done on the fly .
first , when the kidney is initially incised , it will decompress with venous bleeding .
oozing is common for a few minutes , but should stop after the stored blood leaks out .
if oozing continues for more than a few minutes , then the surgeon must question if there is ongoing flow to the kidney .
obviously , visualization of the margins is impaired with bleeding , and the surgeon must troubleshoot this situation immediately . the five major causes for ongoing bleeding are described below : an accessory artery is still perfusing the kidney : occurs most commonly if the upper pole is not released from the adrenal gland and a small accessory artery is undiscovered.a branch of the main artery is located behind the renal vein , and it has been missed .
this occurs if the hilar dissection is performed too laterally after the main artery has branched.to treat conditions ( a ) and ( b ) , the assistant places a large bulldog across the entire hilum and places another long bulldog along the fat between the adrenal gland and the kidney.(c)the third cause of bleeding is a faulty arterial clamp , which does not occlude the artery fully .
the assistant should place a second bulldog on the hilum.(d)the fourth cause is an unrecognized venous branch which is back - bleeding .
this often occurs on the left side , when the vein is clamped too medially and the gonadal , lumbar , and/or adrenal vein still enter the renal vein lateral to the clamp .
replacement of the clamp is recommended.(e)the fifth cause of bleeding is from the collecting system , which may be caused by from collateral vessels from the ureter .
oversewing the collecting system results in hemostasis.if arterial bleeding continues and all of the above maneuvers are unsuccessful , then the author would recommend unclamping a small portion of the renal vein , to allow venous outflow to counterbalance the arterial inflow .
an accessory artery is still perfusing the kidney : occurs most commonly if the upper pole is not released from the adrenal gland and a small accessory artery is undiscovered .
a branch of the main artery is located behind the renal vein , and it has been missed . this occurs if the hilar dissection is performed too laterally after the main artery has branched .
the third cause of bleeding is a faulty arterial clamp , which does not occlude the artery fully .
this often occurs on the left side , when the vein is clamped too medially and the gonadal , lumbar , and/or adrenal vein still enter the renal vein lateral to the clamp .
the fifth cause of bleeding is from the collecting system , which may be caused by from collateral vessels from the ureter . oversewing the collecting system results in hemostasis .
after excision , the renal parenchyma can be treated with an energy source , such as cautery ( monopolar scissors ) or an argon beam coagulator ( assistant ) .
care should be taken with the argon beam to have veins clamped , as argon can induce gas embolism.step 8 : reconstruction ( fig .
8the renal parenchymal is reapproximated to close the defect step 8 : reconstruction ( fig .
8the renal parenchymal is reapproximated to close the defect the renal parenchymal is reapproximated to close the defect working arms ( ce and la ) : needle driver and prograspfourth arm ( ca ) : dual - blade retractor or robotic clip applier.assistant : needle driver to pass / extract needles , scissors to cut suture , clip applier to tighten suture . working arms ( ce and la ) : needle driver and prograsp fourth arm ( ca ) : dual - blade retractor or robotic clip applier .
assistant : needle driver to pass / extract needles , scissors to cut suture , clip applier to tighten suture .
only one needle driver is used since the prograsp is a much stronger grasper and is effective when tightening sutures .
additionally , the prograsp can easily be used to drive needles if the nondominant hand is needed .
the suture may be tied or a clip may be used to secure the suture .
individual larger veins or arteries can be sutured with figure - of - eight sutures if needed on central tumors .
the parenchyma is then closed every 1 cm with a # 1 vicryl suture on a ct needle .
the stitches are approximately 7 cm long , and a clip has been preplaced on the distal end of the suture . after passing the suture through the parenchyma , the console surgeon grabs the stitch with the prograsp instrument and tenses the suture .
next , another clip ( placed by the assistant or the robotic clip applier ) is used to sandwich the renal parenchyma to ensure hemostasis .
this technique has been used very effectively in the laparoscopic approach , and a similar approach is used robotically .
the author usually places several sutures to completely close the parenchyma . after placing several stitches ,
the artery can be unclamped as well , but if there is bleeding , more stitches need to be placed .
bolster of thrombogenic material such as surgicel or gel foam to fill the renal defect .
floseal may be placed at anytime during this step , depending on the surgeon s preference and the size of the resection.step 9 : drain placement , extraction of specimen , and exiting the abdomenonce hemostasis is achieved , the robot is undocked and the console surgeon scrubs into the operative field .
one of the robotic trocars can be removed and that site can be used for the entrapment sack . after specimen entrapment , a drain may be placed if desired .
the drain is placed near the resection bed and exits out of a new puncture in the flank .
if one of the existing trocar sites is used for the drain , patients will often experience leakage around the drain which soils the bed and gown of the patient .
the author checks a complete blood count and a creatinine level in the recovery room , and often a second check is performed within 12 h of surgery with larger resections .
patients are started on liquids the day after surgery , and advanced when tolerating liquids . since
essential hypertension is fairly prevalent in the author s population , blood pressure is monitored to minimize hypertensive crisis .
the drain fluid is sent for creatinine level test and the drain is removed prior to discharge if no urine leak is detected .
patients may be seen back in the office to discuss pathology results 12 weeks after surgery , depending on travel arrangements .
the four - arm approach has been used in patients with tumors in all locations except posterior hilar midpole lesions .
as this communication represents a surgical atlas , the detailed assessment of our series is under separate submission .
this submission and atlas may be useful for the surgeon who is interested in exploring minimally invasive partial nephrectomy in lieu of open partial nephrectomy or ablative technology .
the technique was devised on the da vinci s system , and certainly the original da vinci platform possesses mobility limitations which may result in tighter angles and reduced effectiveness of the fourth arm . to accommodate such limitations ,
although lateralization of the camera may aid in mobility , dissection of the upper pole may become more challenging . in practice
further limitations of robotic partial nephrectomy include the inherent need for the assistant to become an active participant in the surgery .
the assistant may be required to achieve vascular control , biopsy the tumor bed , and place clips to sandwich the renal parenchyma .
such maneuvers are usually performed by the operating surgeon in open and laparoscopic partial nephrectomy . as instrumentation matures
clearly a retroperitoneal approach would be more direct , but alternatively , the kidney could be mobilized and rotated so the posterior surface is visible .
the author has performed this procedure with lateralization of all the trocars , and with a three - trocar approach instead of a four - trocar approach .
such a discussion is beyond the scope of this atlas , but the major criticisms have been warm ischemia and the possibility that the laparoscopic approach may harbor more complications [ 1 , 7 ] . | da vinci robotic surgery is becoming a standard alternative to open and laparoscopic surgical techniques .
robotic partial nephrectomy has been described in limited numbers . in this article , a surgical atlas of the transperitoneal four - arm approach to robotic partial nephrectomy
is outlined .
surgical pearls , pitfalls , and limitations are reviewed . | Introduction
Methods
Postoperative orders
Results
Discussion | radical nephrectomy , partial nephrectomy , and ablative modalities are well described [ 1 , 2 ] . the open , laparoscopic , and percutaneous approaches are prevalent techniques [ 1 , 2 ] . robotic partial nephrectomy is in a fledgling state , but the complexity and reconstructive nature of laparoscopic partial nephrectomy may theoretically be well suited to a robotic assisted approach . there have been a few reports of the procedure , with differing techniques [ 35 ] . as evidenced with robotic prostatectomy , there are several advantages of da vinci assisted laparoscopic surgery . the robotic approach subjectively allows better visualization , improved control via multiple effector arms , and reduced surgeon fatigue . the goal of this publication is to describe one method to perform a da vinci four - arm approach to partial nephrectomy using a parallelogram port placement . this approach has been validated in tumors in all locations except directly posterior midpole lesions . robotic partial nephrectomy will be outlined in several steps , with a similar format as a recently published communication regarding robotic prostatectomy . for the purposes of this article
, the instruments in the working arms may be interchanged depending on whether or the surgeon is right or left handed.step 1 : trocar placement with parallelogram arrangement and docking ( fig . 1).fig . 1four - arm approach to da vinci partial nephrectomy . umbilicus is visualized step 1 : trocar placement with parallelogram arrangement and docking ( fig . umbilicus is visualized four - arm approach to da vinci partial nephrectomy . 1 cephalad arm . assistant port is not visualized , but is usually placed between the camera port and the cephalad arm . extensive ex vivo work was performed to create a trocar arrangement that could be used in most renal tumors . several challenges were present , especially since the kidney is many times larger than the prostate , and thus , a larger range of motion is necessary . the trocar arrangement was optimized for hilar dissection and retraction , with the acceptance of some minor collisions in the extreme upper and lower parts of the surgical field . the camera and the cephalad arm are always paired on one side of the robot ( with the joint on the camera arm towards the cephalad arm ) , and the caudal and fourth arms are paired on the other side of the robot . after the abdomen is inspected , the remaining robotic trocars are placed , as seen in fig . the trocars are placed 812 cm apart depending on the size of the patient s torso . for the purposes of this article
, the ports will be referred to as cephalad ( ce ) , lateral ( la ) , caudal ( ca ) , assistant ( as ) , and camera ( cam ) . in this communication
the ce and la arms are classified as the working arms . in older three - arm da vinci systems , the assistant could place a second trocar in lieu of the fourth arm . docking is performed diagonally so that the center of the robot is in a line with the camera port and the renal hilum ( usually off the tip of the 11th rib ) . after insufflation and port placement , the robot is docked , and the primary surgeon engages the console . for obese patients ,
all ports should be shifted laterally.step 2 : reflection of the colon ( fig . 2colon is reflected over the anterior surface of the kidney step 2 : reflection of the colon ( fig . 2colon is reflected over the anterior surface of the kidney colon is reflected over the anterior surface of the kidney working arms ( ce and la ) : monopolar scissor ( 35 w ) and prograsp ( alternative : bipolar or plasmakinetic forceps).caudal arm ( ca ) : dual - blade atrial retractor.assistant : suction / irrigator.scope : 30 downward orientation . working arms ( ce and la ) : monopolar scissor ( 35 w ) and prograsp ( alternative : bipolar or plasmakinetic forceps ) . caudal arm ( ca ) : dual - blade atrial retractor . reflection of the colon is a critical step , as it confers exposure for the remainder of the operation . when beginning ones experience with robotic renal surgery , the surgeon should reflect the colon as much as possible to allow wide access to the retroperitoneum . the peritoneum should be incised and the colon reflected medially until gerota s fascia is visible along the medial length of the kidney . note that the left colon lies quite laterally in comparison to the right colon , and more mobilization may be needed on the left . the proper plane between the peritoneum and retroperitoneum is often avascular , so the colo - renal attachments may be swept downwards or cut with an athermal technique when the plane is reached . the cephalad extent of the dissection should occur up to the liver edge or splenic edge and then laterally along the edge of the spleen or liver . the surgeon can easily provide countertraction with the prograsp , and the dual - blade retractor may be used to push the colon medially . on the right ,
on the left , the ureter and the gonadal vein may be identified , but the aorta is often not directly seen , since it is surrounded by more adherent lymphatics and fat . caution is advised near the iliac vessels and appendix when incising the peritoneum near the pelvis ; one usually does not need to dissect so caudally . on the left , it is critical to avoid the pancreas by performing the dissection directly on the perinephric fat.step 3 : lateral retraction of the lower pole of the kidney to put hilum on stretch ( fig . 3the kidney is retracted laterally to allow exposure and dissection of the renal hilum step 3 : lateral retraction of the lower pole of the kidney to put hilum on stretch ( fig . 3the kidney is retracted laterally to allow exposure and dissection of the renal hilum the kidney is retracted laterally to allow exposure and dissection of the renal hilum working arms ( ce and la ) : monopolar scissor and prograsp.fourth arm ( ca ) : dual - blade retractor.assistant : suction / irrigator . working arms ( ce and la ) : monopolar scissor and prograsp . fourth arm ( ca ) : dual - blade retractor . assistant : suction / irrigator . lateral retraction of the kidney , originally popularized by kavoussi laparoscopically , suspends the kidney to allow the renal hilum to be on stretch . after reflection of the colon and identification of the ureter , the psoas muscle is identified medial to the ureter . once a glimpse of the psoas muscle is identified , it is exploited with upward retraction of the kidney and perinephric fat . the retractor is placed under the lower pole of the kidney and is used to elevate the kidney away from the great vessels . as such , the renal hilum is stretched and working space is created between the kidney and the great vessels . once the hilum is on stretch , there are several maneuvers to find the renal vein : one may trace the gonadal vein to the cava and then trace the cava to the renal vein ( or , on the left , trace the gonadal vein directly to the renal vein ) . with experience
this tracing becomes unnecessary , and one may actually visualize the large biphasic venous pulsation . the console surgeon may use the scissors to gently push the fat off the anterior surface of the renal vein to gain exposure . once the renal vein is identified , the artery is then dissected with the help of the assistant who can retract the vein cranially and caudally . on the right side ,
the artery is often easier to identify since the right renal vein is rarely tethered by other branches.step 4 : dissection of kidney off of the adrenal gland ( fig . 4the upper pole is mobilized from the adrenal attachments step 4 : dissection of kidney off of the adrenal gland ( fig . 4the upper pole is mobilized from the adrenal attachments the upper pole is mobilized from the adrenal attachments working arms ( ce and la ) : monopolar scissor and prograsp.fourth arm ( ca ) : dual - blade retractor or robotic clip applier.assistant : suction / irrigator . fourth arm ( ca ) : dual - blade retractor or robotic clip applier . assistant : suction / irrigator . dissection of the kidney s upper pole from the adrenal gland is useful for tumors in the upper part of the kidney , in which the adrenal resection is not anticipated . often , there are significant unnamed blood vessels traveling in between the adrenal and the kidney . the area between the kidney parenchyma and adrenal gland can be divided with careful use of monopolar cautery . the assistant can thin out the fat with blunt dissection and strands are divided by the console surgeon . the prograsp is useful to give countertraction to the assistant s suction device to allow dissection of the area . the fourth arm ( ca ) with the dual - blade retractor should be repositioned on the anterior surface of the kidney to retract the whole kidney caudally . alternatively , the dual - blade retractor could be exchanged for a robotic clip applier . if the upper pole renal tumor is very large , then the surgeon may elect to resect the adrenal gland en bloc with the mass . thus , the surgeon will be dissecting along the lateral border of the vena cava or aorta . to gain additional exposure for adrenal resection , the colon may need additional medialization . on the right , the colon should be reflected such that the infrahepatic cava is visualized ; this involves incising the peritoneum along the edge of the liver . on the left side , the surgeon may incise the lateral splenic attachments near the diaphragm and the splenorenal ligaments . the spleen will then fall medially with the colon , gaining exposure to the medial aspect of the left adrenal gland . the surgeon should generally not take down the splenocolic attachments , as several blood vessels travel in this area . if collisions ensue or mobility is impaired , the scissors are switched to the cephalad arm and the fourth - arm instrument is removed . on the right , superior retraction of the liver is often needed with an extra 5 mm port in the subxyphoid location.step 5 : remove fat around lesion to expose renal parenchyma ( fig . 5the parenchymal margins of the kidney are exposed by removing the fat around the lesion step 5 : remove fat around lesion to expose renal parenchyma ( fig . 5the parenchymal margins of the kidney are exposed by removing the fat around the lesion the parenchymal margins of the kidney are exposed by removing the fat around the lesion working arms ( ce and la ) : monopolar scissors and prograsp.fourth arm ( ca ) : dual - blade retractor.assistant : laparoscopic ultrasound probe , suction / irrigator , 10 mm spoon . exposure of the parenchyma is important , as this edge will be sewn later in the case . if fat is totally detached , the assistant may extract the tissue with a 10 mm spoon . technically , removal of adipose tissue around the tumor is not difficult except in patients with abundant adherent perinephric fat . if the perinephric fat does not easily slide off the kidney , this step becomes very tedious and time consuming , with numerous entries into vessels in gerota s fascia . during exposure of the renal parenchyma
all anticipated staff changes should occur during this step to maintain consistency of staff during tumor excision . thrombogenic agents should be prepared so there are no distractions during clamping and tumor excision . the deep margins should be mentally visualized with three - dimensional spatial orientation.step 6 : clamping the renal hilum ( fig . some bulldogs are controlled by the console surgeon ; most are placed by the assistant . mannitol ( 12.5 or 25 gm ) and furosemide ( 1020 mg ) may be given to the patient prior to vascular occlusion . the hilum is exposed by lifting the lower pole of the kidney with the dual - blade retractor . the console surgeon may use the prograsp to retract the vein slightly to expose the artery . clamping the vein is optional , but the author prefers to clamp the vein to ensure a bloodless field and to help identify problems during resection ( see step 6 , below ) . theoretically , the kidney should only have one arterial vessel , but in practice , multiple arteries are not uncommon . accessory upper pole arteries may be overlooked if the upper pole is not freed from the adrenal gland . arterial branches may also be missed if the hilar dissection is performed too laterally , as the main artery may have branched . several pearls may aid in this step : 1 . if the assistant is inexperienced , the console surgeon may scrub into the field and clamp the hilum him / herself . some bulldogs may be handled by the prograsp and may be manipulated by the console surgeon ; if this is desired , then rehearse ex vivo prior to the operation . oncological principles should be followed ; a negative margin is the paramount goal , and collecting system or hilar structures should be entered with impunity if needed . the author does not use a ureteral catheter , as the robotic camera offers superb visualization in the bloodless field , and the collecting system is identifiable . nevertheless , a ureteral catheter , with retrograde indigo carmine injection , may be employed if desired . after excision of the tumor , the author sends frozen sections of the base of the nephrotomy . bleeding from the nephrotomy while clamped is a major source of anxiety during resection , and troubleshooting must be done on the fly . first , when the kidney is initially incised , it will decompress with venous bleeding . obviously , visualization of the margins is impaired with bleeding , and the surgeon must troubleshoot this situation immediately . the five major causes for ongoing bleeding are described below : an accessory artery is still perfusing the kidney : occurs most commonly if the upper pole is not released from the adrenal gland and a small accessory artery is undiscovered.a branch of the main artery is located behind the renal vein , and it has been missed . the assistant should place a second bulldog on the hilum. (d)the fourth cause is an unrecognized venous branch which is back - bleeding . replacement of the clamp is recommended. (e)the fifth cause of bleeding is from the collecting system , which may be caused by from collateral vessels from the ureter . oversewing the collecting system results in hemostasis.if arterial bleeding continues and all of the above maneuvers are unsuccessful , then the author would recommend unclamping a small portion of the renal vein , to allow venous outflow to counterbalance the arterial inflow . an accessory artery is still perfusing the kidney : occurs most commonly if the upper pole is not released from the adrenal gland and a small accessory artery is undiscovered . a branch of the main artery is located behind the renal vein , and it has been missed . this occurs if the hilar dissection is performed too laterally after the main artery has branched . care should be taken with the argon beam to have veins clamped , as argon can induce gas embolism.step 8 : reconstruction ( fig . 8the renal parenchymal is reapproximated to close the defect the renal parenchymal is reapproximated to close the defect working arms ( ce and la ) : needle driver and prograspfourth arm ( ca ) : dual - blade retractor or robotic clip applier.assistant : needle driver to pass / extract needles , scissors to cut suture , clip applier to tighten suture . only one needle driver is used since the prograsp is a much stronger grasper and is effective when tightening sutures . individual larger veins or arteries can be sutured with figure - of - eight sutures if needed on central tumors . the parenchyma is then closed every 1 cm with a # 1 vicryl suture on a ct needle . the stitches are approximately 7 cm long , and a clip has been preplaced on the distal end of the suture . this technique has been used very effectively in the laparoscopic approach , and a similar approach is used robotically . floseal may be placed at anytime during this step , depending on the surgeon s preference and the size of the resection.step 9 : drain placement , extraction of specimen , and exiting the abdomenonce hemostasis is achieved , the robot is undocked and the console surgeon scrubs into the operative field . one of the robotic trocars can be removed and that site can be used for the entrapment sack . after specimen entrapment , a drain may be placed if desired . the drain is placed near the resection bed and exits out of a new puncture in the flank . if one of the existing trocar sites is used for the drain , patients will often experience leakage around the drain which soils the bed and gown of the patient . the author checks a complete blood count and a creatinine level in the recovery room , and often a second check is performed within 12 h of surgery with larger resections . patients are started on liquids the day after surgery , and advanced when tolerating liquids . patients may be seen back in the office to discuss pathology results 12 weeks after surgery , depending on travel arrangements . the four - arm approach has been used in patients with tumors in all locations except posterior hilar midpole lesions . as this communication represents a surgical atlas , the detailed assessment of our series is under separate submission . this submission and atlas may be useful for the surgeon who is interested in exploring minimally invasive partial nephrectomy in lieu of open partial nephrectomy or ablative technology . the technique was devised on the da vinci s system , and certainly the original da vinci platform possesses mobility limitations which may result in tighter angles and reduced effectiveness of the fourth arm . to accommodate such limitations ,
although lateralization of the camera may aid in mobility , dissection of the upper pole may become more challenging . in practice
further limitations of robotic partial nephrectomy include the inherent need for the assistant to become an active participant in the surgery . the assistant may be required to achieve vascular control , biopsy the tumor bed , and place clips to sandwich the renal parenchyma . such maneuvers are usually performed by the operating surgeon in open and laparoscopic partial nephrectomy . as instrumentation matures
clearly a retroperitoneal approach would be more direct , but alternatively , the kidney could be mobilized and rotated so the posterior surface is visible . the author has performed this procedure with lateralization of all the trocars , and with a three - trocar approach instead of a four - trocar approach . such a discussion is beyond the scope of this atlas , but the major criticisms have been warm ischemia and the possibility that the laparoscopic approach may harbor more complications [ 1 , 7 ] . | [
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on november 12 1990 , bill gates delivered his keynote address entitled information at your fingertips at fall comdex and depicted the promising future of the emerging information era.1 nearly 20 years later , we are surrounded by an invisible computing environment and interconnected pervasively by either wired or wireless networks .
we are already used to enjoying everything in our daily life conveniently , wherever we are .
we benefit from online shopping and information exchange with people from the other side of the earth in an instant by just clicking the mouse on our desk .
our lifestyle has changed dramatically , and we face many unprecedented problems in the health domain .
one of the greatest obsessions is the increase in chronic illness that has occurred concurrently with accompanying lifestyle changes .
figure 1 shows the change in mortality structure for different reasons , including diseases and natural decrepitude , over the past 100 years in japan .
mortality due to conventional causes such as accidents , aging , hepatic disease , and pneumonia is under control , so there is no large change .
the leading causes of death are cerebral disease , cardiovascular disease , and cancer , which account for 60% of all deaths .
the world health organization ( who ) also reported that chronic diseases are the leading causes of deaths worldwide and their impact is steadily growing . approximately 17 million people die prematurely each year because of the global epidemic of chronic diseases.2 today , health care , in relation to chronic conditions , is a more pressing challenge than ever before . to facilitate effective health care , an innovative strategy and action plan
every country should consider health care as a long - term investment for future national dividend .
they need to change policies in order to establish a stepwise framework for offering a flexible and practical public health care approach .
they have to take steps toward discovering new knowledge and achieving significant improvements in prevention and control of chronic disease .
health care is neither a new desire nor a modish topic . pursuing a healthier life and longevity through various means , using alchemies to discover the panacea ,
qin shi huang ( 259 bc to 210 bc ) , the first emperor of a unified china , mobilized national resources to pursue his dream of immortality in 219 bc .
he dispatched a court sorcerer , xu fu , and his fleet , including 3,000 young men and women , to search for the elixir of life.3 an antiquity of the third century bc , known as the daoyin picture , shown in figure 2 , was unearthed in 1973 from a grave named
mawangdui in hunan province , china.4 it told a similar story of how health care among civilians dates back to the han dynasty ( 206 bc to 220 ad ) and was thought to be the earliest existing color painting concerning health care in chinese history .
the painting illustrates 22 men and 22 women from different age groups on a piece of silk , 100 cm long and 50 cm high , showing 44 postures , such as squatting and kneeling , but most are in the standing position .
brief marginalia beside each posture indicate the action name and its effectiveness in preventing chronic conditions , such as arthritis , digestive system ailments , and problems with the sensory organs ( eyes and ears ) and immune system .
daoyin exercise imitates the motion of animals , such as apes and monkeys , to stretch , incline , twist , rotate , and bend the limbs and trunk , as well as to play with sticks and balls .
such kinds of stretching and relaxing gymnastics aim to enhance cardiopulmonary function and improve health through various body and breathing movements ( inhalation and exhalation , especially hypogastric breath ) .
daoyin is academically accredited as being the root of taijiquan and qigong , both of which theories hold that circulating qi ( subtle energy ) through body movements can not only improve the health but also slow down the aging process and increase physical strength and mental acumen.5 in approximately the same historical period , around the second century bc , maharishi patanjali ( there are no authentic records on his birth and death ) , an indian philosopher known as the father of yoga , compiled a collection of aphorisms on yoga practice in the book the yoga sutra .
a healthy body and mind can be attained through the practice of relaxation , exercise , breathing , diet , and meditation , the so - called five yoga principles , by following the 8 stages in yoga in order to reunite with the divine energy in the universe.6 by synchronizing deep , slow , and rhythmic breathing with proper exercise through the yoga postures , or asana , can systematically stretch and tone the muscles and ligaments to help enhance the flexibility of the spine and the joints , improve blood circulation , and rejuvenate the various systems of the body . as body and mind
are intimately interconnected , meditation and physical relaxation can ease the tension in the muscles , rest the whole body , and in the end , help to conserve energy , revitalize bodily systems , achieve inner peace , and release worries and fears .
moreover , a well - balanced diet of natural foods nourishes both mind and body and keeps the body supple , the mind calm , and the immune system strong .
although only sages in ancient india practiced yoga , its benefits for both physical and mental health are widely recognized today .
similarly , in ancient greece , herodicus ( circa fifth century bc ; there are no authentic records of his birth and death ) , a gymnastics master , was respected as the father of sports medicine.7 he was credited with the first use of therapeutic exercise for the treatment of disease and the maintenance of health .
he considered poor health to be the result of an imbalance between diet and physical activity , and therefore recommended a strict diet , regular physical activity , and massage using beneficial herbs and oils .
herodicus first laid down the principles for rational and mechanical methods of treatment and was one of the first to describe the technique of massage .
he advised that movements should be slow and gentle at first , then rapid in combination with increased pressure , followed by gentle movements again .
he believed that this combination was the ideal way to maintain good standards of health .
one of herodicus disciples , hippocrates ( circa 460 bc to circa 377 bc ) , who was considered the father of medicine , observed that some diseases were contracted by the manner of life and concluded that the constitutions and habits of people followed the nature of the land where they lived .
he left the earliest definite information relating to the beneficial effects of massage and the general therapeutic application of mechanical vibration .
hippocrates suggested that applying massage with supplemental mechanical and physical processes is helpful to aid circulation , relieve stasis , and consequently , quicken metabolic processes.8 a rich variety of beliefs and practices were fostered over several millennia in the east and west based on distinct culture and philosophy . through combining elements of qigong , yoga , meditation , massage , and martial arts to regulate breathing and mental activity ,
these finally evolved into hundreds of styles of medical sports and medical gymnastic exercises , which are widely used today to prevent disease and promote good health .
these ancestral heritages are our precious assets . standing on the shoulders of giants enables us to fight diseases and manage health more wisely .
chronic conditions are now recognized as a compelling challenge to all of the communities rather than just a private matter .
although the risk of outbreaks , such as a new influenza pandemic , will require constant vigilance , it is the
invisible epidemics of heart disease , stroke , diabetes , cancer , and other chronic diseases that , for the foreseeable future , will take the greatest toll in terms of deaths and disability .
the who offers the health community a new global goal : to reduce death rates from all chronic diseases by 2% per year over and above the existing trends during the next 10 years .
this global action to prevent chronic diseases would result in saving the lives of 36 million people who would otherwise have died due to chronic diseases by 2015 .
the achievement would also result in appreciable economic benefits for all countries.2 a rapid response , above all , must be forward - looking . to raise all citizens awareness of the importance of health promotion and disease prevention , the involvement of all areas of society and a creative approach are indispensable .
a variety of policies , strategies , and activities are now being initiated nationwide in japan at all levels , from administration and community groups down to the individual . a 12-year government - sponsored campaign for health promotion , known as
healthy japan 21 , has been advocated nationwide since the year 2000.9 in addition , the japanese parliament10 issued a health promotion law to define individual responsibility and interactive coordination among citizens , communities , organizations , and government administrations .
the law reconfirms that the national goal of medical insurance reconstruction is health promotion and disease prevention .
founded in 2005 , the japan mibyou system association ( jmsa ) defined mibyou ( hypohealth ) as a state between health and disease.11 the jmsa is committed to the better control of the hypohealth state in order to improve human wellness .
it has established a series of educational programmes and an official accreditation system for health promoters that aim to provide wholesome and secure care to all citizens .
more and more professional communities , research institutions , and industrial sectors across the world have been committing to daily health care .
many studies place high priority on personal lifelong health information infrastructure for the mass population in daily application .
the health data bank application service provider service platform was released as a multifaceted aid for the management of corporate employee s medical examination results.12 the service supplies health care personnel with a set of tools for effective employee health guidance and counseling .
it takes into account factors such as an employee s current physical condition , living habits , environment , and age - related changes in longitudinal management .
each employee can browse his or her personal data through internet channels and can view self - records of check - ups and graph detailing historical changes over a long period .
health vault is a universal hub of networks to connect personal health devices and other services .
it helps to store and manage personal medical information in a single central site on the world wide web.13 it aims to provide a seamless connection interface for various home health and wellness monitoring devices , such as sport watches , blood glucose monitors , and blood pressure monitors , marketed by different manufacturers worldwide .
google health gathers personal medical records from doctors , hospitals , and pharmacies ; manages medical history profiles , such as health conditions , medications , allergies , and laboratory results ; and shares personal health information with family members , doctors , or caregivers.14 philips proposed the complete care cycle from prevention to telemedicine , aiming to cover paradigms of prevention , diagnosis , treatment , rehabilitation , and management .
its goal is to build a secure and personalized health care platform that provides a remote connection between chronically ill patients and their health care providers .
it also aims to promote patients behavior change , expand care providers reach into homes , and improve clinical staff efficiency and effectiveness.15 to provide more effective vital sign monitoring suitable for daily use , a variety of pervasive computing technologies have been widely developed for health care applications . since the first accurate recording of an electrocardiogram ( ecg ) reported by willem einthoven in 1895 , heartbeat measurement has come a long way and are now available for use in various situations .
whenever a person sits on a chair16 or on a toilet,17 sleeps in a bed,18,19 sits in a bathtub,20 or even takes a shower,21 the heartbeat can be monitored conveniently , without disturbing the person . over 50 home - oriented devices , such as pulse oximeters , ecg monitors , glucose monitors , insulin pumps , blood pressure monitors , spirometers , pedometers , body composition meters , and weight scales ,
most of them are for both general wellness and chronic conditions , such as congestive heart failure , chronic obstructive pulmonary disorder , asthma , hypertension , diabetes , and obesity .
some of them include medstar,22 viterion telehealth life center,23 commander home monitor,24 carematix wellness system,25 lifestar,26 housecall plus,27 medicompass,28 sensewear,29 heartview,30 motiva,31 sentry telehealth monitor,32 health buddy,33 vitelcare,34 advanced care and alert portable telemedical monitor,35 wealthy outfit,36 and the wellness phone.37 these products commonly characterize a home terminal connected to the internet or a telephone line and one or more wearable or portable devices that are able to measure various physiological data , such as body weight ( bw ) , blood pressure , blood glucose , peak flow , oxygen saturation , heart rate and respiration rate , body fat and body temperature ( bt ) , sleep , and energy expenditure .
a canadian company , care in motion technology inc , has released an appealing solution called care in motion .
it includes distributed earphone - like devices and a central data server to provide a seamless health care service suitable for various daily life scenarios . instead of using multiple disparate sensors , the earphone - like device adopts a unique scheme to monitor up to 20 physiological parameters , such as pulse rate , respiratory rate , spo2 , bt , sleep , and activity , continuously in real time .
it is so small that it is promoted as wear and forget.38 most current efforts are directed toward developing a series of pervasive vital sign monitoring technologies , such as plug and play,36 wear and forget,38 and plug is all.18 however , an investigation from the who reported that most current health care systems still have common problems that need to be addressed : a ) the difference between acute and chronic care is not sufficiently emphasized ; b ) patients are rarely supported by either a simple means of involving themselves in self - management or essential information to enable them to manage their condition to the greatest extent possible ; c ) patients are seldom provided with a long - term management plan for chronic conditions to ensure the best outcomes.39 such problems remain to be solved by a systematic scheme .
this system should stress several aspects in 1 ) how to facilitate physiological data collection seamlessly and persistently in the daily environment , 2 ) how to apply data mining algorithms to perform comprehensive interpretation of multifarious long - term data fusion , and ultimately , 3 ) how to build a scalable health care integrated platform for individualized lifelong health management.40
since 1994 , west aizu village had pioneered the challenge to 100 years of age project , with public financial support of circa 2.4 billion ( us$28.13 million).41 the fundamental goal is to promote healthier life and longevity by providing a total care solution package to villagers .
the village built a wired network infrastructure , improved the soil , delivered enhanced educational programs on the importance of a nutritionally balanced diet and good lifestyle practices , and initiated a health promotion campaign .
homecare devices were distributed to 687 of 2,819 families in the village . the device can measure systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , bt , and bw ; record a one - minute ecg and heart rate ( hr ) ; and collect answers to a daily questionnaire shown on a liquid crystal display screen .
the time of the daily measurement is not strictly stipulated : either morning or afternoon is fine , depending on the users preference .
seven health care nurses are in charge of the data analysis and responses to inquiries from users .
figure 3 shows 2 data samples measured during a 2-year period from a couple ; a ) and b ) indicate the wife and husband , respectively .
it is observed that the temporal structure of vital signs in the wife demonstrates the wax and wane corresponding to the seasonal ebb and flow .
sbp , dbp , and hr tend to decline in the summer and rise in the winter .
however , the seasonal biorhythmicity in the husband shows a different pattern . by analyzing the data collected from hundreds of persons over several years
, it is found that individual biorhythmicity differs in period and mesor , amplitude and phase , and zenith and nadir . comprehensive interpretation and proper visualization of these data from different standpoints
are relevant to users pathophysiological conditions , health state ( hs ) , and longevity .
figure 4 represents a demonstration of data visualization in a transformed space , which illustrates the monthly change in the sbp profile over 2 years . the reference profile for comparison
monthly change is evaluated by calculating the similarity between a monthly profile and the reference profile using the dynamic time warping ( dtw ) method.42 results are derived using the data shown in figures 3a and 3b , respectively . comparing the remarkable rhythmic alternations in figure 3a , only smaller changes in the monthly sbp profile can be seen in figure 4a . on the contrary ,
the indistinctive seasonal fluctuation in figure 3b exhibits a larger diversity among monthly repetitive patterns in figure 4b .
epidemiological analysis show that the villagers longevity increased from 73.1 years ( 80.0 years for females ) in 1985 to 77.6 years ( 84.1 years for females ) in 2000 , and mortality from stomach cancer decreased from 138.9% ( 125.4% for females ) in 1988 to 91.7% ( 66.7% for females ) in 2002 referring to national average as 100%.41 more insights into the outcomes of the causalities among many relevant factors are likely to be obtained by further mining this mountain of data and will be reported in separate papers .
still , many questions , such as how to make sense of these results obtained from analyzing huge amounts of collected data , how to infer a person s hs based on these results , and how to predict the future propensity to hs based on the current and past health conditions , remain to be answered .
study in these domains relies on proper mathematical approaches and data fusion models , which will perhaps require an appropriate definition of health and establishment of a new discipline for quantifying the hs comprehensively .
metrology of health aims to quantify the overall hs from an integrative standpoint.43 health evaluation was conventionally conducted by demographic investigation and epidemiological analysis based on large population data accumulated over a long period . in this way , a statistical confidence interval for discriminating between healthy and unhealthy was determined . on the other hand , the definition of health from the who advocates that ,
health is a state of complete physical , mental and social well - being and not merely the absence of disease or infirmity.44 this is commonly referred to as the health triangle .
a three - dimensional ( 3d ) phase space is proposed to express the changing hs over a lifetime , as shown in figure 5 .
the 3 dimensions are physical condition ( x - axis ) , mental status ( y - axis ) , and social well - being ( z - axis ) . an individual lifelong health dynamics is described by a unique locus in the phase space continuum .
the space continuum is divided into 5 spheriform regions indicating 5 hss : inactive ( blue ) , healthy ( green ) , hypohealthy ( yellow ) , unhealthy ( red ) , and infinite ( white ) .
the boundaries of zones vary and depend on individual adaptation and long - term evolution .
the blue zone indicates the inactive or unconscious state , such as a baby in infancy or an elder in senile dementia or a feeble state . the green zone is a state in which the overall function in all aspects works properly .
it can easily be ameliorated to a healthy state when treated earlier , but can possibly deteriorate to an unhealthy state if left untreated .
the red zone indicates an unhealthy or morbid state , from which one is able to either recover to hypohealthy or deteriorate to death .
hs dynamics over the course of a life is described as a continuous trace in the 3d space continuum unless interrupted by a paroxysmal event , such as a traffic accident , a natural disaster , or a murder .
each point in the space indicates a hs at a temporal moment . a person s hs is mostly located in the green zone , with an occasional aberration to yellow , and even the red zone if he or she falls sick .
every life is created from a fertilized ovum through a programmed cellular division , but no two bodily existences exhibit identical patterns .
each life begins at the origin , but then grows at different initial speed in diverse directions in the space continuum .
however , death , as a destination of the life course , is much more complicated .
death may be caused by bodily functions ceasing because of the natural caducity or cytoclasis or due to an accident or other cause leading to sudden death . from the standpoint of the health metrology framework ,
demise due to natural aging without disease is an ideal life process , which is expressed as converging to the origin as shown in figure 5a .
if death is due to accident or disease , the trace will diverge in the infinite , as shown in figure 5b .
the difference is that the former will be continuous , while the latter would have discontinuity .
the physical condition of a human body can now be investigated on multiple scales , from molecules to cells , through tissues and organs , up to the whole body .
mathematical model simulation from the microscale to the macroscale for the biological continuum provides a systematic framework for interpreting the wealth of findings obtained from biological , physiological , and clinical research .
various instrumentation techniques can detect and quantify organic lesions by investigating various biomarkers and physiological signs .
moreover , mathematical models can simulate pathophysiological processes and predict how and when diseases will develop.45,46 mental status describes a level of either cognitive or emotional health and is clinically assessed by mental status examination through a mental status exam ( mse ) form in psychiatric practice.47 the mse form includes 12 items ( appearance , attitude , behavior , speech , affect , mood , thought processes , thought content , perception , orientation , memory / concentration , insight / judgment ) to observe and aims to quantify a person s current state of mind .
objective descriptions of a person s mental status are now supported by interpreting neurophysiological evidence from advanced neuropsychiatric diagnostic tools , such as positron emission tomography and magnetic resonance imaging.48 the term social well - being has been used mainly in sociology studies and involves particularly complicated issues .
many attempts have been made to quantify social well - being by a numerical measure .
indicators , such as public and private consumption per head ; income level ; unemployment rate ; infant survival rate ; life expectancy at birth ; accessibility to social , medical , and educational services ; and quality of life , are usually used to measure social well - being , to evaluate economic activity and living standard , and to develop better economic policies.49 on the other hand , social well - being in the health triangle framework should describe personality from the point of view of social interconnection , personal contribution , and the ability to function in social activities.50 it must include some features pertaining to personal characteristics , such as social credits ( sc ) , eg , credit card and tax payment records , police files , and public reputation ( pr ) from the surroundings . it is well known that , as a general rule , physical ailments , mental disorders , and social malfunctions are interconnected .
an unhealthy body can trigger an unhealthy mind , and mental disorder can worsen diseases in the body and lead to malfunction in social activity . fixing a problem in one area can often help the others . in summary ,
hs is a function h ( x , y , z ) of collective factors in 3 dimensions .
( 1)hs = h(x , y , z)where x , y , and z are the coordinates in the 3d phase space and can be further expressed as given below .
( 2)x = fx[hr(t ) , bp(t ) , bt(t ) ,
, efx(t ) , icxyz(t ) ]
( 3)y = fy[mse(t ) , , efy(t ) , icyxz(t ) ]
( 4)z = fz[sc(t ) , pr(t ) , , efz(t ) , iczxy(t ) ] the terms ef ( t ) and ic ( t ) describe the environmental factors and the interconnection among the 3 dimensions , respectively .
modern chaos theory has been applied to a wide variety of biological phenomena.51 deterministic behavior characterizes chaos , where irregular patterns obey mathematical equations and are critically dependent on initial conditions .
little disparity in the initial condition can lead to an entirely different trajectory in the phase space.52 in the health triangle framework , all possible states in the phase space can be described as a chaotic function of time showing the health dynamics in various zones .
cloud - like dot clusters illustrate the health dynamics with focal stability and random perturbation . during the juvenile and teenage phases
in contrast , from middle age , fluctuations become smaller and recovery is usually slower .
its shape and demeanor elucidate life dynamics in terms of hs over a lifelong course from birth to death .
further quantitative exploration on the metrology of health is open to the health care communities for extensive discussion and intense collaborations among multiple disciplines .
from the ancient greek philosophers , like plato ( circa 429 bc to circa 347 bc ) and aristotle ( circa 384 bc to circa 322 bc ) , to german philosopher , georg wilhelm friedrich hegel ( 17701831 ) , one statement continues to reverberate ; what is rational is actual , and what is actual is rational . despite 2400 million sperms
are released in a single ejaculation to travel along the female s vagina through the cervix and the uterus to meet a mature ovum , only 1 sperm can fertilize this ovum , creating a unique new life .
although the preventative mechanisms of polyspermy are believed to be essential in inhibiting fertilization by more than 1 sperm , additional details concerning changes in the zona pellucida remain unknown.53 nevertheless , we can imagine how fierce the competition is among the sperms in the uterus .
still , it can not be concluded that the successful sperm is the strongest among all the competitors , like a marathon winner , but it seems that there is no doubt that this unique new life must be created most adaptively and optimally among all possible options from the point of view of natural selection and evolution . because the process of natural selection and evolution shapes most of the vulnerabilities in organisms , disease is considered inevitable yet an effective protection mechanism in the human body , responding to hostile factors in order to render the body immune from further harm.54 ancient wisdom pointed out that diseases arise from either a lack or surfeit of use of the body.55 it implies that when a body is kept operating within the proper range of self - adjustment and adaptability , the body works steadily and can recover from ailments via its intrinsic defense mechanisms . if it is forced to work outside its proper range , illness follows .
many illnesses , including mental disorders and physical diseases , and especially lifestyle - related chronic conditions , such as hypertension , hyperlipidemia , diabetes , obesity , arteriosclerosis , osteoporosis , asymptomatic stroke , potential heart attack , and fatty liver are due to years of bodily abuse and mental overstress .
traditional chinese medicine ( tcm ) considers that human moods , health , and behavior are modulated by the ebb and flow of yin and yang , and an imbalance between the yin and yang states is the essential cause of the incidence and exacerbation of a disease .
the goal of treatment is , in principle , to restore and maintain the balance between yin and yang among the visceral organs.55 similarly , ancient western medicine claims that health is the equal distribution of the forces from moist , dry , cold , hot , bitter , sweet , and so on .
health , by contrast , is a harmonious blending of these factors.56 recent medicinal studies show that modern epidemics are most likely to arise from the mismatch between the physiological design of our bodies and novel aspects of our environment.54 acute and chronic conditions manifest through different mechanisms and over varying time spans , and therefore the approach to deal with each should be different .
bian que ( 407 bc to 310 bc ) was a famous chinese physician during the period of warring states in china .
when the king of the feudal state of wei asked him , you and your two brothers are all skilled in medicine , which of you is the best ? bian que answered ,
my eldest brother is the best , second brother is the next , and i am the last . the king puzzled and asked , then how is it that you are the most famous ?
my eldest brother takes care of persons before they feel any symptoms , my second brother cures diseases in their early stage .
people thus mistake them as not special or only capable of treating minor ailments , while i treat diseases when they are already well developed or are very serious .
people therefore look upon me as well versed in medical skill , and know my fame all over the country.57,58 the prevention of disease is of primary importance in tcm doctrine and practice .
it insists that a highly skilled doctor usually focuses on preventing the incidence of diseases instead of treating diseases , just as a wise monarch governs his country by satisfying his citizens rather than suppressing their probable riot before they feel desperation .
this is simply metaphorized that we should avoid digging a well after feeling thirsty or forging weapons after war has begun.55 a dualism that considered mind and body separately prevailed until ren dscartes in the 17th century . over the past few decades , researchers have conducted hundreds of scientific studies in an attempt to measure the effects of spirituality on health from a holistic point of view .
a weakened body can lead to a frazzled mind , whereas depression can trigger or worsen diseases in the body . fixing a problem in one aspect , moreover , can often help the other.59 nevertheless , we still know very little about spirituality , although it performs potently , because its measurement is often beyond the scope of current scientific research .
moreover , health is usually improved slowly through daily activities conducted on a regular basis , and many significant benefits of improved health may have no obvious signs within a short term .
although meditation can not manifest significantly the slow cancer growth or improve recovery from acute illness , one of the clearest health benefits of churchgoers is that they live longer than nonattendees .
in other words , churchgoers have a 25% reduction in mortality.60 mysteries such as why some people are vulnerable and others are not , and why spirituality has different effects on different persons and different diseases remain a puzzle .
even if religion and spirituality were shown convincingly to be related to better health , some critics briefly analyzed many studies assessing the effects of church attendances and prayers on health and asserted that those studies fail to control for factors such as behavioral and genetic differences , age , sex , education , and hs .
they concluded that the evidence of an association between spirituality and health is weak and lacks consistency , even among well - conducted studies .
it is still premature to promote faith and religion as adjunctive medical treatments.61,62 because contradictory conclusions from the large amount of data collected using different experimental protocols and interpreted using various methods often confuse rather than clarifying the issue , establishment of a metrological theory to evaluate the hs systematically is invaluable .
two of the most important goals in the metrology of health are to quantify the personal hs through comprehensive interpretation of multifarious data collected over a period of years in the health triangle framework and to untangle the causal connections among longevity and biorhythmicities ; pathogeny and immunity ; temporal and environmental factors ; and social , behavioral , and psychoneurotic aspects
as long as life on earth continues , health care is an everlasting challenge affecting the sustainable development and prosperity of human beings in the future and no longer just a personal matter .
immortality is impossible biologically because all the biological systems have the apoptosis mechanism genetically built - in .
however , we can strive to conduct a healthier life , to slow down the aging process , and to live longer .
health care is an effective way to prevent and control chronic diseases and has emerged as a key part of a growing global awareness of the need for significant investments of time , enthusiasm , and money .
the paradigm must shift from an intermittent hospital - based system to a lifelong home - based scheme , where every home should be a hub for health care deployment in order for individuals to be involved on a daily basis in an entire care cycle from prevention to diagnosis and from treatment to follow - up .
although an omnipotent elixir enabling all people to recover from disease and maintain good health does not exist , rhythmic daily lifestyle , intake of nutritionally balanced meals , daily exercise , positive attitude , and mental wellness should be elements of a common formula .
just as diseases need time to breed , good health needs not only time to cultivate but also time to maintain .
on the coming november 9 at the 2010 mhealth summit , 20 years later after his speech information at your fingertips , bill gates will be presenting a keynote address to discuss how modern information and communication technologies can save lives and promote health by connecting leaders in industry , government , academia , philanthropic organizations and the private sector.63 in 20 years time , november 6 2030 , what further surprises will bill gates have brought to the world ? | health care is an everlasting challenge that requires effort at all levels , from national and organizational to individual domains , over a long - term period .
this article reviews the historical development of health care - related issues worldwide and accentuates the necessity of health care in the current era . through investigating the historical background in both the occidental and the oriental worlds and the latest achievements from academic and industrial endeavors
, many lessons can be learnt , and as a result , an integrative strategy is proposed to meet today s pressing needs . a government - funded project , challenge to 100 years of age , which involved more than 600 residents in west aizu village , lasted over 15 years .
after reviewing its outcomes , a metrology of health based upon the three - dimensional phase space , which integrates with social well - being , mental status , and physical condition for a lifelong span , is advocated as a measure of the holistic view of health state . | Introduction
Health care in the past
Health care worldwide today
The Challenge to 100 years of age project in west Aizu village
Metrology of health
Discussion
Conclusion
Epilogue | our lifestyle has changed dramatically , and we face many unprecedented problems in the health domain . figure 1 shows the change in mortality structure for different reasons , including diseases and natural decrepitude , over the past 100 years in japan . mortality due to conventional causes such as accidents , aging , hepatic disease , and pneumonia is under control , so there is no large change . the leading causes of death are cerebral disease , cardiovascular disease , and cancer , which account for 60% of all deaths . approximately 17 million people die prematurely each year because of the global epidemic of chronic diseases.2 today , health care , in relation to chronic conditions , is a more pressing challenge than ever before . to facilitate effective health care , an innovative strategy and action plan
every country should consider health care as a long - term investment for future national dividend . they need to change policies in order to establish a stepwise framework for offering a flexible and practical public health care approach . health care is neither a new desire nor a modish topic . he dispatched a court sorcerer , xu fu , and his fleet , including 3,000 young men and women , to search for the elixir of life.3 an antiquity of the third century bc , known as the daoyin picture , shown in figure 2 , was unearthed in 1973 from a grave named
mawangdui in hunan province , china.4 it told a similar story of how health care among civilians dates back to the han dynasty ( 206 bc to 220 ad ) and was thought to be the earliest existing color painting concerning health care in chinese history . daoyin is academically accredited as being the root of taijiquan and qigong , both of which theories hold that circulating qi ( subtle energy ) through body movements can not only improve the health but also slow down the aging process and increase physical strength and mental acumen.5 in approximately the same historical period , around the second century bc , maharishi patanjali ( there are no authentic records on his birth and death ) , an indian philosopher known as the father of yoga , compiled a collection of aphorisms on yoga practice in the book the yoga sutra . a healthy body and mind can be attained through the practice of relaxation , exercise , breathing , diet , and meditation , the so - called five yoga principles , by following the 8 stages in yoga in order to reunite with the divine energy in the universe.6 by synchronizing deep , slow , and rhythmic breathing with proper exercise through the yoga postures , or asana , can systematically stretch and tone the muscles and ligaments to help enhance the flexibility of the spine and the joints , improve blood circulation , and rejuvenate the various systems of the body . as body and mind
are intimately interconnected , meditation and physical relaxation can ease the tension in the muscles , rest the whole body , and in the end , help to conserve energy , revitalize bodily systems , achieve inner peace , and release worries and fears . moreover , a well - balanced diet of natural foods nourishes both mind and body and keeps the body supple , the mind calm , and the immune system strong . similarly , in ancient greece , herodicus ( circa fifth century bc ; there are no authentic records of his birth and death ) , a gymnastics master , was respected as the father of sports medicine.7 he was credited with the first use of therapeutic exercise for the treatment of disease and the maintenance of health . he considered poor health to be the result of an imbalance between diet and physical activity , and therefore recommended a strict diet , regular physical activity , and massage using beneficial herbs and oils . hippocrates suggested that applying massage with supplemental mechanical and physical processes is helpful to aid circulation , relieve stasis , and consequently , quicken metabolic processes.8 a rich variety of beliefs and practices were fostered over several millennia in the east and west based on distinct culture and philosophy . through combining elements of qigong , yoga , meditation , massage , and martial arts to regulate breathing and mental activity ,
these finally evolved into hundreds of styles of medical sports and medical gymnastic exercises , which are widely used today to prevent disease and promote good health . chronic conditions are now recognized as a compelling challenge to all of the communities rather than just a private matter . although the risk of outbreaks , such as a new influenza pandemic , will require constant vigilance , it is the
invisible epidemics of heart disease , stroke , diabetes , cancer , and other chronic diseases that , for the foreseeable future , will take the greatest toll in terms of deaths and disability . to raise all citizens awareness of the importance of health promotion and disease prevention , the involvement of all areas of society and a creative approach are indispensable . a variety of policies , strategies , and activities are now being initiated nationwide in japan at all levels , from administration and community groups down to the individual . a 12-year government - sponsored campaign for health promotion , known as
healthy japan 21 , has been advocated nationwide since the year 2000.9 in addition , the japanese parliament10 issued a health promotion law to define individual responsibility and interactive coordination among citizens , communities , organizations , and government administrations . founded in 2005 , the japan mibyou system association ( jmsa ) defined mibyou ( hypohealth ) as a state between health and disease.11 the jmsa is committed to the better control of the hypohealth state in order to improve human wellness . more and more professional communities , research institutions , and industrial sectors across the world have been committing to daily health care . the health data bank application service provider service platform was released as a multifaceted aid for the management of corporate employee s medical examination results.12 the service supplies health care personnel with a set of tools for effective employee health guidance and counseling . it takes into account factors such as an employee s current physical condition , living habits , environment , and age - related changes in longitudinal management . each employee can browse his or her personal data through internet channels and can view self - records of check - ups and graph detailing historical changes over a long period . its goal is to build a secure and personalized health care platform that provides a remote connection between chronically ill patients and their health care providers . it also aims to promote patients behavior change , expand care providers reach into homes , and improve clinical staff efficiency and effectiveness.15 to provide more effective vital sign monitoring suitable for daily use , a variety of pervasive computing technologies have been widely developed for health care applications . whenever a person sits on a chair16 or on a toilet,17 sleeps in a bed,18,19 sits in a bathtub,20 or even takes a shower,21 the heartbeat can be monitored conveniently , without disturbing the person . some of them include medstar,22 viterion telehealth life center,23 commander home monitor,24 carematix wellness system,25 lifestar,26 housecall plus,27 medicompass,28 sensewear,29 heartview,30 motiva,31 sentry telehealth monitor,32 health buddy,33 vitelcare,34 advanced care and alert portable telemedical monitor,35 wealthy outfit,36 and the wellness phone.37 these products commonly characterize a home terminal connected to the internet or a telephone line and one or more wearable or portable devices that are able to measure various physiological data , such as body weight ( bw ) , blood pressure , blood glucose , peak flow , oxygen saturation , heart rate and respiration rate , body fat and body temperature ( bt ) , sleep , and energy expenditure . a canadian company , care in motion technology inc , has released an appealing solution called care in motion . it is so small that it is promoted as wear and forget.38 most current efforts are directed toward developing a series of pervasive vital sign monitoring technologies , such as plug and play,36 wear and forget,38 and plug is all.18 however , an investigation from the who reported that most current health care systems still have common problems that need to be addressed : a ) the difference between acute and chronic care is not sufficiently emphasized ; b ) patients are rarely supported by either a simple means of involving themselves in self - management or essential information to enable them to manage their condition to the greatest extent possible ; c ) patients are seldom provided with a long - term management plan for chronic conditions to ensure the best outcomes.39 such problems remain to be solved by a systematic scheme . this system should stress several aspects in 1 ) how to facilitate physiological data collection seamlessly and persistently in the daily environment , 2 ) how to apply data mining algorithms to perform comprehensive interpretation of multifarious long - term data fusion , and ultimately , 3 ) how to build a scalable health care integrated platform for individualized lifelong health management.40
since 1994 , west aizu village had pioneered the challenge to 100 years of age project , with public financial support of circa 2.4 billion ( us$28.13 million).41 the fundamental goal is to promote healthier life and longevity by providing a total care solution package to villagers . seven health care nurses are in charge of the data analysis and responses to inquiries from users . sbp , dbp , and hr tend to decline in the summer and rise in the winter . however , the seasonal biorhythmicity in the husband shows a different pattern . comprehensive interpretation and proper visualization of these data from different standpoints
are relevant to users pathophysiological conditions , health state ( hs ) , and longevity . figure 4 represents a demonstration of data visualization in a transformed space , which illustrates the monthly change in the sbp profile over 2 years . comparing the remarkable rhythmic alternations in figure 3a , only smaller changes in the monthly sbp profile can be seen in figure 4a . epidemiological analysis show that the villagers longevity increased from 73.1 years ( 80.0 years for females ) in 1985 to 77.6 years ( 84.1 years for females ) in 2000 , and mortality from stomach cancer decreased from 138.9% ( 125.4% for females ) in 1988 to 91.7% ( 66.7% for females ) in 2002 referring to national average as 100%.41 more insights into the outcomes of the causalities among many relevant factors are likely to be obtained by further mining this mountain of data and will be reported in separate papers . still , many questions , such as how to make sense of these results obtained from analyzing huge amounts of collected data , how to infer a person s hs based on these results , and how to predict the future propensity to hs based on the current and past health conditions , remain to be answered . study in these domains relies on proper mathematical approaches and data fusion models , which will perhaps require an appropriate definition of health and establishment of a new discipline for quantifying the hs comprehensively . metrology of health aims to quantify the overall hs from an integrative standpoint.43 health evaluation was conventionally conducted by demographic investigation and epidemiological analysis based on large population data accumulated over a long period . on the other hand , the definition of health from the who advocates that ,
health is a state of complete physical , mental and social well - being and not merely the absence of disease or infirmity.44 this is commonly referred to as the health triangle . a three - dimensional ( 3d ) phase space is proposed to express the changing hs over a lifetime , as shown in figure 5 . the 3 dimensions are physical condition ( x - axis ) , mental status ( y - axis ) , and social well - being ( z - axis ) . an individual lifelong health dynamics is described by a unique locus in the phase space continuum . the space continuum is divided into 5 spheriform regions indicating 5 hss : inactive ( blue ) , healthy ( green ) , hypohealthy ( yellow ) , unhealthy ( red ) , and infinite ( white ) . the boundaries of zones vary and depend on individual adaptation and long - term evolution . hs dynamics over the course of a life is described as a continuous trace in the 3d space continuum unless interrupted by a paroxysmal event , such as a traffic accident , a natural disaster , or a murder . a person s hs is mostly located in the green zone , with an occasional aberration to yellow , and even the red zone if he or she falls sick . each life begins at the origin , but then grows at different initial speed in diverse directions in the space continuum . however , death , as a destination of the life course , is much more complicated . death may be caused by bodily functions ceasing because of the natural caducity or cytoclasis or due to an accident or other cause leading to sudden death . from the standpoint of the health metrology framework ,
demise due to natural aging without disease is an ideal life process , which is expressed as converging to the origin as shown in figure 5a . the physical condition of a human body can now be investigated on multiple scales , from molecules to cells , through tissues and organs , up to the whole body . moreover , mathematical models can simulate pathophysiological processes and predict how and when diseases will develop.45,46 mental status describes a level of either cognitive or emotional health and is clinically assessed by mental status examination through a mental status exam ( mse ) form in psychiatric practice.47 the mse form includes 12 items ( appearance , attitude , behavior , speech , affect , mood , thought processes , thought content , perception , orientation , memory / concentration , insight / judgment ) to observe and aims to quantify a person s current state of mind . objective descriptions of a person s mental status are now supported by interpreting neurophysiological evidence from advanced neuropsychiatric diagnostic tools , such as positron emission tomography and magnetic resonance imaging.48 the term social well - being has been used mainly in sociology studies and involves particularly complicated issues . many attempts have been made to quantify social well - being by a numerical measure . indicators , such as public and private consumption per head ; income level ; unemployment rate ; infant survival rate ; life expectancy at birth ; accessibility to social , medical , and educational services ; and quality of life , are usually used to measure social well - being , to evaluate economic activity and living standard , and to develop better economic policies.49 on the other hand , social well - being in the health triangle framework should describe personality from the point of view of social interconnection , personal contribution , and the ability to function in social activities.50 it must include some features pertaining to personal characteristics , such as social credits ( sc ) , eg , credit card and tax payment records , police files , and public reputation ( pr ) from the surroundings . it is well known that , as a general rule , physical ailments , mental disorders , and social malfunctions are interconnected . an unhealthy body can trigger an unhealthy mind , and mental disorder can worsen diseases in the body and lead to malfunction in social activity . ( 1)hs = h(x , y , z)where x , y , and z are the coordinates in the 3d phase space and can be further expressed as given below . little disparity in the initial condition can lead to an entirely different trajectory in the phase space.52 in the health triangle framework , all possible states in the phase space can be described as a chaotic function of time showing the health dynamics in various zones . during the juvenile and teenage phases
in contrast , from middle age , fluctuations become smaller and recovery is usually slower . its shape and demeanor elucidate life dynamics in terms of hs over a lifelong course from birth to death . further quantitative exploration on the metrology of health is open to the health care communities for extensive discussion and intense collaborations among multiple disciplines . from the ancient greek philosophers , like plato ( circa 429 bc to circa 347 bc ) and aristotle ( circa 384 bc to circa 322 bc ) , to german philosopher , georg wilhelm friedrich hegel ( 17701831 ) , one statement continues to reverberate ; what is rational is actual , and what is actual is rational . despite 2400 million sperms
are released in a single ejaculation to travel along the female s vagina through the cervix and the uterus to meet a mature ovum , only 1 sperm can fertilize this ovum , creating a unique new life . although the preventative mechanisms of polyspermy are believed to be essential in inhibiting fertilization by more than 1 sperm , additional details concerning changes in the zona pellucida remain unknown.53 nevertheless , we can imagine how fierce the competition is among the sperms in the uterus . because the process of natural selection and evolution shapes most of the vulnerabilities in organisms , disease is considered inevitable yet an effective protection mechanism in the human body , responding to hostile factors in order to render the body immune from further harm.54 ancient wisdom pointed out that diseases arise from either a lack or surfeit of use of the body.55 it implies that when a body is kept operating within the proper range of self - adjustment and adaptability , the body works steadily and can recover from ailments via its intrinsic defense mechanisms . many illnesses , including mental disorders and physical diseases , and especially lifestyle - related chronic conditions , such as hypertension , hyperlipidemia , diabetes , obesity , arteriosclerosis , osteoporosis , asymptomatic stroke , potential heart attack , and fatty liver are due to years of bodily abuse and mental overstress . traditional chinese medicine ( tcm ) considers that human moods , health , and behavior are modulated by the ebb and flow of yin and yang , and an imbalance between the yin and yang states is the essential cause of the incidence and exacerbation of a disease . the goal of treatment is , in principle , to restore and maintain the balance between yin and yang among the visceral organs.55 similarly , ancient western medicine claims that health is the equal distribution of the forces from moist , dry , cold , hot , bitter , sweet , and so on . health , by contrast , is a harmonious blending of these factors.56 recent medicinal studies show that modern epidemics are most likely to arise from the mismatch between the physiological design of our bodies and novel aspects of our environment.54 acute and chronic conditions manifest through different mechanisms and over varying time spans , and therefore the approach to deal with each should be different . when the king of the feudal state of wei asked him , you and your two brothers are all skilled in medicine , which of you is the best ? even if religion and spirituality were shown convincingly to be related to better health , some critics briefly analyzed many studies assessing the effects of church attendances and prayers on health and asserted that those studies fail to control for factors such as behavioral and genetic differences , age , sex , education , and hs . two of the most important goals in the metrology of health are to quantify the personal hs through comprehensive interpretation of multifarious data collected over a period of years in the health triangle framework and to untangle the causal connections among longevity and biorhythmicities ; pathogeny and immunity ; temporal and environmental factors ; and social , behavioral , and psychoneurotic aspects
as long as life on earth continues , health care is an everlasting challenge affecting the sustainable development and prosperity of human beings in the future and no longer just a personal matter . health care is an effective way to prevent and control chronic diseases and has emerged as a key part of a growing global awareness of the need for significant investments of time , enthusiasm , and money . the paradigm must shift from an intermittent hospital - based system to a lifelong home - based scheme , where every home should be a hub for health care deployment in order for individuals to be involved on a daily basis in an entire care cycle from prevention to diagnosis and from treatment to follow - up . on the coming november 9 at the 2010 mhealth summit , 20 years later after his speech information at your fingertips , bill gates will be presenting a keynote address to discuss how modern information and communication technologies can save lives and promote health by connecting leaders in industry , government , academia , philanthropic organizations and the private sector.63 in 20 years time , november 6 2030 , what further surprises will bill gates have brought to the world ? | [
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chronic low back pain ( clbp ) is one of the most common reasons for all physician visits in the usa and is a leading contributor to job - related disability and missed work ( chou and shekelle , 2010 ; hart et al . , 1995 ) .
non - specific clbp , which represents the majority of clbp patients , is characterized by a lack of recognizable pathology ( chou et al .
, 2007 ; ehrlich , 2003 ; savigny et al . , 2009 ) . although clbp is a serious health concern , treatment for clbp has achieved limited success ( bogduk , 2004 ) .
increasing evidence suggests a crucial role of central nervous system plasticity in the development and maintenance of non - specific clbp . to develop more effective treatments , it is crucial to understand the underlying neurobiology of clbp in the brain .
a prevailing theory in the pathogenesis of chronic pain is that the nociceptive afferents become sensitized in such a way that the signaling of these nociceptive afferents increases perceived pain disproportionately to the pain stimulus ( coderre et al . , 1993 ) .
this process is facilitated by a dysfunction of the descending pain modulatory circuits ( woolf and doubell , 1994 ; zimmermann , 2001 ) .
the periaqueductal gray ( pag ) is a key region involved in endogenous pain inhibition ( fields , 2004 ) .
previous studies have shown that pag stimulation can significantly inhibit behavioral responses to noxious stimuli in both animals ( mayer et al . , 1971 ; reynolds , 1969 ) and humans ( baskin et al . , 1986
recent studies have shown that the functional connectivity fluctuations and structural connectivity between the pag and the ventral medial prefrontal cortex ( vmpfc)/rostral anterior cingulate cortex ( racc ) predicted mind wandering away from pain , i.e. , spontaneous disengagement of attention from pain ( kucyi et al . , 2013 ) . the structural connectivity between these two regions also predicted individual difference in placebo analgesia ( stein et al . , 2012 ) .
it is now believed that the brainstem ( pag ) receives direct projections from regions within the limbic forebrain such as the anterior cingulate cortex ( acc ) and limbic - related areas such as the insula and amygdala and modulates pain by descending modulation of the spinal cord neurons ( brooks and tracey , 2005 ; fields , 2004 ; heinricher et al .
, 2009 ; ploner et al . , 2010 ; tracey and mantyh , 2007 ) . a recent study has shown that the pag is functionally connected to the vmpfc / racc , insula and amygdala during resting state ( kong et al . , 2010b ) .
the insula is a key region in pain process ( bernard et al . , 1992 ; chudler et al . , 1993 ; craig , 2002 ; craig et al
, 2000 ; kong et al . , 2013a ; kong et al . , 2006
a previous study suggested that prestimulus functional connectivity between the insula and pain - modulatory brain regions ( e.g. , pag ) differed between physically identical trials that were rated as painful and trials perceived as non - painful ( ploner et al . , 2010 ) .
the amygdala has a central role in regulating emotional responses during acute and persistent pain ( martikainen et al . , 2013 ; neugebauer et al . ,
amygdala and their role in pain perception and modulation ( ploner et al . , 2010 ) , it is possible that alterations in these pathways may also contribute to the development or maintenance of chronic pain .
recent neuroimaging studies have shown that clbp is associated with alterations in resting state brain activity ( apkarian et al .
, 2009 ; apkarian et al . , 2004 ; baliki et al . , 2011 ;
baliki et al . , 2006 ; kobayashi et al . , 2009 ; tagliazucchi et al .
however , the role of the pag and the associated networks detected by resting state fmri in clbp is still unclear . in the present study
, we investigated pag centered brain resting state functional connectivity ( fc ) differences between clbp patients and matched hcs and fc differences when clbp patients experienced different levels of pain intensity .
we hypothesized that clbp would be associated with abnormal fc between the pag and other brain regions including the vmpfc , insula , and amygdala , given the close link between these regions and their role in pain modulation .
please also see a previous published study ( kong et al . , 2013b ) for more details on the experimental procedure .
the data have been used in this previous publication ( kong et al . , 2013b ) , but
, we compared structure and function difference between the clbp and controls using structural imaging data with morphometric analysis and resting state mri data with degree centrality ( dc ) analysis ( kong et al . , 2013b ) .
degree centrality is a measure of local network connectivity and identifies the most connected nodes by counting the number of direct connections to all other nodes ( buckner et al . , 2009 ) .
there is no overlap on the results between the previous paper and the current paper .
eighteen clbp patients and 18 healthy controls , matched for age and gender , completed the study ( see table 1 for demographic details ) .
the institutional review board at massachusetts general hospital approved the study and all subjects gave written informed consent .
all subjects were clinically diagnosed with nonspecific clbp with a duration of at least 6 months by a clinical evaluation , including the use of x - ray / mri reports , when available .
only those patients meeting the quebec low back pain task force classification criteria for class i or ii ( axial lbp with possible occasional radiation to the thigh , and no sensory or motor complaints ) were enrolled ( werneke and hart , 2004 ) .
subjects were classified as having non - specific back pain ( i.e. , patients with specific diagnoses , non - spinal etiology , or radicular symptoms were not included in the study ) .
subjects were also excluded if they reported major systemic diseases or history of head injury or coma .
clbp patients were asked to rate their current pain using a visual analog scale ( 0 no pain , 10 maximum imaginable pain ) , both before and after the resting state fmri scan .
endogenous back pain intensity during the resting state was defined as the average pain rating before and after the scan .
the brief pain inventory ( bpi ) was used to assess the severity of pain ( the sensory dimension ) and the impact of pain on daily feelings and functions ( the reactive dimension ) in the preceding week , i.e. , a week before the experiment date ( cleeland and ryan , 1994 ; tan et al . , 2004 ) .
depressive symptoms were assessed using the beck depression inventory ( bdi - ii ) for all participants ( beck et al . , 1961 ) .
healthy controls , matched for gender , age and race , were recruited in the community .
after the first resting state scan , clbp patients were taken out of the scanner to perform exercises for a period lasting up to 10 min to exacerbate their endogenous lower back pain ( see fig .
these exercises were tailored to each patient based on their report of which movements exacerbated their pain .
the exercises determined to exacerbate low back pain included a set of slow movements such as sit - ups , lumbar flexion / extension , and lumbar rotation , where the subject rotated his or her body from side to side at a self - selected speed . during the screening ,
if at the end of the first resting state scan the patient 's clbp pain rating was too strong ( 7 in 010 scale ) and the patient was reluctant to perform exercises to enhance their pain experience , they were asked to wait for 10 min in a comfortable position before starting the second half of the scanning session . during the exercises , clbp patients were intermittently asked to report their level of pain using the 010 pain scale ; the exercises were repeated until subjects reported an increase in pain of approximately 3 points on the pain scale .
once this level of pain was achieved , subjects were placed back in the scanner to repeat the same fmri scans that were acquired before the pain - inducing maneuvers .
all maneuvers were applied slowly at a pace that the patients feel acceptable . for healthy controls , structural and resting state scans
were only collected once . before and immediately after each 6 minute resting state mri scan
, subjects were asked to rate the intensity of their lbp using a 010 pain scale . the average self - reported pain rating before and after resting state fmri scanning
medication use per self - report was limited to non - steroidal anti - inflammatory drugs ( nsaids , e.g. , ibuprofen , motrin , advil , and naproxen ) and acetaminophen ( e.g. , tylenol ) .
additional non - pharmacological methods of self - reported pain management included chiropractic massages , physical therapy , exercises , and acupuncture .
all mri data were acquired with a 3 t siemens whole - body scanner with echo - planar imaging capability using a 32-channel radio - frequency head coil at the martinos center for biomedical imaging . during the resting state
fmri scan , subjects were asked to keep their eyes open and look at a darkened screen for 6 min .
the bold fmri scan acquisition included 47 slices with slice thickness of 3 mm , tr = 3000 ms , te = 30 ms , and a 3 3 mm in - plane spatial resolution .
t1-weighted mprage structural images were acquired using the following parameters : voxel size 1.2 1.2 1.2 mm , tr = 2.2 s , te = 1.54 ms , flip angle = 7 , slices = 144 ; field of view = 230 .
the fmri data were then preprocessed using spm8 software ( available at : http://www.fil.ion.ucl.ac.uk/spm ) implemented in a matlab suite ( mathworks , inc . ,
images were realigned to correct for motion , corrected for errors in slice timing , spatially transformed to standard stereotaxic space ( based on the montreal neurologic institute coordinate system ) .
a recent study shows that the motion induced artifacts occur with movements on the order of a few tenths of a millimeter or less and produce systematic but spurious correlations in functional connectivity , such that long distance correlations are decreased by subject motion , whereas many short - distance correlations are increased ( power et al . , 2012 ) .
anova on each movement parameter with clbp patients in high pain condition and in low pain condition and healthy control as between - subjects factor revealed no significant differences ( p > 0.1 ) . in table 2 , we show that the means for six movement parameters are small and there was no significant group difference on movement parameters .
there were no participants with movement greater than 3 mm of translation or 3 degrees of rotation .
there were also no significant differences between the total range of movement across any axis of translation or rotation between groups ( see table 2 ) .
the data were then smoothed with a 6-mm full - width half - maximum ( fwhm ) gaussian kernel .
the smooth kernel size was chosen because fwhm resolution usually equals or is greater than twice the voxel size ( mikl et al . , 2008 ) .
data were then bandpass filtered from 0.01 to 0.08 hz to remove low frequency noise ( including slow scanner drifts ) and influences of higher frequencies reflecting cardiac and respiratory signals ( cordes et al . , 2001 ) .
to address head motion concerns in resting - state fmri analyses , we calculated the voxel - specific mean framewise displacement ( fd ) for accounting head motion at group - level analysis ( power et al .
, 2012 , 2013 ; power et al . , 2014 ; van dijk et al . ,
2012 ) . fd measure indexes the movement of the head from one volume to the next and is calculated as the sum of the absolute values of the differentiated realignment estimates ( by backward differences ) at every time point ( power et al . , 2012 ) .
then , we repeated the above analyses after removing frames with fd > 0.5 mm ( scrubbing ) .
bad time points and two time points after bad time points were deleted .
functional connectivity analysis was carried out by applying a seed - region approach using the right ventrolateral pag ( x = 4 , y = 26 , z = 14 , with 2 mm radius ) as the fc seed ( see fig .
the rationale for choosing this location as a seed is : 1 ) in a previous study , we found that increased levels of heat pain can evoke a significant fmri signal increase in this region ( kong et al . , 2010a ) ; 2 ) it is located within the ventrolateral pag , which is believed to be important for opioid antinociception ( bandler and shipley , 1994 ) ; 3 ) it is consistent with previous pag seed based fc study showing that the pag is functionally connected to the acc / vmpfc and the insula ( kong et al . , 2010b ) .
one concern is that signals from other regions may confound the findings due to partial volume effect .
we plotted the time course from the pag seed for all subjects and found no cyclic trends in the data ( see fig .
given that the pag seed region sits adjacent to a ventricle with significant pulsatility effect , we also used a seed from the third ventricle ( x = 0 , y = 3 , z = 7 , with 2 mm radius ) as a control region . for each seed region , individual participant analyses were carried out using the general linear model ( glm ) with the time series for the seed region as well as for the nuisance covariates ( white matter , cerebrospinal fluid , and six motion parameters ) as predictors .
these nuisance signals are typically adjusted for in resting - state fc studies because they reflect signal fluctuations of nonneuronal origin ( e.g. , physiological artifacts associated with variables such as cardiac and respiratory cycles , csf motion , and scanner drift ) ( fox and raichle , 2007 ) .
contrast images were generated for each subject by estimating the regression coefficient between all brain voxels and each seed 's time series , respectively .
these images were then included in second - level group random effect analyses , adopting a t - test design .
we also used regression analyses to examine whether illness duration and/or endogenous pain intensity were related to pag functional connectivity , when considering the clbp participants alone .
endogenous pain intensity was defined as the average self - reported pain rating immediately before and after resting state fmri scanning .
the threshold was family - wise alpha level set to p = 0.05 , family - wise error ( fwe ) correction for multiple comparisons for rois using small volume correction ( svc ) .
based on the anatomical connection of pag and previous studies ( brooks and tracey , 2005 ; fields , 2004 ; heinricher et al . , 2009 ; ploner et al . , 2010
; tracey and mantyh , 2007 ) , the rois include the vmpfc / acc , anterior insula , posterior insula , and amygdala , derived from automated anatomical labeling ( aal , see fig .
a threshold of p < 0.05 family - wise error ( fwe ) corrected at the cluster level across the whole brain was used for non - roi brain regions .
a total of 18 clbp patients ( 6 males ) and 18 age- and gender - matched hcs ( 6 males ) completed the study .
demographics , clinical assessments and characteristics for clbp patients and hcs are presented in table 1 .
the age difference between the two groups was not significant ( mean se 36.1 2.3 in the patient group versus 37.1 2.2 in the control group ) , p = 0.71 .
the duration of illness in the patient group is 5.3 0.9 years .
the bpi was used to measure the pain in the preceding week in the patient group , 6.5 1.7 . of the 18 patients who completed the study , one patient had strong chronic pain at baseline and
the patient received the exact same set of scan procedures before and after the 10 minute rest period , comparable to the healthy control condition .
the self - reported endogenous lbp intensity recorded before and after resting state fmri scanning was averaged and then used as an index of pain during scanning .
after slow clinical pain - inducing maneuver , the self - reported pain intensity was significantly increased from 3.79 0.58 ( mean se ) before pain - inducing maneuver to 6.65 0.46 , t = 5.38 , p < 0.001 . in our data analysis , we define the scanning period during which patients ' low back pain ratings are lower as the low pain ( lp ) condition and the scanning period during which patients ' low back pain ratings are higher as the high pain ( hp ) condition . during the lp condition ( fig .
2c ) , we found predominantly positive correlations between pag activity and activity in nearby structures , including the brainstem ( mostly the midbrain ) , thalamus , parahippocampus , amygdala , and cerebellum , and with distant regions , including the anterior cingulate ( not in control group ) and temporal cortex .
these findings were consistent with our previous pag centered fc study applied in healthy subjects ( kong et al . , 2010b ) and a previous diffusion tensor imaging study on pag ( hadjipavlou et al . , 2006 ) .
whole brain voxel - by - voxel functional connectivity in hcs and clbp patients during the lp condition was compared using a two - sample t - test .
results showed that clbp patients in the lp condition had significantly greater fc between the pag and the left vmpfc / racc , x = 6 , y = 45 , z = 6 , z = 3.20 , voxels = 11 , pfwe < 0.05 svc and right vmpfc / acc , x = 6 , y = 42 , z = 12 , z = 3.18 , voxels = 36 , pfwe < 0.05 svc , as well as other regions including the superior temporal gyrus and the precentral gyrus ( table 3 and fig .
the opposite contrast showed no fc differences above threshold . compared with hcs , patients in the hp condition also showed enhanced fc between the pag and the left vmpfc , x = 6 , y = 42 , z = 9 , z = 2.96 , voxels = 11 , pfwe < 0.05 svc , as well as other regions including the lingual gyrus , superior temporal gyrus , precentral gyrus , dorsal cingulate cortex , and posterior insula ( table 3 and fig .
the opposite contrast showed no fc differences above threshold . when we compared clbp patients in the hp and lp conditions , we did not find a significant difference using the prior threshold ( fig .
, we applied a regression analysis between fc and low back pain ratings using the average pain rating scores in the lp condition .
we believe that this measure captures the immediate pain level during scanning since participants rated their pain before and immediately after each 6 minute resting state mri scan .
for the resting state scan in the hp condition , we found significant negative correlations between fc and lbp ratings in the hp condition at the left vmpfc x = 9 , y = 57 , z = 12 , z = 3.83 , voxels = 9 , pfwe < 0.05 svc , and no positive correlations were found ( fig .
3a ) . to test the association between functional connectivity with the pag and clbp illness duration
, we applied a regression analysis between fc in the lp condition and clbp duration .
the results showed significant negative correlations between fc and clbp illness duration at the right posterior insula , x = 39 , y = 12 , z = 9 , z = 3.04 , voxels = 19 , pfwe < 0.05 svc ( fig .
3b ) and left amygdala , x = 27 , y = 9 , z = 18 , voxels = 3 , pfwe < 0.05 svc ( fig .
3c ) . adding age as covariate of no interest did not change the results . no other significant brain
behavior associations were found . for resting state in the hp condition , we found no significant correlations . using a seed from the third ventricle ( x = 0 , y = 3 , z = 7 , with 2 mm radius ) as a control region showed no group difference in fc with the prefrontal cortex even at lower threshold , p < 0.05 , uncorrected .
pag findings are specific to the pag seed and are unlikely be confounded by signals from the adjacent ventricle area . for scrubbed data , the length of data included in analyses on average was 103.7 3.9 volumes for hp condition , 107.9 2.1 volumes for lp condition , and 111.3 1.9 volumes for healthy controls .
there was no significant difference between the hp and lp conditions ( p > 0.2 ) and between hp and hc or lp and hc ( p values > 0.2 ) .
the repeated analyses without removing frames with framewise displacement ( fd ) > 0.5 mm ( scrubbing ) yielded similar results ( results not reported here ) .
in this study , we investigated differences in the pag centered resting state functional connectivity in clbp patients relative to age and gender matched controls .
interestingly , we found that the functional connectivity between the pag and the vmpfc / racc decreased as endogenous back pain intensity increased after pain - inducing maneuver , suggesting the dynamic character of functional connectivity at the pag .
these functional changes of pag point out that the pag in particular may play an important role in the pathophysiology of clbp .
abnormal vmpfc / racc activity has been found in clbp in previous studies . compared to hcs
, clbp patients showed increased high - frequency bold oscillations ( 0.120.20 hz ) circumscribed mainly to the vmpfc and brain regions within the default network ( baliki et al . , 2011 ) .
more recently , using arterial spin labeling , it has been found that provoked increases in endogenous lbp ratings were positively associated with statistically significant increases in regional cerebral blood flow in a widespread network of cortical areas , including the bilateral vmpfc in clbp patients ( wasan et al . , 2011 ) .
in addition , compared with healthy controls , patients demonstrated stronger default mode network connectivity to the pregenual anterior cingulate cortex , the left inferior parietal lobule , and the right insula ( loggia et al . , 2013 ) .
it has been shown that the prefrontal cortex exerts active control on pain perception by modulating corticosubcortical and corticocortical pathways ( lorenz et al . , 2003 ) .
the current study further highlights the role of vmpfc / racc functional connectivity abnormality in clbp and links the vmpfc / racc to the pag . in a previous study on intrinsic connectivity patterns of the pag using resting - state fmri in a large cohort of 100 healthy subjects
, it has been reported that pag activity is positively correlated with surrounding subcortical brain regions as well as with cortical regions , including the anterior cingulate and anterior insula ( kong et al . , 2010b ) .
multiple studies suggest that pag activity tends to be connected with the racc during pain perception and modulation ( bingel et al . , 2006 ; petrovic et al . , 2002 ;
2005 ) . increased levels of heat pain can evoke a significant fmri signal increase in the pag ( kong et al . , 2010a ) . moreover , using midbrain / brainstem specific imaging , an increased pag activity in response to pain has been demonstrated ( eippert et al . , 2009 ) .
the involvement of the pag in pain processing and modulation has been known for a long time .
brain imaging studies ( bushnell et al . , 1999 ; eippert et al . ,
kong et al . , 2010a ; kong et al . , 2006 ; teutsch et al . , 2008 ; wasan et al . ,
2011 ) have found that the pag is activated during the presentation of noxious stimuli as well as in association with pathological pain states such as chronic low back pain .
studies have shown that distraction tasks reduce the subjective pain sensation ( tracey et al . , 2002 ; valet et al . , 2004 ) .
activation in the periaqueductal gray was significantly increased during the distraction condition ( tracey et al . , 2002 ; valet et al . , 2004 ) , and the total increase in activation was predictive of changes in perceived pain intensity ( tracey et al . , 2002 ) .
the pag also plays a role in pain facilitation . in accordance with a pain facilitation role ,
the magnitude of pag activation has been shown to correlate with the degree of patients ' neuropathic pain symptoms ( freynhagen et al . , 2006 ) .
in irritable bowel syndrome ( ibs ) patients , activity in the vmpfc has been shown to disrupt a functional connection between the lateral pfc and the pag , suggesting that the vmpfc and the pag are involved in enhancing clinical pain ( mayer et al . , 2005 ) .
previous studies have shown a general hypersensitivity to painful stimuli in clbp patients ( farasyn and meeusen , 2005 ; giesecke et al . , 2004 ; puta et al . ,
thus , the enhanced pag vmpfc / racc fc in clbp patients may indicate an enhanced pain inhibition or facilitation .
further studies are needed to clarify the role of this fc change . in the present study , in the vmpfc
, the strength of intrinsic connectivity with the pag was negatively correlated with pain intensity in high pain condition .
the vmpfc has been implicated in regulating affective responses by manipulating the contextual evaluation of sensory events ( rolls and grabenhorst , 2008 ) .
vmpfc activation was associated with decreases in pain unpleasantness ratings induced by mindfulness meditation in healthy subjects ( zeidan et al . , 2011 ) .
in this context , we speculate that a decrease in the intrinsic correlations between the pag and the vmpfc in relation to the pain intensity may indicate a failed pain modulation in the brain in response to worsening of pain .
an alternative explanation is that both the vmpfc and the pag reflect arousal , which is high in hp condition .
interestingly , such correlation was significant only in the hp condition in which the pain level was enhanced by clinical pain - inducing maneuvers , suggesting that the vmpfc
pag network is more related with higher pain states . whether the observed association between spontaneous pain intensity and changes in pag connectivity reflects either an adaptive mechanism or an abnormal state in cortical excitability that predisposes individuals to chronic pain needs to be further clarified .
a previous study has found that the prestimulus fc between the insula and the pag predicted perceived painfulness ( ploner et al . , 2010 ) , suggesting that these two regions interact to determine pain perception .
it has been reported that activation in the contralateral posterior insula was positively correlated with temperature level , whereas subjective intensity related more to activation of the right anterior insula ( craig et al . , 2000 ) .
it has been suggested that the posterior insula may provide a primary interoceptive cortex , specialized for perception of internal bodily states incorporating pain , temperature , and autonomic arousal ( craig , 2003 ; critchley et al . , 2002 ) .
we found that the longer an individual is in the clbp state , the weaker the functional connectivity between the pag and the posterior insula .
we speculate that this may suggest that after long - term clbp suffering , the body is adapted to the situation , and thus the modulation mechanism is somehow weakened .
a previous study also found that verum acupuncture induced a higher level of correlations among the amygdala - associated network including the insula and the pag ( qin et al . , 2008 ) , suggesting that this network may be involved in pain modulation .
however , it is currently unknown whether altered fc with the pag is the consequence of or the cause of clbp .
insula fc was altered in high pain condition . in a recent study of migraine pain patients ( mainero et al . , 2011 ) , investigators have found that patients had greater fc between the pag and the ventral prefrontal cortex compared to the control group .
migraineurs who develop pain in response to normally innocuous stimulation ( i.e. , migraineurs with a history of allodynia ) exhibited decreased fc with the pag in the insula and the mpfc , compared with migraineurs without allodynia .
this result is consistent with findings observed in lbp patients . in another study on a fibromyalgia patient
, investigators have found that patients with fibromyalgia ( a chronic pain disorder characterized by chronic widespread pain and allodynia ( a heightened and painful response to pressure ) ) display less functional connectivity between the acc and the pag ( jensen et al . , 2012 ) .
we speculate that this may reflect the heterogeneity of different chronic pain conditions . taken together
, these findings may suggest that different roles of pain descending control system underlie the localized chronic pain ( e.g. , lbp , migraine ) and widespread pain ( e.g. , fibromyalgia pain ) .
the first potential limitation is the order effects between the high endogenous lbp and low endogenous lbp conditions .
one challenge of clbp studies is that once lbp is provoked , it is hard to control without any pharmacological intervention . in this study , we used exercise to provoke the patients ' lbp ; thus , the high pain condition tended to follow the low pain condition . secondly , our fmri data was not acquired with cardiac - gating which minimizes physiological motion artifact due to pressure wave pulsatility in arteries within and around the brain ( napadow et al . , 2009 ) .
third , a control for the movement exercises corresponding to the pain - inducing maneuvers is missing since healthy controls underwent only one resting state session .
the pain - inducing maneuvers include a set of slow movements such as sit - ups , lumbar flexion / extension , and lumbar rotation .
these daily life activities are not intense for healthy controls , although they can elicit pain in patients with clbp .
thus , it is unlikely that movement exercises in healthy controls would evoke low back pain or induce functional connectivity changes in pain modulation network since there would be no pain expected in healthy controls .
previous studies also indicate that the intrinsic resting state functional connectivity is reliable across different sessions ( birn et al . , 2013 ; liao et al . , 2013 ; shehzad et al . ,
one concern is that the signal to noise ratio ( snr ) might not be good enough to detect pag activity .
however , previous resting state mri studies have successfully used small seed regions , such as amygdala subregions ( roy et al . , 2009 ) , nucleus accumbens ( cauda et al . , 2011 ) , and red nucleus ( nioche et al . , 2009 ) , and found valid fc results .
finally , we did not include medication in the model , and although it is unlikely that medication exposure accounts for our results , we can not completely rule out the effect of medication .
it is important to note , however , that we excluded all patients using opioids in the study , as a previous study found that daily oral administration of morphine for 1 month can cause anatomical changes in the brain ( younger et al . , 2011 ) .
vmpfc fc and that the fc between the pag and the vmpfc decreases as endogenous pain intensity increases in high pain condition .
these findings may not only deepen our understanding of pain modulation and the development of chronic pain but also ultimately help inform mechanism - based therapies for treating different types of acute and chronic pain . | chronic low back pain is a common neurological disorder .
the periaqueductal gray ( pag ) plays a key role in the descending modulation of pain . in this study , we investigated brain resting state pag functional connectivity ( fc ) differences between patients with chronic low back pain ( clbp ) in low pain or high pain condition and matched healthy controls ( hcs ) .
pag seed based functional connectivity ( fc ) analysis of the functional mr imaging data was performed to investigate the difference among the connectivity maps in the clbp in the low or high pain condition and hc groups as well as within the clbp at differing endogenous back pain intensities .
results showed that fc between the pag and the ventral medial prefrontal cortex ( vmpfc)/rostral anterior cingulate cortex ( racc ) increased in clbp patients compared to matched controls .
in addition , we also found significant negative correlations between pain ratings and pag vmpfc / racc fc in clbp patients after pain - inducing maneuver .
the duration of clbp was negatively correlated with pag insula and pag amygdala fc before pain - inducing maneuver in the patient group .
these findings are in line with the impairments of the descending pain modulation reported in patients with clbp .
our results provide evidence showing that clbp patients have abnormal fc in pag centered pain modulation network during rest . | Introduction
Materials and methods
Results
Discussion | chronic low back pain ( clbp ) is one of the most common reasons for all physician visits in the usa and is a leading contributor to job - related disability and missed work ( chou and shekelle , 2010 ; hart et al . non - specific clbp , which represents the majority of clbp patients , is characterized by a lack of recognizable pathology ( chou et al . to develop more effective treatments , it is crucial to understand the underlying neurobiology of clbp in the brain . a prevailing theory in the pathogenesis of chronic pain is that the nociceptive afferents become sensitized in such a way that the signaling of these nociceptive afferents increases perceived pain disproportionately to the pain stimulus ( coderre et al . this process is facilitated by a dysfunction of the descending pain modulatory circuits ( woolf and doubell , 1994 ; zimmermann , 2001 ) . the periaqueductal gray ( pag ) is a key region involved in endogenous pain inhibition ( fields , 2004 ) . , 1986
recent studies have shown that the functional connectivity fluctuations and structural connectivity between the pag and the ventral medial prefrontal cortex ( vmpfc)/rostral anterior cingulate cortex ( racc ) predicted mind wandering away from pain , i.e. it is now believed that the brainstem ( pag ) receives direct projections from regions within the limbic forebrain such as the anterior cingulate cortex ( acc ) and limbic - related areas such as the insula and amygdala and modulates pain by descending modulation of the spinal cord neurons ( brooks and tracey , 2005 ; fields , 2004 ; heinricher et al . a recent study has shown that the pag is functionally connected to the vmpfc / racc , insula and amygdala during resting state ( kong et al . the insula is a key region in pain process ( bernard et al . , 2006
a previous study suggested that prestimulus functional connectivity between the insula and pain - modulatory brain regions ( e.g. the amygdala has a central role in regulating emotional responses during acute and persistent pain ( martikainen et al . recent neuroimaging studies have shown that clbp is associated with alterations in resting state brain activity ( apkarian et al . however , the role of the pag and the associated networks detected by resting state fmri in clbp is still unclear . in the present study
, we investigated pag centered brain resting state functional connectivity ( fc ) differences between clbp patients and matched hcs and fc differences when clbp patients experienced different levels of pain intensity . we hypothesized that clbp would be associated with abnormal fc between the pag and other brain regions including the vmpfc , insula , and amygdala , given the close link between these regions and their role in pain modulation . , 2013b ) , but
, we compared structure and function difference between the clbp and controls using structural imaging data with morphometric analysis and resting state mri data with degree centrality ( dc ) analysis ( kong et al . there is no overlap on the results between the previous paper and the current paper . eighteen clbp patients and 18 healthy controls , matched for age and gender , completed the study ( see table 1 for demographic details ) . only those patients meeting the quebec low back pain task force classification criteria for class i or ii ( axial lbp with possible occasional radiation to the thigh , and no sensory or motor complaints ) were enrolled ( werneke and hart , 2004 ) . subjects were classified as having non - specific back pain ( i.e. , patients with specific diagnoses , non - spinal etiology , or radicular symptoms were not included in the study ) . clbp patients were asked to rate their current pain using a visual analog scale ( 0 no pain , 10 maximum imaginable pain ) , both before and after the resting state fmri scan . endogenous back pain intensity during the resting state was defined as the average pain rating before and after the scan . the brief pain inventory ( bpi ) was used to assess the severity of pain ( the sensory dimension ) and the impact of pain on daily feelings and functions ( the reactive dimension ) in the preceding week , i.e. healthy controls , matched for gender , age and race , were recruited in the community . after the first resting state scan , clbp patients were taken out of the scanner to perform exercises for a period lasting up to 10 min to exacerbate their endogenous lower back pain ( see fig . the exercises determined to exacerbate low back pain included a set of slow movements such as sit - ups , lumbar flexion / extension , and lumbar rotation , where the subject rotated his or her body from side to side at a self - selected speed . during the screening ,
if at the end of the first resting state scan the patient 's clbp pain rating was too strong ( 7 in 010 scale ) and the patient was reluctant to perform exercises to enhance their pain experience , they were asked to wait for 10 min in a comfortable position before starting the second half of the scanning session . during the exercises , clbp patients were intermittently asked to report their level of pain using the 010 pain scale ; the exercises were repeated until subjects reported an increase in pain of approximately 3 points on the pain scale . once this level of pain was achieved , subjects were placed back in the scanner to repeat the same fmri scans that were acquired before the pain - inducing maneuvers . for healthy controls , structural and resting state scans
were only collected once . anova on each movement parameter with clbp patients in high pain condition and in low pain condition and healthy control as between - subjects factor revealed no significant differences ( p > 0.1 ) . there were also no significant differences between the total range of movement across any axis of translation or rotation between groups ( see table 2 ) . the rationale for choosing this location as a seed is : 1 ) in a previous study , we found that increased levels of heat pain can evoke a significant fmri signal increase in this region ( kong et al . , 2010a ) ; 2 ) it is located within the ventrolateral pag , which is believed to be important for opioid antinociception ( bandler and shipley , 1994 ) ; 3 ) it is consistent with previous pag seed based fc study showing that the pag is functionally connected to the acc / vmpfc and the insula ( kong et al . we plotted the time course from the pag seed for all subjects and found no cyclic trends in the data ( see fig . given that the pag seed region sits adjacent to a ventricle with significant pulsatility effect , we also used a seed from the third ventricle ( x = 0 , y = 3 , z = 7 , with 2 mm radius ) as a control region . for each seed region , individual participant analyses were carried out using the general linear model ( glm ) with the time series for the seed region as well as for the nuisance covariates ( white matter , cerebrospinal fluid , and six motion parameters ) as predictors . we also used regression analyses to examine whether illness duration and/or endogenous pain intensity were related to pag functional connectivity , when considering the clbp participants alone . based on the anatomical connection of pag and previous studies ( brooks and tracey , 2005 ; fields , 2004 ; heinricher et al . the age difference between the two groups was not significant ( mean se 36.1 2.3 in the patient group versus 37.1 2.2 in the control group ) , p = 0.71 . the duration of illness in the patient group is 5.3 0.9 years . the bpi was used to measure the pain in the preceding week in the patient group , 6.5 1.7 . of the 18 patients who completed the study , one patient had strong chronic pain at baseline and
the patient received the exact same set of scan procedures before and after the 10 minute rest period , comparable to the healthy control condition . the self - reported endogenous lbp intensity recorded before and after resting state fmri scanning was averaged and then used as an index of pain during scanning . after slow clinical pain - inducing maneuver , the self - reported pain intensity was significantly increased from 3.79 0.58 ( mean se ) before pain - inducing maneuver to 6.65 0.46 , t = 5.38 , p < 0.001 . in our data analysis , we define the scanning period during which patients ' low back pain ratings are lower as the low pain ( lp ) condition and the scanning period during which patients ' low back pain ratings are higher as the high pain ( hp ) condition . 2c ) , we found predominantly positive correlations between pag activity and activity in nearby structures , including the brainstem ( mostly the midbrain ) , thalamus , parahippocampus , amygdala , and cerebellum , and with distant regions , including the anterior cingulate ( not in control group ) and temporal cortex . these findings were consistent with our previous pag centered fc study applied in healthy subjects ( kong et al . whole brain voxel - by - voxel functional connectivity in hcs and clbp patients during the lp condition was compared using a two - sample t - test . results showed that clbp patients in the lp condition had significantly greater fc between the pag and the left vmpfc / racc , x = 6 , y = 45 , z = 6 , z = 3.20 , voxels = 11 , pfwe < 0.05 svc and right vmpfc / acc , x = 6 , y = 42 , z = 12 , z = 3.18 , voxels = 36 , pfwe < 0.05 svc , as well as other regions including the superior temporal gyrus and the precentral gyrus ( table 3 and fig . compared with hcs , patients in the hp condition also showed enhanced fc between the pag and the left vmpfc , x = 6 , y = 42 , z = 9 , z = 2.96 , voxels = 11 , pfwe < 0.05 svc , as well as other regions including the lingual gyrus , superior temporal gyrus , precentral gyrus , dorsal cingulate cortex , and posterior insula ( table 3 and fig . when we compared clbp patients in the hp and lp conditions , we did not find a significant difference using the prior threshold ( fig . , we applied a regression analysis between fc and low back pain ratings using the average pain rating scores in the lp condition . for the resting state scan in the hp condition , we found significant negative correlations between fc and lbp ratings in the hp condition at the left vmpfc x = 9 , y = 57 , z = 12 , z = 3.83 , voxels = 9 , pfwe < 0.05 svc , and no positive correlations were found ( fig . to test the association between functional connectivity with the pag and clbp illness duration
, we applied a regression analysis between fc in the lp condition and clbp duration . the results showed significant negative correlations between fc and clbp illness duration at the right posterior insula , x = 39 , y = 12 , z = 9 , z = 3.04 , voxels = 19 , pfwe < 0.05 svc ( fig . for resting state in the hp condition , we found no significant correlations . using a seed from the third ventricle ( x = 0 , y = 3 , z = 7 , with 2 mm radius ) as a control region showed no group difference in fc with the prefrontal cortex even at lower threshold , p < 0.05 , uncorrected . pag findings are specific to the pag seed and are unlikely be confounded by signals from the adjacent ventricle area . for scrubbed data , the length of data included in analyses on average was 103.7 3.9 volumes for hp condition , 107.9 2.1 volumes for lp condition , and 111.3 1.9 volumes for healthy controls . there was no significant difference between the hp and lp conditions ( p > 0.2 ) and between hp and hc or lp and hc ( p values > 0.2 ) . in this study , we investigated differences in the pag centered resting state functional connectivity in clbp patients relative to age and gender matched controls . interestingly , we found that the functional connectivity between the pag and the vmpfc / racc decreased as endogenous back pain intensity increased after pain - inducing maneuver , suggesting the dynamic character of functional connectivity at the pag . these functional changes of pag point out that the pag in particular may play an important role in the pathophysiology of clbp . abnormal vmpfc / racc activity has been found in clbp in previous studies . compared to hcs
, clbp patients showed increased high - frequency bold oscillations ( 0.120.20 hz ) circumscribed mainly to the vmpfc and brain regions within the default network ( baliki et al . more recently , using arterial spin labeling , it has been found that provoked increases in endogenous lbp ratings were positively associated with statistically significant increases in regional cerebral blood flow in a widespread network of cortical areas , including the bilateral vmpfc in clbp patients ( wasan et al . in addition , compared with healthy controls , patients demonstrated stronger default mode network connectivity to the pregenual anterior cingulate cortex , the left inferior parietal lobule , and the right insula ( loggia et al . the current study further highlights the role of vmpfc / racc functional connectivity abnormality in clbp and links the vmpfc / racc to the pag . in a previous study on intrinsic connectivity patterns of the pag using resting - state fmri in a large cohort of 100 healthy subjects
, it has been reported that pag activity is positively correlated with surrounding subcortical brain regions as well as with cortical regions , including the anterior cingulate and anterior insula ( kong et al . increased levels of heat pain can evoke a significant fmri signal increase in the pag ( kong et al . the involvement of the pag in pain processing and modulation has been known for a long time . ,
2011 ) have found that the pag is activated during the presentation of noxious stimuli as well as in association with pathological pain states such as chronic low back pain . activation in the periaqueductal gray was significantly increased during the distraction condition ( tracey et al . the pag also plays a role in pain facilitation . in accordance with a pain facilitation role ,
the magnitude of pag activation has been shown to correlate with the degree of patients ' neuropathic pain symptoms ( freynhagen et al . in irritable bowel syndrome ( ibs ) patients , activity in the vmpfc has been shown to disrupt a functional connection between the lateral pfc and the pag , suggesting that the vmpfc and the pag are involved in enhancing clinical pain ( mayer et al . previous studies have shown a general hypersensitivity to painful stimuli in clbp patients ( farasyn and meeusen , 2005 ; giesecke et al . ,
thus , the enhanced pag vmpfc / racc fc in clbp patients may indicate an enhanced pain inhibition or facilitation . in the present study , in the vmpfc
, the strength of intrinsic connectivity with the pag was negatively correlated with pain intensity in high pain condition . in this context , we speculate that a decrease in the intrinsic correlations between the pag and the vmpfc in relation to the pain intensity may indicate a failed pain modulation in the brain in response to worsening of pain . an alternative explanation is that both the vmpfc and the pag reflect arousal , which is high in hp condition . interestingly , such correlation was significant only in the hp condition in which the pain level was enhanced by clinical pain - inducing maneuvers , suggesting that the vmpfc
pag network is more related with higher pain states . a previous study has found that the prestimulus fc between the insula and the pag predicted perceived painfulness ( ploner et al . it has been reported that activation in the contralateral posterior insula was positively correlated with temperature level , whereas subjective intensity related more to activation of the right anterior insula ( craig et al . we found that the longer an individual is in the clbp state , the weaker the functional connectivity between the pag and the posterior insula . a previous study also found that verum acupuncture induced a higher level of correlations among the amygdala - associated network including the insula and the pag ( qin et al . however , it is currently unknown whether altered fc with the pag is the consequence of or the cause of clbp . insula fc was altered in high pain condition . , 2011 ) , investigators have found that patients had greater fc between the pag and the ventral prefrontal cortex compared to the control group . , migraineurs with a history of allodynia ) exhibited decreased fc with the pag in the insula and the mpfc , compared with migraineurs without allodynia . in another study on a fibromyalgia patient
, investigators have found that patients with fibromyalgia ( a chronic pain disorder characterized by chronic widespread pain and allodynia ( a heightened and painful response to pressure ) ) display less functional connectivity between the acc and the pag ( jensen et al . taken together
, these findings may suggest that different roles of pain descending control system underlie the localized chronic pain ( e.g. in this study , we used exercise to provoke the patients ' lbp ; thus , the high pain condition tended to follow the low pain condition . third , a control for the movement exercises corresponding to the pain - inducing maneuvers is missing since healthy controls underwent only one resting state session . the pain - inducing maneuvers include a set of slow movements such as sit - ups , lumbar flexion / extension , and lumbar rotation . these daily life activities are not intense for healthy controls , although they can elicit pain in patients with clbp . thus , it is unlikely that movement exercises in healthy controls would evoke low back pain or induce functional connectivity changes in pain modulation network since there would be no pain expected in healthy controls . previous studies also indicate that the intrinsic resting state functional connectivity is reliable across different sessions ( birn et al . however , previous resting state mri studies have successfully used small seed regions , such as amygdala subregions ( roy et al . finally , we did not include medication in the model , and although it is unlikely that medication exposure accounts for our results , we can not completely rule out the effect of medication . it is important to note , however , that we excluded all patients using opioids in the study , as a previous study found that daily oral administration of morphine for 1 month can cause anatomical changes in the brain ( younger et al . vmpfc fc and that the fc between the pag and the vmpfc decreases as endogenous pain intensity increases in high pain condition . these findings may not only deepen our understanding of pain modulation and the development of chronic pain but also ultimately help inform mechanism - based therapies for treating different types of acute and chronic pain . | [
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with advances in health care , many children with developmental disabilities and neurologic disorders are living longer lives , and increasingly require diagnostic and therapeutic interventions .
pediatricians and pediatric subspecialists are increasing being called upon to safely sedate and provide analgesia for these children for diagnostic procedures ( ct , mri , angiogram , endoscopy , and bronchoscopy ) and for therapeutic interventions ( interventional radiology , intracranial injury , and emergency stabilization ) .
this paper will focus on children with developmental disabilities and neurologic injury , and will highlight the risks involved with these patients , and the effects of common sedatives and analgesic agents on the central nervous system .
the purpose of this paper is to provide the pediatrician and pediatric subspecialist a better understanding on the neurologic effects of different sedative and analgesic medications so that rational and safe choices can be used in children with developmental disabilities and neurologic disorders without causing further neurologic compromise .
we performed an extensive review of the medical literature regarding sedation analgesia in children with developmental disability and neurologic disorders utilizing pubmed .
analgesia , pediatric , child , neonate , brain , developmental disabilities , neurologic , autism , epilepsy , seizure , stroke , hydrocephalus , traumatic brain injury , intracranial hemorrhage , hypoxia - ischemia , and encephalopathy and the period of search was from 19602010 .
the authors are pediatric neurocritical care specialists and have extensive clinical experience caring for pediatric patients with developmental disabilities and neurologic disorders and research experience in experimental animal models of pediatric neurologic injury .
sedation and analgesia for the pediatric patient with developmental disabilities and neurologic disorder require a thorough understanding of potential adverse events , and the knowledge and skill to avoid potentially life - threatening complications from the administration of sedative and analgesic medications .
in addition , the practitioner must focus particular attention on the entire periprocedural period including presedation evaluation , sedation / analgesia administration , and recovery .
the american academy of pediatrics , section on anesthesiology has published guidelines for the pediatric perioperative anesthesia environment , which includes suggestions for age categorization , need for intensive care following sedation for recovery , and presence of coexisting disease .
since these guidelines were published , sedation outside of the operating room continues to increase , along with the varied practitioner 's disciplines that are delivering sedation . with this practice increasing ,
the debate about safety and the practitioner core competency requirements to provide sedation and/or analgesia to the complex pediatric patient with developmental disabilities and neurologic disorders has also increased and several policy statements have been published by different professional societies , with no clear evidence of practice standards and incidence of adverse outcomes . to aid in the investigation of the practice and potential adverse outcomes associated with the delivery of sedation outside the operating room , the pediatric sedation research consortium ( psrc ) , a collection of 37 institutions that share information on sedation practices within their individual institutions , has created a self - reporting prospective , observational database .
this database has provided vital information to define the frequency and nature of adverse events during pediatric sedation from a multispecialty perspective .
large psrc studies have shown a relatively low risk to pediatric sedation by practitioners other than anesthesiologists . however , despite zero deaths in the 49,836 sedation encounters , one in 65 of these sedation encounters was associated with stridor , wheezing , airway obstruction , laryngospasm , or central apnea , conditions that all have the potential to deteriorate to respiratory failure and death .
airway obstruction and pulmonary complications were the most frequently cited adverse event . in subsequent analysis , factors that related to higher rates of pulmonary complications were young patients , use of adjunctive opiates , and patients with a higher american society of anesthesiology ( asa ) status ( iii ) , a large proportion with neurologic conditions .
this continues to emphasize that pediatric patients with neurologic disorder and developmental disabilities receiving sedation continue to be at increased risk for adverse events with the most prevalent concerns for airway obstruction and altered respiratory mechanics .
unfortunately , extensive studies have not been performed to identify specific patients at risk and aid in the development of evidence - based clinical protocols for patients with neurologic pathology and developmental disabilities .
most reported experience refers to scattered case reports of specific syndromes ( butler et al .
so what are the actual added risks associated with sedation of the pediatric patient with developmental disabilities or neurologic disorders ?
brain mri has become an important diagnostic and management tool for these children and is being increasingly used in many pediatric centers .
kannikeswaran and colleagues recently published a retrospective review of children , 118 years of age , sedated for brain mri with and without developmental disability .
developmental disability is defined by these investigators as delay in one of the following : fine / gross motor , cognitive , speech / language , social / personal , and activities of daily living .
pentobarbital and fentanyl were the two most common medications used with no difference in mean dosages between children classified as
however , the patients classified as having developmental disability had a threefold increased incidence of hypoxia ( 11.9% versus 4.9% ; p < .01 ) .
these findings seem to recapitulate the findings described in the psrc studies : an increase in adverse events , most notably airway compromise , for children with developmental disabilities and those with neurologic disorders . in this study ,
the most common diagnosis for the cause of developmental disability was autism ( 36% ) .
in addition , the authors included patients with attention deficit hyperactivity disorder ( 20% ) as a diagnosis for developmental disability . in another study , a small observational chart review performed by elwood et al .
suggests that the anteroposterior oropharyngeal airway diameter was smaller in children with developmental delay than in those without developmental delay , in static mri images .
the limitations in this study were the varied diagnoses within groups of patients diagnosed with developmental delay without specific recommendations for certain patient populations .
however , it does reinforce the idea that sedation practitioners need to exhibit marked vigilance for airway patency in patients with developmental disabilities .
in addition to a baseline risk for airway compromise in patients with developmental disability , cortellazzi and colleagues showed that the risk for airway obstruction significantly increased in neurologically impaired children undergoing mri who were administered a combination of sedative medications .
thus , as practitioners escalate pharmacologic intervention a patient with developmental disabilities or neurologic disorders is at increased risk for airway obstruction and may need higher level of care , including the potential need for a pediatric emergency medicine specialist , anesthesiologist , or intensivist .
protocols are based on agent pharmacokinetic and pharmacodynamic profiles , with an attempt to maintain a proper plane of sedation and analgesia without respiratory and hemodynamic compromise .
however , virtually no protocol exists on the use of different sedatives and analgesic medications with the focus on preventing neurologic compromise .
while there is not enough evidence - based data to support specific clinical guidelines for sedation and analgesia in children with developmental disabilities and neurologic disorders , the authors ' hope is that the sedating practitioner will have a better understanding to safely administer these medications without promoting further neuronal injury .
the practitioner must have a well - developed understanding of the effects that different sedative and analgesic agents will have on cerebral vasculature , metabolism , autoregulation ( maintaining constant cerebral blood flow despite changes in perfusion pressure ) , intracranial pressure ( icp ) , and cerebral perfusion pressure ( the perfusion pressure that causes blood flow to the brain ) ; see table 1 that provides the route and dosages of the different sedative and analgesic medications that are commonly administered .
opioids , such as morphine , fentanyl , and remifentanil , have long been considered effective adjuvant medications for analgesia of patients with developmental disabilities and neurologic disorders .
opioids are very useful in the treatment of nociceptive pain , and are crucial in developing a balanced sedation plan when analgesia is a concern , for example , intubation and comorbid injuries .
higher doses of opioids can also have some degree of sedation and even hypnosis ; however , sedation is a side effect and not the intended pharmacodynamic purpose .
furthermore , opioids lack amnestic properties , and therefore are rarely used as sole agents for sedation in children .
opioids are commonly coadministered with benzodiazepines , because of their ability to provide sedation , amnesia , and hypnosis .
side effects of all opioids are similar , and include : constipation , urinary retention , sedation , nausea , vomiting , respiratory depression , bradycardia , hypotension , and pruritis .
cerebral metabolic oxygen rate , cerebral blood flow ( cbf ) , and icp all decrease with the administration of opioids if a patient 's arterial carbon dioxide ( paco2 ) remains unchanged .
an increase in paco2 relaxes smooth muscle , dilates cerebral vessels , decreases cerebrovascular resistance , and increases cbf .
however , opioids have minimal effects on cerebral hemodynamics in adequately resuscitated patients with controlled ventilation .
the use of short - acting and ultra - short acting iv narcotics ( fentanyl , sufentanil , and remifentanil ) via bolus infusion and/or continuous infusion reported conflicting data on icp effects [ 1315 ] .
opioids have a direct effect on the respiratory centers in the medulla , and decrease minute ventilation by decreasing respiratory rate and produce a dose - dependent depression of ventilatory response to carbon dioxide levels ; therefore , opioids decrease the apneic threshold which may lead to hypoxia and respiratory failure .
this side effect may be exacerbated in children with developmental disabilities and neurologic disorders , who commonly have hypotonia , central apnea , and inadequate airway reflexes .
morphine , like most narcotics tend to decrease heart rate , depending on the level of sympathetic output , through central vagal stimulation .
in addition to a negative chronotropic effect , morphine can lower mean arterial blood pressure via arterial and venous dilation . venodilation lasts longer than arterial dilation and at increasing dosages will decrease cardiac output and lower myocardial oxygen demands .
because of these properties morphine is commonly administered in adults with myocardial ischemia ; however , in patients with traumatic brain injury a substantial decrease in cardiac output and cerebral perfusion pressure may lead to cerebral ischemia .
morphine administration results in elevated histamine levels released from non - ige - mediated stimulation of mast cells .
histamine can result in decreased systemic vascular resistance and an increased incidence of pruritis in children . in children with developmental disabilities and neurologic disorders
it may be difficult to differentiate between increasing levels of agitation due to pain or pruritis , and other side effects .
the terminal half - life of morphine is higher in neonates , especially preterm neonates , and decreases with age ; however , there is significant individual variability in children . on average the terminal half - life ( t1/2 ) is approximately 9 hours in preterm infants , 6.5 hours in full - term neonates , and 2 hours in infants and children .
fentanyl has several advantages over morphine as an adjuvant medication for analgesia in children with developmental disabilities and neurologic disorders .
fentanyl crosses the blood brain barrier quickly and has a rapid onset and relatively short offset . in lower doses
, fentanyl has minimal effects on cardiac output or respiratory depression unless used in combination with other medications such as benzodiazepines .
fentanyl is highly lipophilic and can be administered by intranasal , transmucosal , or transdermal routes .
one of the major side effects of fentanyl is that rapid iv bolus can cause chest wall rigidity . in our intensive care unit ,
fentanyl is the most common opioid used to provide analgesia in postoperative patients with developmental disabilities and neurologic disorders and is administered over 3 to 5 minutes when administered by the iv route .
we also commonly administer fentanyl in combination with midazolam to provide sedation and analgesia for intubation and mechanical ventilation .
remifentanil is a potent ultra - short acting synthetic opioid that has become a common component of total intravenous anesthetics ( tivas ) especially for procedures requiring neuromonitoring of somatosensory evoked potentials ( ssep ) and motor evoked potentials ( mep ) in children for neurosurgery and spinal surgery .
the half life is 4 minutes , and unlike other synthetic opioids which are metabolized by hepatic elimination , remifentanil is metabolized by nonspecific tissue and plasma esterases , thereby eliminating accumulation .
remifentanil is also commonly used in combination with other sedatives such as propofol or midazolam for short painful procedures .
significant dose - dependent bradycardia can be associated with remifentanil administration . due to the lack of effect on neuromonitoring , lack of accumulation , and short half life ,
remifentanil can be used with great success by allowing the practitioner the ability to quickly adjust the depth of sedation for patients with neurologic disorders .
benzodiazepines are particularly useful for sedation in pediatric patients with developmental disabilities and neurologic disorders because of their pleiotropic effects : sedation , anxiolysis , muscle relaxation , and anterograde amnesia .
benzodiazepines also have anticonvulsant effects by enhancing the effect of the neurotransmitter gamma - aminobutyric acid ( gaba ) and is ideal for sedating children with epilepsy .
significant effects on organ systems include : decrease in blood pressure , depressed ventilation ( transient apnea , especially in combination with opioids ) , and a decrease in cerebral metabolic rate .
it is very interesting to note that basic science research continues to try to elucidate the effect of anesthetics , including benzodiazepines , on the developing brain and neurocognitive function .
no traumatic brain injury studies , solely involving pediatric patients , exist on the administration of commonly used benzodiazepines ( midazolam , lorazepam , diazepam ) .
one case series studied the effects of diazepam in 7 adults and 1 adolescent with severe traumatic brain injury and revealed a reduction in cerebral blood flow and cerebral metabolic rate with no effect on blood pressure . in our icu , if the fentanyl fails to control intracranial hypertension , and as long as the hemodynamics are adequate , we will then commonly administer a bolus iv dose of midazolam and start a continuous iv infusion in a critically ill patient who is intubated and mechanically ventilated .
midazolam is a short - acting ( unlike lorazepam and diazepam ) , water - soluble benzodiazepine commonly used for sedation in children with developmental disabilities and neurologic disorders .
midazolam does not cause local irritation after injection ( unlike diazepam ) and can be readily mixed with other medications for intravenous , intramuscular , oral , intranasal , or rectal administration .
rapid redistribution from the brain to other tissues , and rapid metabolism by the liver , accounts for a short duration of action and an elimination half life of 1 to 2 hours .
barbiturates , such as thiopental and pentobarbital , have long been considered effective sedatives for patients with neurologic disorders .
barbiturates decrease cerebral blood flow ( cbf ) and cerebral metabolic rate ( cmro2 ) in a dose - dependent fashion but preserves autoregulation .
reductions in cbf and cmro2 result in a reduction in icp which can be useful for traumatic brain injury patients as well as acute stroke or intracranial hemorrhage patients with intracranial hypertension . in animal studies ,
barbiturates ' neuroprotective characteristics are also related to reductions in ischemia - induced glutamate release and inhibition of intracellular calcium release [ 23 , 24 ] .
recovery time can be prolonged following administration which may be a concern if subtle changes in neurologic exam need to be detected such as in the acute pediatric stroke patient .
some pediatric patients may also experience severe agitation during recovery which can confound sequential neurologic examinations .
high - dose pentobarbital is also used as a third tiered therapy to control intracranial hypertension following severe traumatic brain injury in children but the practitioner must be aware of the association with hemodynamic instability and the need for blood pressure support with intravenous fluids and inotropic infusions .
besides myocardial depression and hypotension , barbiturates also have the side effect of respiratory depression which can lead to hypoxia .
this side effect may be exacerbated in children with developmental disabilities . in a retrospective study , 260 children with developmental disabilities and 226 children without developmental disabilities undergoing brain mri with sedation were reviewed .
no difference in dosages of pentobarbital were observed between the two groups but there was a threefold increased incidence of hypoxia in children with developmental disability ( 11.9% versus 4.9% ) . in summary
, pentobarbital is commonly used for sedation in children undergoing diagnostic radiologic procedures such as mri or ct scan , but in the child with developmental disabilities or neurologic disorders the practitioner must be aware of the important side effects of systemic hypotension and respiratory depression and be prepared to respond to them quickly to avoid further neurologic injury .
thiopental is an ultrashort - acting barbiturate which in its iv form is commonly used as an induction agent for intubation . the fast onset for induction and short duration of effect make it an attractive agent for rapid sequence intubation in pediatrics .
it still poses some of the risks associated with barbiturates including myocardial depression and hypotension .
the use of rectal thiopental ( 1525 mg / kg ) for sedation of pediatric patients undergoing diagnostic ct imaging of the head has been reported to be effective . due to the short duration of action ,
thiopental is generally not used for sedation during mri imaging due to longer scan times .
etomidate is a short - acting iv drug that produces sedation , anxiolysis , and amnesia .
etomidate has the benefits of decreasing icp by reductions in cbf and cmro2 and has the advantage of producing less cardiovascular depression than barbiturates or propofol , and preserving cerebral perfusion pressure [ 28 , 29 ] .
these neuroprotective qualities are counterbalanced by its ability to increase cerebral vascular resistance by a greater magnitude than its reduction of cmro2 resulting in an increased metabolic deficit [ 30 , 31 ] .
the increased metabolic deficit has the potential to expand the ischemic core and penumbra in brain - injured tissue .
this increase in cerebrovascular tone is thought to be attributed to etomidate 's inhibition of nitric oxide synthase .
previous reports on the efficacy of etomidate for pediatric sedation during diagnostic imaging have been mixed .
performed a prospective randomized trial comparing etomidate versus pentobarbital for head and neck ct imaging in children 6 months to 6 years .
sedation success rate was significantly lower in the etomidate group ( 57%76% versus 97% ) but the duration of sedation was not surprisingly shorter in the etomidate group .
prospective data collected from the psrc also compared sedation with pentobarbital versus etomidate for diagnostic ct .
only 1 of 446 children sedated with etomidate was deemed not ideal sedation compared to 11 of the 396 children who received pentobarbital and duration of sedation was shorter with etomidate ( 34 versus 144 minutes ) .
etomidate may have a role for sedation during diagnostic ct imaging but etomidate 's short duration of sedation is disadvantageous for the longer scan times required for mri .
of further note , etomidate 's role in the sedation of the pediatric stroke and intracranial hemorrhage patient may be limited and caution should be used to avoid further expansion of the penumbra and ischemic core .
ketamine is a phenylcyclidine derivative typically formulated as a mixture of two enantiomers in a hydrochloride salt form .
it possess low ph of around 4 which can produce pain at the injection site when administered intramuscularly or intravenously .
ketamine is a n - methyl - d - aspartate ( nmda ) antagonist which produces increases in cbf and cmro2 [ 35 , 36 ] .
early studies in patients with obstructed csf pathways reported ketamine administration increased icp with reductions in cerebral perfusion pressure [ 37 , 38 ] .
more recent studies in adult patients with severe head injury have demonstrated improvements in cerebral perfusion pressure and minimal increases in icp with ketamine [ 3941 ] .
one recent report of 30 intubated pediatric head injury patients observed that single doses of ketamine lowered icp without producing decreases in blood pressure or cerebral perfusion pressure .
however , it is still unclear regarding the effect of ketamine on icp in patients where ventilation is not being tightly controlled . at the present time
, there is not enough data to recommend the use of ketamine in the pediatric population at risk for intracranial hypertension , but further studies to assess the role of ketamine in this patient population are warranted .
ketamine is sometimes used as a continuous infusion in intubated patients as an anti - convulsant for children with refractory epilepsy . while ketamine is also a bronchodilator and
is helpful in children with asthma , it increases oropharyngeal and airway secretions which may be problematic in children with neurologic disorders who have difficulty handling respiratory secretions . as a result , pretreatment with an antisialogogue such as glycopyrrolate or atropine before the administration of ketamine may be beneficial . in older children and adolescents , hallucinations and delirium can occur ; these patients are often premedicated with a short - acting benzodiazepine such as midazolam .
propofol is a short - acting sedative - hypnotic iv agent that can be used to provide moderate or deep sedation .
propofol can induce a deep state of sedation rapidly , provide a short duration of effect , and have a pleasant recovery phase .
propofol is a very popular agent for sedating pediatric patients with neurologic conditions for noninvasive diagnostic imaging , such as a ct scan or mri . due to the fast onset and recovery following administration , repeated neurologic examinations
are easy to assess such as a child with sickle cell disease who comes in with altered mental status due to a stroke .
propofol also has anticonvulsant properties and reduces icp which can be advantageous in sedating a patient with epilepsy or a patient with concerns for obstructive hydrocephalus due to a malfunctioning ventriculoperitoneal shunt to obtain diagnostic neuroradiologic imaging [ 4446 ] .
while there have been cases of propofol providing adequate sedation and successfully treating intracranial hypertension [ 47 , 48 ] , several pediatric traumatic brain injury case reports have reported metabolic acidosis and death in patients on prolonged ( 24 hrs ) continuous infusion of propofol [ 4953 ] . in the
2003 published guidelines for the care of severe pediatric traumatic brain - injured patients , continuous infusion of propofol is not recommended .
adverse effects of propofol include pain at the injection site , apnea or respiratory depression , hypotension , and bradycardia which can be detrimental in a patient at risk for brain ischemia
. propofol does not provide any analgesia . as already discussed , a rare but potentially fatal
hyperlipidemia and multiorgan system failure was first described in pediatric patients who received prolonged ( 24 hours ) continuous infusion and at higher dosages ( > 4.5 mg / kg / hr ) [ 51 , 55 , 56 ] .
dexmedetomidine , a centrally acting 2-adrenergic agonist , is a recently fda approved agent used for short term ( < 24 hours ) continuous iv sedation of adults who are tracheally intubated . like propofol , it has a rapid onset and a relatively rapid elimination half life and is administered as a loading dose followed by continuous iv infusion .
one of the advantages is that it provides sedation with a lower risk of respiratory depression than many other sedative medications .
there is increased interest with this agent as a sedative during non - invasive neuroradiologic imaging studies in children who are not intubated . in one study ,
dexmedetomidine was compared to propofol in children undergoing mri studies . while the onset of sedation and recovery time were significantly shorter in the children that received propofol , hypotension , respiratory depression and desaturation were more common compared to the children receiving dexmedetomidine .
there is increased interest in the use of dexmedetomidine as a sedative and potential neuroprotective agent in both adults and children , as animal studies revealed neuroprotection from hypoxia - ischemia and decreased apoptosis and adult human studies in healthy volunteers demonstrated parallel decrease in cmro2 and cbf , which may temporarily be helpful in briefly sedating patients who are at risk for intracranial hypertension such as head trauma , brain tumor , and obstructive hydrocephalus [ 59 , 60 ] . in pediatric traumatic brain injury case reports , no detrimental effects on their icp was observed .
however , systemic hypertension was observed in one child who were receiving dexmedetomidine with other sedatives , while bradycardia was observed in 2 other children who was receiving dexmedetomidine , other sedatives , and therapeutic hypothermia [ 59 , 60 ] .
further studies are warranted on the potential use and side effects of this agent in children at risk for intracranial hypertension .
hypotension has been reported as well as hypertension , the latter thought to be due to peripheral 2b agonism with peripheral vasoconstriction .
there are conflicting reports on the effects of ventilatory function , with some studies suggesting mild respiratory depression , while others show no effect .
while icp does not appear to increase , cerebral perfusion pressure and cbf have been shown to decrease .
while a variety of sedative and analgesic medications have been used in pediatric patients with developmental disabilities and neurologic disorders , it is clear that further studies are needed to determine the optimal agent(s ) that will maximize good outcome and will minimize or prevent respiratory , circulatory , and further neurologic compromise . | sedation and analgesia performed by the pediatrician and pediatric subspecialists are becoming increasingly common for diagnostic and therapeutic purposes in children with developmental disabilities and neurologic disorders ( autism , epilepsy , stroke , obstructive hydrocephalus , traumatic brain injury , intracranial hemorrhage , and hypoxic - ischemic encephalopathy ) .
the overall objectives of this paper are ( 1 ) to provide an overview on recent studies that highlight the increased risk for respiratory complications following sedation and analgesia in children with developmental disabilities and neurologic disorders , ( 2 ) to provide a better understanding of sedatives and analgesic medications which are commonly used in children with developmental disabilities and neurologic disorders on the central nervous system . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions | with advances in health care , many children with developmental disabilities and neurologic disorders are living longer lives , and increasingly require diagnostic and therapeutic interventions . pediatricians and pediatric subspecialists are increasing being called upon to safely sedate and provide analgesia for these children for diagnostic procedures ( ct , mri , angiogram , endoscopy , and bronchoscopy ) and for therapeutic interventions ( interventional radiology , intracranial injury , and emergency stabilization ) . this paper will focus on children with developmental disabilities and neurologic injury , and will highlight the risks involved with these patients , and the effects of common sedatives and analgesic agents on the central nervous system . the purpose of this paper is to provide the pediatrician and pediatric subspecialist a better understanding on the neurologic effects of different sedative and analgesic medications so that rational and safe choices can be used in children with developmental disabilities and neurologic disorders without causing further neurologic compromise . we performed an extensive review of the medical literature regarding sedation analgesia in children with developmental disability and neurologic disorders utilizing pubmed . analgesia , pediatric , child , neonate , brain , developmental disabilities , neurologic , autism , epilepsy , seizure , stroke , hydrocephalus , traumatic brain injury , intracranial hemorrhage , hypoxia - ischemia , and encephalopathy and the period of search was from 19602010 . the authors are pediatric neurocritical care specialists and have extensive clinical experience caring for pediatric patients with developmental disabilities and neurologic disorders and research experience in experimental animal models of pediatric neurologic injury . sedation and analgesia for the pediatric patient with developmental disabilities and neurologic disorder require a thorough understanding of potential adverse events , and the knowledge and skill to avoid potentially life - threatening complications from the administration of sedative and analgesic medications . in addition , the practitioner must focus particular attention on the entire periprocedural period including presedation evaluation , sedation / analgesia administration , and recovery . the american academy of pediatrics , section on anesthesiology has published guidelines for the pediatric perioperative anesthesia environment , which includes suggestions for age categorization , need for intensive care following sedation for recovery , and presence of coexisting disease . with this practice increasing ,
the debate about safety and the practitioner core competency requirements to provide sedation and/or analgesia to the complex pediatric patient with developmental disabilities and neurologic disorders has also increased and several policy statements have been published by different professional societies , with no clear evidence of practice standards and incidence of adverse outcomes . in subsequent analysis , factors that related to higher rates of pulmonary complications were young patients , use of adjunctive opiates , and patients with a higher american society of anesthesiology ( asa ) status ( iii ) , a large proportion with neurologic conditions . this continues to emphasize that pediatric patients with neurologic disorder and developmental disabilities receiving sedation continue to be at increased risk for adverse events with the most prevalent concerns for airway obstruction and altered respiratory mechanics . unfortunately , extensive studies have not been performed to identify specific patients at risk and aid in the development of evidence - based clinical protocols for patients with neurologic pathology and developmental disabilities . so what are the actual added risks associated with sedation of the pediatric patient with developmental disabilities or neurologic disorders ? brain mri has become an important diagnostic and management tool for these children and is being increasingly used in many pediatric centers . developmental disability is defined by these investigators as delay in one of the following : fine / gross motor , cognitive , speech / language , social / personal , and activities of daily living . these findings seem to recapitulate the findings described in the psrc studies : an increase in adverse events , most notably airway compromise , for children with developmental disabilities and those with neurologic disorders . in another study , a small observational chart review performed by elwood et al . suggests that the anteroposterior oropharyngeal airway diameter was smaller in children with developmental delay than in those without developmental delay , in static mri images . the limitations in this study were the varied diagnoses within groups of patients diagnosed with developmental delay without specific recommendations for certain patient populations . however , it does reinforce the idea that sedation practitioners need to exhibit marked vigilance for airway patency in patients with developmental disabilities . in addition to a baseline risk for airway compromise in patients with developmental disability , cortellazzi and colleagues showed that the risk for airway obstruction significantly increased in neurologically impaired children undergoing mri who were administered a combination of sedative medications . thus , as practitioners escalate pharmacologic intervention a patient with developmental disabilities or neurologic disorders is at increased risk for airway obstruction and may need higher level of care , including the potential need for a pediatric emergency medicine specialist , anesthesiologist , or intensivist . protocols are based on agent pharmacokinetic and pharmacodynamic profiles , with an attempt to maintain a proper plane of sedation and analgesia without respiratory and hemodynamic compromise . however , virtually no protocol exists on the use of different sedatives and analgesic medications with the focus on preventing neurologic compromise . while there is not enough evidence - based data to support specific clinical guidelines for sedation and analgesia in children with developmental disabilities and neurologic disorders , the authors ' hope is that the sedating practitioner will have a better understanding to safely administer these medications without promoting further neuronal injury . the practitioner must have a well - developed understanding of the effects that different sedative and analgesic agents will have on cerebral vasculature , metabolism , autoregulation ( maintaining constant cerebral blood flow despite changes in perfusion pressure ) , intracranial pressure ( icp ) , and cerebral perfusion pressure ( the perfusion pressure that causes blood flow to the brain ) ; see table 1 that provides the route and dosages of the different sedative and analgesic medications that are commonly administered . opioids , such as morphine , fentanyl , and remifentanil , have long been considered effective adjuvant medications for analgesia of patients with developmental disabilities and neurologic disorders . opioids are very useful in the treatment of nociceptive pain , and are crucial in developing a balanced sedation plan when analgesia is a concern , for example , intubation and comorbid injuries . higher doses of opioids can also have some degree of sedation and even hypnosis ; however , sedation is a side effect and not the intended pharmacodynamic purpose . furthermore , opioids lack amnestic properties , and therefore are rarely used as sole agents for sedation in children . opioids are commonly coadministered with benzodiazepines , because of their ability to provide sedation , amnesia , and hypnosis . side effects of all opioids are similar , and include : constipation , urinary retention , sedation , nausea , vomiting , respiratory depression , bradycardia , hypotension , and pruritis . cerebral metabolic oxygen rate , cerebral blood flow ( cbf ) , and icp all decrease with the administration of opioids if a patient 's arterial carbon dioxide ( paco2 ) remains unchanged . an increase in paco2 relaxes smooth muscle , dilates cerebral vessels , decreases cerebrovascular resistance , and increases cbf . the use of short - acting and ultra - short acting iv narcotics ( fentanyl , sufentanil , and remifentanil ) via bolus infusion and/or continuous infusion reported conflicting data on icp effects [ 1315 ] . opioids have a direct effect on the respiratory centers in the medulla , and decrease minute ventilation by decreasing respiratory rate and produce a dose - dependent depression of ventilatory response to carbon dioxide levels ; therefore , opioids decrease the apneic threshold which may lead to hypoxia and respiratory failure . this side effect may be exacerbated in children with developmental disabilities and neurologic disorders , who commonly have hypotonia , central apnea , and inadequate airway reflexes . morphine , like most narcotics tend to decrease heart rate , depending on the level of sympathetic output , through central vagal stimulation . in addition to a negative chronotropic effect , morphine can lower mean arterial blood pressure via arterial and venous dilation . because of these properties morphine is commonly administered in adults with myocardial ischemia ; however , in patients with traumatic brain injury a substantial decrease in cardiac output and cerebral perfusion pressure may lead to cerebral ischemia . histamine can result in decreased systemic vascular resistance and an increased incidence of pruritis in children . in children with developmental disabilities and neurologic disorders
it may be difficult to differentiate between increasing levels of agitation due to pain or pruritis , and other side effects . the terminal half - life of morphine is higher in neonates , especially preterm neonates , and decreases with age ; however , there is significant individual variability in children . on average the terminal half - life ( t1/2 ) is approximately 9 hours in preterm infants , 6.5 hours in full - term neonates , and 2 hours in infants and children . fentanyl has several advantages over morphine as an adjuvant medication for analgesia in children with developmental disabilities and neurologic disorders . in lower doses
, fentanyl has minimal effects on cardiac output or respiratory depression unless used in combination with other medications such as benzodiazepines . in our intensive care unit ,
fentanyl is the most common opioid used to provide analgesia in postoperative patients with developmental disabilities and neurologic disorders and is administered over 3 to 5 minutes when administered by the iv route . we also commonly administer fentanyl in combination with midazolam to provide sedation and analgesia for intubation and mechanical ventilation . remifentanil is a potent ultra - short acting synthetic opioid that has become a common component of total intravenous anesthetics ( tivas ) especially for procedures requiring neuromonitoring of somatosensory evoked potentials ( ssep ) and motor evoked potentials ( mep ) in children for neurosurgery and spinal surgery . the half life is 4 minutes , and unlike other synthetic opioids which are metabolized by hepatic elimination , remifentanil is metabolized by nonspecific tissue and plasma esterases , thereby eliminating accumulation . remifentanil is also commonly used in combination with other sedatives such as propofol or midazolam for short painful procedures . due to the lack of effect on neuromonitoring , lack of accumulation , and short half life ,
remifentanil can be used with great success by allowing the practitioner the ability to quickly adjust the depth of sedation for patients with neurologic disorders . benzodiazepines are particularly useful for sedation in pediatric patients with developmental disabilities and neurologic disorders because of their pleiotropic effects : sedation , anxiolysis , muscle relaxation , and anterograde amnesia . benzodiazepines also have anticonvulsant effects by enhancing the effect of the neurotransmitter gamma - aminobutyric acid ( gaba ) and is ideal for sedating children with epilepsy . significant effects on organ systems include : decrease in blood pressure , depressed ventilation ( transient apnea , especially in combination with opioids ) , and a decrease in cerebral metabolic rate . it is very interesting to note that basic science research continues to try to elucidate the effect of anesthetics , including benzodiazepines , on the developing brain and neurocognitive function . no traumatic brain injury studies , solely involving pediatric patients , exist on the administration of commonly used benzodiazepines ( midazolam , lorazepam , diazepam ) . one case series studied the effects of diazepam in 7 adults and 1 adolescent with severe traumatic brain injury and revealed a reduction in cerebral blood flow and cerebral metabolic rate with no effect on blood pressure . in our icu , if the fentanyl fails to control intracranial hypertension , and as long as the hemodynamics are adequate , we will then commonly administer a bolus iv dose of midazolam and start a continuous iv infusion in a critically ill patient who is intubated and mechanically ventilated . midazolam is a short - acting ( unlike lorazepam and diazepam ) , water - soluble benzodiazepine commonly used for sedation in children with developmental disabilities and neurologic disorders . rapid redistribution from the brain to other tissues , and rapid metabolism by the liver , accounts for a short duration of action and an elimination half life of 1 to 2 hours . barbiturates , such as thiopental and pentobarbital , have long been considered effective sedatives for patients with neurologic disorders . reductions in cbf and cmro2 result in a reduction in icp which can be useful for traumatic brain injury patients as well as acute stroke or intracranial hemorrhage patients with intracranial hypertension . high - dose pentobarbital is also used as a third tiered therapy to control intracranial hypertension following severe traumatic brain injury in children but the practitioner must be aware of the association with hemodynamic instability and the need for blood pressure support with intravenous fluids and inotropic infusions . this side effect may be exacerbated in children with developmental disabilities . in a retrospective study , 260 children with developmental disabilities and 226 children without developmental disabilities undergoing brain mri with sedation were reviewed . no difference in dosages of pentobarbital were observed between the two groups but there was a threefold increased incidence of hypoxia in children with developmental disability ( 11.9% versus 4.9% ) . in summary
, pentobarbital is commonly used for sedation in children undergoing diagnostic radiologic procedures such as mri or ct scan , but in the child with developmental disabilities or neurologic disorders the practitioner must be aware of the important side effects of systemic hypotension and respiratory depression and be prepared to respond to them quickly to avoid further neurologic injury . thiopental is an ultrashort - acting barbiturate which in its iv form is commonly used as an induction agent for intubation . etomidate is a short - acting iv drug that produces sedation , anxiolysis , and amnesia . etomidate has the benefits of decreasing icp by reductions in cbf and cmro2 and has the advantage of producing less cardiovascular depression than barbiturates or propofol , and preserving cerebral perfusion pressure [ 28 , 29 ] . the increased metabolic deficit has the potential to expand the ischemic core and penumbra in brain - injured tissue . previous reports on the efficacy of etomidate for pediatric sedation during diagnostic imaging have been mixed . performed a prospective randomized trial comparing etomidate versus pentobarbital for head and neck ct imaging in children 6 months to 6 years . prospective data collected from the psrc also compared sedation with pentobarbital versus etomidate for diagnostic ct . etomidate may have a role for sedation during diagnostic ct imaging but etomidate 's short duration of sedation is disadvantageous for the longer scan times required for mri . of further note , etomidate 's role in the sedation of the pediatric stroke and intracranial hemorrhage patient may be limited and caution should be used to avoid further expansion of the penumbra and ischemic core . more recent studies in adult patients with severe head injury have demonstrated improvements in cerebral perfusion pressure and minimal increases in icp with ketamine [ 3941 ] . one recent report of 30 intubated pediatric head injury patients observed that single doses of ketamine lowered icp without producing decreases in blood pressure or cerebral perfusion pressure . at the present time
, there is not enough data to recommend the use of ketamine in the pediatric population at risk for intracranial hypertension , but further studies to assess the role of ketamine in this patient population are warranted . ketamine is sometimes used as a continuous infusion in intubated patients as an anti - convulsant for children with refractory epilepsy . while ketamine is also a bronchodilator and
is helpful in children with asthma , it increases oropharyngeal and airway secretions which may be problematic in children with neurologic disorders who have difficulty handling respiratory secretions . propofol is a short - acting sedative - hypnotic iv agent that can be used to provide moderate or deep sedation . propofol can induce a deep state of sedation rapidly , provide a short duration of effect , and have a pleasant recovery phase . propofol also has anticonvulsant properties and reduces icp which can be advantageous in sedating a patient with epilepsy or a patient with concerns for obstructive hydrocephalus due to a malfunctioning ventriculoperitoneal shunt to obtain diagnostic neuroradiologic imaging [ 4446 ] . while there have been cases of propofol providing adequate sedation and successfully treating intracranial hypertension [ 47 , 48 ] , several pediatric traumatic brain injury case reports have reported metabolic acidosis and death in patients on prolonged ( 24 hrs ) continuous infusion of propofol [ 4953 ] . in the
2003 published guidelines for the care of severe pediatric traumatic brain - injured patients , continuous infusion of propofol is not recommended . adverse effects of propofol include pain at the injection site , apnea or respiratory depression , hypotension , and bradycardia which can be detrimental in a patient at risk for brain ischemia
. there is increased interest with this agent as a sedative during non - invasive neuroradiologic imaging studies in children who are not intubated . in one study ,
dexmedetomidine was compared to propofol in children undergoing mri studies . while the onset of sedation and recovery time were significantly shorter in the children that received propofol , hypotension , respiratory depression and desaturation were more common compared to the children receiving dexmedetomidine . there is increased interest in the use of dexmedetomidine as a sedative and potential neuroprotective agent in both adults and children , as animal studies revealed neuroprotection from hypoxia - ischemia and decreased apoptosis and adult human studies in healthy volunteers demonstrated parallel decrease in cmro2 and cbf , which may temporarily be helpful in briefly sedating patients who are at risk for intracranial hypertension such as head trauma , brain tumor , and obstructive hydrocephalus [ 59 , 60 ] . in pediatric traumatic brain injury case reports , no detrimental effects on their icp was observed . however , systemic hypertension was observed in one child who were receiving dexmedetomidine with other sedatives , while bradycardia was observed in 2 other children who was receiving dexmedetomidine , other sedatives , and therapeutic hypothermia [ 59 , 60 ] . further studies are warranted on the potential use and side effects of this agent in children at risk for intracranial hypertension . there are conflicting reports on the effects of ventilatory function , with some studies suggesting mild respiratory depression , while others show no effect . while a variety of sedative and analgesic medications have been used in pediatric patients with developmental disabilities and neurologic disorders , it is clear that further studies are needed to determine the optimal agent(s ) that will maximize good outcome and will minimize or prevent respiratory , circulatory , and further neurologic compromise . | [
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myeloid cells are unique cell types as regards their content of high amounts of esterified arachidonic acid ( aa ) and the enzymes necessary to metabolize free aa into different products via cyclooxygenase and lipoxygenase pathways . however , unlike mast cell , which readily respond to cross - linking fcri receptors , and platelets , which release aa in response to classical aggregating agents , less information is available about the physiologically relevant stimuli in polymorphonuclear leukocytes ( pmn ) and macrophages , since most experiments have been carried out either with xenobiotics or combining chemoattractants with chemicals and priming agents .
an example of the second situation has been the use of formylated peptides in combination with cytochalasin b and thapsigargin , which extend the time span of calcium transients and allow the occurrence of ca dependent events such as translocation of the cytosolic phospholipase a2 ( cpla2 ) from the cytosol to lipid bilayers [ 14 ] .
however , this scenario has suddenly changed with the emergence of new views on the function of the immune system based on the recognition of microbial patterns .
pmn are the first blood cell type able to migrate into tissues following microbial invasion .
pmn respond to a large set of stimuli , including inflammatory mediators and microbial products .
this group of stimuli is most relevant , since microorganisms have unique molecules , termed pathogen - associated molecular patterns ( pamps ) , which are recognized through pattern recognition receptors ( prrs ) by the host innate immune system .
the toll - like receptor family ( for review , see [ 5 , 6 ] ) and nucleotide - binding oligomerization domain family proteins ( nod ) ( for review , see [ 7 , 8 ] ) are representative of what janeway first called prr .
c - lectin type receptors are also prr that may interact with structural signatures expressed in microorganisms .
experiments in human pmn using as stimuli a set of pamp signatures including the mannose polymer mannan and peptidoglycan ( pgn ) , a polymer of sugars and amino acids that forms a mesh - like layer outside the plasma membrane of bacteria , showed a robust release of [ h]aa ( figures 1(a ) and 1(b ) ) and the production of leukotriene ( lt ) b4 and prostaglandin ( pg ) e2 ( figures 1(c ) and 1(d ) ) .
the release of [ h]aa observed under these conditions was not blunted by calpeptin , an inhibitor of the formation of microvesicles , but was inhibited by treatment with the cpla2 inhibitor pyrrolidine-1 . the released product was identified as genuine unesterified aa by thin layer chromatography analysis , since the radioactivity detected in the supernatants showed a rf distinct from that associated with [ h]triglycerides and [ h]phospholipids , which were only observed associated with the cell pellets .
the release of aa obtained under these conditions was comparable to that elicited by the so far considered reference stimuli such as the formylated peptide combined with thapsigargin or cytochalasin b , and complement - coated zymosan particles . in sharp contrast , stimuli mimicking other bacterial pamp , that is , lipoteichoic acid , bacterial lipopolysaccharide ( lps ) , muramyldipeptide ( mdp ) , and the tlr2 agonist pam3csk4 did not induce aa release .
the effect of pgn was observed with pgn from both s. aureus and b. subtilis , thus indicating that pgn displaying the structural features of both gram + and gram bacteria are equally active .
preincubation of pmn with anti - tlr2 mab prior to the addition of pgn did not inhibit aa release , thus suggesting that tlr2 receptors are not involved in the response to pgn .
reported tlr2-independent cysteinyl - lt release from mouse bone marrow dendritic cells stimulated with pgn , since the response was intact in tlr2 mice .
the assignment of the biological effect of pgn to definite prr has been a matter of debate [ 12 , 13 ] .
both tlr and nod receptors have been involved and the controversy stems from the assignment of the biological properties to either the pgn polymer or the block elements mdp and d - glutamic acid - meso diaminopimelic acid .
molecular weight fractionation of s. aureus pgn showed the association of aa - releasing activity with fractions of molecular weight > 30 kda , whereas no activity was detected in the < 30 kda ultrafiltrate , which is consistent with the mr of soluble pgn .
the biological significance of the lipid mediators release by pmn in response to tlr ligands was recently underscored in an in vitro model of migration through endothelial cell monolayers . in this system ,
pmn migration was inhibited by ltb4 receptor antagonist and platelet - activating factor ( paf ) receptor antagonists and was associated with the production of these mediators .
current understanding of pmn biology has been modified by recent findings indicating that the life span of pmn can be prolonged by proinflammatory agonists , and also by the depiction of mechanisms of translational control of the expression of specific proteins that endow the pmn with the potential for rapid protein synthesis from constitutive mrna without requiring new transcript generation [ 1618 ] .
the possibility that this mechanism could be operative in pamp - dependent responses and might influence aa metabolism through the expression of cox-2 , was a challenging hypothesis .
since pge2 is a major product resulting from aa in the pmn ( figures 1(c ) and 1(d ) ) that can be produced both by cox-1 , the constitutive isoform of cyclooxygenase , and cox-2 , the inducible isoform , the effect of a set of pamp signatures on the expression of cox-2 was addressed .
unexpectedly , preformed mrna encoding for cox-2 was detected in resting pmn , whereas cox-2 protein was only detectable after stimulation with either mannan or pgn .
cox-1 protein showed the same level of expression in the absence and presence of several stimuli , but well below the level detected in platelets , which are the archetypal source of cox-1 .
pam3csk4 showed a less robust effect and lipoteichoic acid , an agonist of tlr2/tlr6 heterodimers , did not elicit cox-2 protein induction .
mdp , which is the archetypal ligand for nod2 , also failed to induce cox-2 expression . since interaction between nod2 and specific tlr pathways has been reported as a mechanism of cooperation in the innate immune response that lead to the synergistic activation of host cells [ 2022 ] , the effect of the combined addition of both s. aureus pgn and mdp was assessed .
the induction of cox-2 protein by pgn was observed as soon as 30 minutes after addition of the stimulus and remained almost unchanged from 1 to 18 hours .
a similar trend was observed for both c3bi - coated zymosan and mannan , although a decreasing tendency was observed around 18 hours in response to these ligands .
these results indicate that pgn contains a structural signature not acting on nod2 nor mimicked by lipoteichoic acid and pam3csk4 , which could act via the tlr route in combination with an additional catch - up receptor(s ) and/or by an as yet ill - defined tlr2-independent route .
since pmn are terminally differentiated cells that contain regulators of transcriptional control and show signal - dependent activation of mrna translation [ 17 , 18 ] , the hypothesis that cox-2 mrna could be one of those mrna controlled in the same manner was put forward .
contrary to this view , one could argue that the calculation of the predicted secondary structure energy of the 5-untraslated region ( utr ) of cox-2 mrna is 36.94 kca / mol , as judged from the application of rnafold software [ 23 , 24 ] to sequences available in data banks ( figure 2(a ) ) .
this value is lower than that usually associated with transcriptional regulation ( 50 kcal / mol ) ; however , it fits well with those reported for many transcripts detected using cdna library arrays which are regulated at the transcriptional level in human monocytes adherent to p - selectin .
moreover , the presence of four tracts of 58 consecutive pyrimidine bases is an additional feature strongly suggesting the possibility of translational control by mammalian target of rapamycin ( mtor ) .
the presence of the 5-utr in cox-2 transcripts in human pmn was confirmed by rt - pcr with a set of primers spanning the first 20 nucleotides of exon 1 and exon 2 of cox-2 , which gave similar results to pcr reactions using the primers selected from exons 5 and 7 ( figure 2(b ) ) .
preincubation of pmn with 100 nm rapamycin inhibited the induction of cox-2 elicited by complement - coated zymosan , pgn , and mannan , thereby suggesting that the mtor route is implicated in the translational regulation of cox-2 protein induction . given that mtor is integrated in a signalling cascade , the proximal component of which is phosphoinositide-3 kinase ( pi3k ) , the effect of the pi3k inhibitor wortmannin was addressed .
a significant inhibition of cox-2 induction was produced by wortmannin as well as by the translation inhibitor cycloheximide .
this provides further evidence of the involvement of the mtor route , since the phosphorylation of this translation inhibitor by mtor disrupts its binding to eif4e and activates cap - dependent translation
actinomycin d did not influence the induction of cox-2 protein elicited by mannan and pgn , whereas it fully inhibited the response to lps .
since cox-2 mrna stability in some cell types is regulated at the 3-utr , pmn were incubated in the presence and absence of 1 g / ml actinomycin d for 30 minutes before the addition of pgn to address the half - life of cox-2 mrna . in the absence of actinomycin d ,
53 7% of the starting cox-2 mrna was detected in control cells versus 70 9% ( mean sd ,
n = 3 ) in cells treated with pgn for 2 hours after addition of the stimulus . actinomycin d treatment induced a further drop of the remaining mrna in vehicle treated cells , whereas this additional drop was hardly observed in pgn - treated pmn .
further assessment of transcriptional regulation of cox-2 expression was carried out by looking at the effect of 2-hydroxy-4-trifluoromethylbenzoic acid , an inhibitor of both nf-b and nf - at [ 27 , 28 ] , which is a useful tool to address in a single step transcriptional regulation since both transcription factors have been involved in cox-2 regulation in different cell types [ 29 , 30 ] .
hydroxy-4-trifluoromethylbenzoic acid lacked a significant effect on cox-2 protein expression in response to all of the stimuli tested .
taken collectively , these data suggest that transcriptional regulation is not the main mechanism whereby cox-2 expression is regulated in human pmn . to ascertain whether the above described mechanisms are either a unique property of pmn or are also operative in other myeloid cells , cox-2 protein expression was assayed in monocytes ; however , the time course was somewhat different from that observed in pmn , since cox-2 protein steadily increased up to 48 hours . unlike pmn , rapamycin did not influence cox-2 protein expression in monocytes nor in macrophages , whereas actinomycin d significantly blocked cox-2 protein induction expression in response to zymosan , mannan , pgn , and the soluble -glucan laminarin .
these results strongly suggest that different mechanisms can be involved in cox-2 regulation in pmn and mononuclear phagocytes .
uptake of phagocytosable particles is strongly dependent on the expression of receptors involved in the recognition of serum proteins displaying opsonic functions such as complement factors and antibodies .
this is relevant to the engulfment of fungi and bacteria since these microbes can be coated by the complement factor 3 derived protein c3b and by opsonic igg class antibodies .
the display of receptors on the different cell types including fcr receptors , complement receptors , and prr is a key factor to determine the inflammatory and phagocytic responses and it can widely vary among different cell types ( figure 3 ) .
in addition , signals elicited upon binding of receptors by their cognate ligands may be balanced by concomitant signals induced by associated pamp or from the environment , or even by the expression of cell - specific adaptors .
this is particularly relevant to mononuclear phagocytes in view of the different patterns of differentiation they may undergo due to the presence of cytokines and growth factors in the inflammatory milieu . aa metabolism was assessed in mononuclear phagocytes stimulated with antigen / antibody immune complexes and zymosan , a cell wall extract of the yeast saccharomyces cerevisiae . since formation of immune complexes ( ic ) in fluids containing complement
is accompanied by the covalent linkage of c3bi onto the ab and because c3bi coating of zymosan is known to increase inflammatory responses and aa release in leukocytes , experiments were carried out with preformed ic treated with normal human serum to allow the formation of adducts between igg -chain and c3b--chain , a process that has been related to the clearance of ic with a limited inflammatory response .
notably , the aa released by c3bi - ic was significantly lower than that induced by ic containing similar amounts of igg , thus suggesting that the reaction of ic with c3bi gives rise to an ic lattice showing a distinct ability to interact with signaling receptors .
the most likely interpretation of these findings is that the ability of c3bi - ic to interact with complement receptor 3 ( cr3 ) blunts fc / fcr interactions and the attendant aa release associated with fcr cross - linking .
treatment of c3bi - ic with anti - c3 igg , but not with an irrelevant rabbit igg , allowed the recovery of aa - releasing ability , thus indicating that masking the c3bi moieties with igg in the c3bi - ic lattice , makes these complexes similar to those formed in the absence of complement .
conversely , removal of the fc portion of anti - ova igg , which preserves the ability of the f(ab)2 fragment to bind covalently c3bi on the ser-132 of the ch1 domain , abrogated aa - releasing activity , thus indicating that fc - fcr interaction is essential for ic - induced aa release and that stimulation through c3bi does not elicit productive binding in this system .
the mannose receptor ( mr ) , first described by stahl et al . has been the object of detailed analysis regarding its ability to initiate the uptake of glycosylated molecules with terminal mannose , fucose , or n - acetylglucosamine moieties .
its capacity for ligand recognition makes this receptor suitable to phagocytose candida albicans , leishmania donovani , and pneumocytis carinni , among other microorganisms [ 3739 ] .
the mr is the prototypic element of a homonymous family of c - type lectin receptors , which includes the secreted phospholipase a2 m - type receptor , the dendritic cell receptor dec-205 , and endo180/urokinase plasminogen - activated receptor - associated protein .
these receptors contain carbohydrate recognition domains , although the chemical structure of the ligands interacting with those domains shows wide differences .
the mr is mainly expressed in alveolar macrophages , peritoneal macrophages , and macrophages derived from blood monocytes and seems to play a role in the early immune response against invading pathogens .
although the mr was shown to participate in intracellular signalling leading to target gene expression , the absence of signaling motifs in its intracellular tail makes it necessary the assistance from other receptors in order to trigger any signalling cascade .
the mr has been found to exert some effect on the induction of effector th17 cells in mixed leukocytes populations and binding of the mannose polymer mannan to the mr induced a mild expression of cox-2 protein above basal levels , whereas treatment with laminarin , zymosan particles , and preformed ic failed to do so .
notably , monocyte - derived macrophages obtained after two weeks of culture showed a prominent induction of cox-2 protein with concentrations of mannan as low as 0.1 mg / ml , thus suggesting that recognition of mannose - based molecular patterns by macrophages might play a central role in the induction of the innate immune response .
the main function of dendritic cells ( dc ) is the detection of pathogens and the initiation of the host response to microbial invasion .
so far , few studies have been dedicated to the analysis of the production of aa metabolites , in spite of the relevant role of eicosanoids in dc function and the prominent changes in lipid metabolism elicited by m - csf and il-4 along the process of monocyte differentiation .
in addition , pge2 is required for human dc migration in response to chemokines [ 45 , 46 ] , and consistent with this pivotal function , failure of dc to produce pge2 has been considered a major obstacle for the successful application of dc in therapy [ 47 , 48 ] .
pg biosynthesis involves several steps catalyzed by different enzymes , but it depends primarily on the availability of free aa selectively released from phospholipids by cpla2 .
cox-2 is involved in the sustained production of prostanoids , the activity of which is necessary for strong ab response following vaccination .
in addition to the cox-2 route for aa metabolism , there are pathways dependent on constitutively expressed 5-lipoxygenase and cox-1 , which are triggered shortly after cell activation .
as regards 5-lipoxygenase products , deficient extracellular export of ltc4 is associated with a decreased migratory response of dc , whereas cysteinyl - lt increase il-10 production by myeloid dc .
recent studies have disclosed lipoxins as a unique class of lipoxygenase interaction metabolites with a strong ability to suppress the production of il-12 and the function of dc . in keeping with the changes in functional parameters observed upon dc differentiation , aa metabolism in dc showed different patterns in mature and immature dc . whereas the release of aa elicited by zymosan and other ligands showed no difference between immature and tnf-mature cells , increased expression of cox-2
unlike pmn and monocytes , zymosan particles were the most potent stimulus for aa release , which was observed with concentrations as low as 0.1 mg / ml . in contrast , mannan induced aa release to a lower extent . unlike the results observed in monocytes , neither c3bi coating nor opsonization with rabbit igg modified the ability of these stimuli to release aa .
this raises important question about the recognition of -glucan particles and the coupled signaling mechanisms in different cell types . in fact , the main receptor involved in -glucan recognition is dectin-1 , which is expressed on the cell surface of pmn , monocytes , and dc ; however , dc display a unique response to zymosan particles . at first glance
, two mechanisms might explain the different responses : ( i ) expression in some myeloid cell types of an inhibitor , for example , tetraspanin cd37 , that restricts dectin-1-card9 signaling , or ( ii ) gain of function of dc by differentiation - induced expression of a receptor cooperating with dectin-1 .
zymosan - induced aa release was inhibited by laminarin , mannan , and anti - dectin-1 and anti - dc - sign mab , specially when the inhibitors were used in combination .
these data would suggest cooperation of both dectin-1 and dc - sign in zymosan - induced aa release and would agree with the aforementioned hypothesis of the selective expression in dc of a receptor not expressed in other myeloid cell types . to obtain further insight into the type of receptors involved in the recognition of zymosan by dc , the binding of alexa - fluor 488 zymosan was studied in the presence of different inhibitors .
mannan , laminarin , anti - dc - sign mab , and anti - dectin-1 mab blocked zymosan binding , but combination of these inhibitors enhanced binding blockade .
taken together , these data show the existence of a cpla2-dependent route for aa release in dc that is triggered by the binding of zymosan to dectin-1 and dc - sign .
protein tyrosine phosphorylation reactions play a central role in cell signaling through both fcr and dectin-1 in murine dc [ 54 , 55 ] .
since these receptors do not possess intrinsic enzymatic activity , their signal transduction pathways must rely on activation of nonreceptor tyrosine kinases .
this explains why the syk / zap70 family member syk has been found to be critical for linking receptor engagement to many early downstream events including calcium mobilization and activation of the ras / mitogen - activated protein kinase pathway .
the involvement of syk in aa release and cox-2 induction in murine macrophages was first reported by suram et al . , who also showed that aa release and ltc4 production stimulated by zymosan and candida albicans were tlr2-independent .
studies in human dc were addressed by examining tyrosine phosphorylation of the kinase ( a measure of syk activation ) and the effect of syk inhibitors .
both ic and zymosan induced the phosphorylation of tyrosines in the activation loop of syk and syk inhibitors significantly blunted aa release .
however , syk inhibitors only partially affected zymosan - induced cpla2 phosphorylation and the syk inhibitor piceatannol blunted the release of aa by 96% and 54% in response to ic and zymosan , respectively .
r406 , a very specific syk inhibitor , also inhibited completely the response to ic and reduced zymosan - induced aa release by 30% .
zymosan - induced syk phosphorylation was also inhibited with the addition of laminarin , but not by anti - dc - sign mab .
taken collectively , these results are consistent with the notion that syk activity is completely necessary for ic - induced aa release , but it is only partially involved in the signalling mechanism whereby zymosan elicits aa release in dc .
the inhibition of aa release by combinations of laminarin / anti - dectin-1 and anti - dc - sign mab suggested cooperation between dc - sign and dectin-1 .
this was confirmed by showing that dectin-1 coimmunoprecipitated with dc - sign , particularly after the stimulation of dc with zymosan ( figure 4 ) .
additional experiments in hek293 cells transfected with vectors encoding dc - sign and myc - dectin-1 showed a robust coimmunoprecipitation of both c - lectin receptors when immunoprecipitation was carried out with either anti - dc - sign mab or anti - myc mab .
these results are consistent with a system for zymosan recognition in dc involving the interaction of dectin-1 and dc - sign . studies by confocal microscopy confirmed these findings by showing dc - sign clusters in areas of contact with zymosan particles , but not around engulfed particles as judged from the analysis of images taken after 10 minutes , where ingested particles were not surrounded by dc - sign staining ( figure 5 ) .
this finding agrees with recent reports indicating that dc - sign is a mannan - inhibitable zymosan receptor , but does not mediate phagocytosis [ 57 , 58 ] .
, these data would suggest that the differentiation of human monocytes into dc is accompanied by the induction of dc - sign , a receptor that cooperates with dectin-1 to elicit an active metabolism of aa .
further support of the role played by changes associated to the process of dc differentiation on aa metabolism is the enhancement of dectin-1-mediated aa release in alveolar macrophages by gm - csf , a cytokine used to promote dc differentiation . in sharp contrast
, rat peritoneal macrophages respond to zymosan particles by promoting the mobilization of both type iia phospholipase a2 and cpla2 into the phagosomes in the absence of growth factors and cytokines [ 60 , 61 ] . taking collectively
, these findings underscore the importance of environmental factors on the ability of mononuclear phagocytes to regulate the catalytic properties of phospholipases a2 . a diagram of the signaling routes involved in aa metabolism in dc stimulated with fungal stimuli is shown in figure 6 .
fungal pamp acting through dectin-1 and dc - sign induce a cytokine response characterized by a high production of il-10 and il-23 , and a low secretion of il-12 p70 [ 55 , 62 , 63 ] , as compared to the effect on il-12 p70 production of archetypal tlr4 agonists .
this fact may have pathophysiological consequences for the persistence of infection and raises the question of the signaling pathways involved in the predominant il-10 response .
the regulation of il-10 production has been the subject of intense research in tlr4-dependent models and both transcriptional and posttranscriptional mechanisms have been reported . as regards transcriptional regulation ,
many transcription factors have been considered as master regulators , namely stat3 [ 6467 ] , sp1 and sp3 [ 68 , 69 ] , c - maf , nf - y , nf-b [ 7274 ] , pbx1b ( pre - b cell leukemia transcription factor-1b ) , c / ebp , nfat [ 77 , 78 ] , and creb [ 79 , 80 ] .
in addition , posttranscriptional regulation of il-10 message has also been proposed because of the high number of au - rich elements in the 3-utr of il-10 mrna and their binding by the rna - binding protein tristetraprolin , which destabilizes the message . after addressing the stability of il-10 mrna in the presence of actinomycin d
, it was concluded that the regulation of il-10 expression is best explained by transcriptional mechanisms .
computer analysis of human and mouse il10 promoters was carried out using the matinspector program and the transfac database to detect binding sites for transcription factors .
in addition , both sequences were aligned with dna block aligner software to define conserved areas , since these regions are more likely to represent functionally relevant elements .
several of the sites detected were previously associated with the transcriptional regulation of il10 , but there have been some discrepancies regarding their functional relevance and studies with fungus - related stimuli have not been reported .
the first approach was to search for the presence of binding activities to the consensus strings of the transcription factors found in the human il10 promoter .
no binding activity to stat and c / ebp consensus sequences was observed in the nuclear extracts of cells treated with zymosan , whereas binding activity to stat1 and stat3 was elicited by ifn-. constitutive binding activity to sp sites compatible with both sp1 and sp3 was detected , as well as binding activity to cre consensus sequences .
nf-b is activated by zymosan and has been associated with the regulation of il10 in mouse macrophages [ 7274 ] and with the regulation of cox-2 .
taking into account that the expression of cox-2 parallels il-10 induction , experiments were conducted using probes containing the b sites from the human cox2 promoter that have found to be of functional relevance .
the response to zymosan was dose - dependent and binding was competed by the unlabeled sequence .
however , as the sequence involved in nf-b - dependent regulation of il10 expression in the mouse is not conserved in the human il10 promoter , the appearance of b - binding activity in the nuclear extracts upon zymosan challenge is not a proof of the involvement of nf-b in the regulation of il-10 expression in human dc .
altogether , the above - mentioned results did not support the involvement of stat1 , stat3 , and c / ebp in the regulation of il-10 induction and further experiments were conducted focusing on the possible involvement of nf-b and creb . since the activity of creb and nf-b can be modulated by pharmacological tools ,
experiments were conducted with 8-br - camp , a cell permeable analogue of cyclic amp , pge2 , and the protein kinase a inhibitor h-89 .
elevation of the intracellular levels of cyclic amp by both pge2 and 8-br - camp had on its own a limited effect on il-10 production , whereas stimulation with zymosan in the presence of these compounds produced a synergistic increase of il-10 production .
notably , the opposite effect was observed in the presence of the protein kinase a inhibitor h-89 .
in contrast with the effect of the chemicals producing inhibition of creb activity , blockade of nf-b activity by two structurally unrelated inhibitors was accompanied by an increase of il-10 production .
since the regulation of creb activity has been related to calcium / calmodulin kinases and cre coactivators , the activity of which depends on a sensor of both ca and cyclic amp levels , the ca - dependence of il-10 production was addressed .
ionomycin induced a limited production of il-10 , thus suggesting that intracellular ca levels are not the only factor determining il-10 production .
moreover , low micromolar concentrations of cyclosporin induced a significant decrease of il-10 production , thus pointing to the involvement of calcineurin in the regulation of il-10 production . since e prostanoid receptors type 2 and 4 are involved in the regulation of the intracellular levels of cyclic amp and zymosan
is a strong inducer of cox-2 and pge2 production , the effect of inhibiting cox-1 with the specific inhibitor sc560 and cox-2 with the specific inhibitor ns398 was addressed .
the isolated addition of any of those compounds at concentrations of ~1 m to preserve their selectivity , did not show any significant effect on il-10 production .
notably , combination thereof produced a significant inhibition , thus suggesting that both cox isoforms might be involved in an autocrine production of pge2 that regulates intracellular cyclic amp levels and zymosan - induced il-10 production .
taken together , these results suggest that the polarization of dc cytokine response versus il-10 production in response to the fungal surrogate zymosan depends on a fine - tuned balance between nf-b and creb activity , and that pge2 plays a role in this balance . to address directly the involvement of the distinct transcription factors on il-10 regulation ,
chromatin immunoprecipitation ( chip ) assays were conducted using antibodies reactive to p - creb , cbp , c - maf , nf - ya , sp1 , and pbx1 .
significant binding of p - creb to the il10 promoter was observed in dc stimulated with zymosan , but not in control cells nor in samples treated with an irrelevant antibody .
notably , this was associated with a 64-fold increase of the amount of cbp associated to the il10 promoter , thus suggesting that zymosan induces both binding of p - creb to cre sites and recruitment of the coactivator cbp .
chip was negative when the pcr reactions were carried out using primers from the il12 p35 promoter , which does not contain cre sites .
p - creb binding was also detected in the cox2 promoter upon zymosan stimulation , which agrees with the presence of two cre sites in this promoter and with the functional relevance of these sites in cox2 transcriptional regulation [ 85 , 86 ] .
binding of p - creb and cbp to the promoters was coincidental with the detection of torc2 ( transducer of regulated creb activity ) , a creb coactivator also known as creb - regulated transcription coactivator ( crtc ) , in the nuclear extracts .
expression of the mrna encoding both the long and the short form of c - maf was detected in dc , thus agreeing with the reported induction of this factor by lps and il-4 in monocytes , but binding to the il10 promoter was not detected by chip assays . as regards sp1 and sp3 , the detection of binding activity in resting cells was not accompanied by binding to the il10 promoter , which agrees with the notion that this family of transcription factors behaves as a constitutive activator of housekeeping genes and tata - less genes .
stat3 has been associated with il10 transcriptional activation , especially in response to ligands of tlr4 , which differ from zymosan because of their capacity to activate the jak / stat pathway by trif ( tir - domain - containing adapter - inducing interferon-)-dependent mechanisms .
stat binding activity and tyrosine - phosphorylated stat1 were not detected in nuclear extracts from zymosan - stimulated dc , whereas they were induced upon lps and ifn- treatment .
these results show a major role for creb in the transcriptional regulation of il10 in response to the fungal stimulus zymosan .
the response of dc to fungal glucans is characterized by a high production of il-23 and the development of a th17 response .
this is of interest because th17 cells have been implicated in a number of inflammatory and autoimmune diseases , including multiple sclerosis , inflammatory bowel disease , asthma and psoriasis .
so far , only preliminary data have suggested the involvement of lipid mediators in the expansion of th17 cells .
the phospholipid mediator paf is released in response to zymosan in many cell types and is found in increased concentrations in inflammatory lesions .
paf has been shown to induce the production of il-6 and the development of th17 cells when added at picomolar concentrations to monocyte - derived langerhans cells and to keratinocytes . moreover , when langerhans cells ( lc ) were pretreated with paf and then cocultured with anti - cd3- and anti - cd28-activated t cells , the latter developed a th17 phenotype , with a 3-fold increase in the expression of the transcriptional regulator rort and enhanced production of il-17 , il-21 and il-22 .
paf - induced th17 development was prevented by the paf receptor antagonist web2086 and by neutralizing antibodies to il-23 and il-6 .
it was also dependent on lc - t cell contact as shown in transwell experiments .
these data suggest that a lipid mediator , the biosynthesis of which is associated to the eicosanoid cascade , can stimulate lc to produce il-6 and il-23 , which , in contact with tcr - activated t cells , can induce their differentiation into th17 cells .
this may constitute a previously unknown stimulus for the development and persistence of inflammatory processes that could be amenable to pharmacologic intervention .
release of aa and the sequential production of eicosanoid are a blatant outcome of prr binding by their cognate ligands .
the amounts of eicosanoids released under these conditions make pamp the most potent and physiologically relevant stimuli of aa metabolism in myeloid cells
. however , there are a number of significant differences regarding the effect of prr ligands on the different cell types , even though the same types of receptors might be expressed .
this raises relevant questions regarding the distinct signaling routes coupled to the receptors , the role of the concomitant expression of other receptors recognizing the same or other pamp present on the same ligand , and the effect of positive and negative regulators .
of particular interest is the fact that pgn is the most relevant stimulus in pmn , thus underscoring the important role played by these cells in the pyogenic infections produced by gram + bacteria .
complement coating of pamp seems essential for the activation of pmn and monocytes by particles mimicking the fungal cell wall , whereas monocyte - derived dc are a cell type specially endowed to respond to fungal patterns , even in the absence of opsonins .
this property depends on the particular set of receptors expressed on their membranes , which cooperate to recognize -glucans and mannose - containing patterns . the signaling network triggered by the combined binding of dectin-1 and dc - sign in dc
is specially suited for the transcriptional regulation of a pattern of cytokines characterized by a low production of il-12 p70 and a high production of il-10 .
this can be explained by the activation of the transcription factor creb through a mechanism involving the coactivators cbp and torc2/crtc2 , and autocrine production of prostaglandin e2 .
these findings emphasize the need of further work to transform these mechanistic data into valuable tools to treat infectious and autoimmune diseases . | the variable array of pattern receptor expression in different cells of the innate immune system explains the induction of distinct patterns of arachidonic acid ( aa ) metabolism .
peptidoglycan and mannan were strong stimuli in neutrophils , whereas the fungal extract zymosan was the most potent stimulus in monocyte - derived dendritic cells since it induced the production of pge2 , pgd2 , and several cytokines including a robust il-10 response .
zymosan activated b - binding activity , but inhibition of nf-b was associated with enhanced il-10 production . in contrast , treatments acting on creb ( cre binding protein ) , including pge2 , showed a direct correlation between creb activation and il-10 production . therefore , in dendritic cells zymosan induces il10 transcription by a cre - dependent mechanism that involves autocrine secretion of pge2 , thus unraveling a functional cooperation between eicosanoid production and cytokine production . | 1. Polymorphonuclear Leukocytes and Eicosanoid Production
2. The Macrophage and Dendritic Cell System
3. Concluding Remarks |
myeloid cells are unique cell types as regards their content of high amounts of esterified arachidonic acid ( aa ) and the enzymes necessary to metabolize free aa into different products via cyclooxygenase and lipoxygenase pathways . however , unlike mast cell , which readily respond to cross - linking fcri receptors , and platelets , which release aa in response to classical aggregating agents , less information is available about the physiologically relevant stimuli in polymorphonuclear leukocytes ( pmn ) and macrophages , since most experiments have been carried out either with xenobiotics or combining chemoattractants with chemicals and priming agents . however , this scenario has suddenly changed with the emergence of new views on the function of the immune system based on the recognition of microbial patterns . this group of stimuli is most relevant , since microorganisms have unique molecules , termed pathogen - associated molecular patterns ( pamps ) , which are recognized through pattern recognition receptors ( prrs ) by the host innate immune system . the toll - like receptor family ( for review , see [ 5 , 6 ] ) and nucleotide - binding oligomerization domain family proteins ( nod ) ( for review , see [ 7 , 8 ] ) are representative of what janeway first called prr . experiments in human pmn using as stimuli a set of pamp signatures including the mannose polymer mannan and peptidoglycan ( pgn ) , a polymer of sugars and amino acids that forms a mesh - like layer outside the plasma membrane of bacteria , showed a robust release of [ h]aa ( figures 1(a ) and 1(b ) ) and the production of leukotriene ( lt ) b4 and prostaglandin ( pg ) e2 ( figures 1(c ) and 1(d ) ) . the release of [ h]aa observed under these conditions was not blunted by calpeptin , an inhibitor of the formation of microvesicles , but was inhibited by treatment with the cpla2 inhibitor pyrrolidine-1 . the released product was identified as genuine unesterified aa by thin layer chromatography analysis , since the radioactivity detected in the supernatants showed a rf distinct from that associated with [ h]triglycerides and [ h]phospholipids , which were only observed associated with the cell pellets . in sharp contrast , stimuli mimicking other bacterial pamp , that is , lipoteichoic acid , bacterial lipopolysaccharide ( lps ) , muramyldipeptide ( mdp ) , and the tlr2 agonist pam3csk4 did not induce aa release . both tlr and nod receptors have been involved and the controversy stems from the assignment of the biological properties to either the pgn polymer or the block elements mdp and d - glutamic acid - meso diaminopimelic acid . the biological significance of the lipid mediators release by pmn in response to tlr ligands was recently underscored in an in vitro model of migration through endothelial cell monolayers . in this system ,
pmn migration was inhibited by ltb4 receptor antagonist and platelet - activating factor ( paf ) receptor antagonists and was associated with the production of these mediators . current understanding of pmn biology has been modified by recent findings indicating that the life span of pmn can be prolonged by proinflammatory agonists , and also by the depiction of mechanisms of translational control of the expression of specific proteins that endow the pmn with the potential for rapid protein synthesis from constitutive mrna without requiring new transcript generation [ 1618 ] . unexpectedly , preformed mrna encoding for cox-2 was detected in resting pmn , whereas cox-2 protein was only detectable after stimulation with either mannan or pgn . cox-1 protein showed the same level of expression in the absence and presence of several stimuli , but well below the level detected in platelets , which are the archetypal source of cox-1 . since interaction between nod2 and specific tlr pathways has been reported as a mechanism of cooperation in the innate immune response that lead to the synergistic activation of host cells [ 2022 ] , the effect of the combined addition of both s. aureus pgn and mdp was assessed . the induction of cox-2 protein by pgn was observed as soon as 30 minutes after addition of the stimulus and remained almost unchanged from 1 to 18 hours . preincubation of pmn with 100 nm rapamycin inhibited the induction of cox-2 elicited by complement - coated zymosan , pgn , and mannan , thereby suggesting that the mtor route is implicated in the translational regulation of cox-2 protein induction . given that mtor is integrated in a signalling cascade , the proximal component of which is phosphoinositide-3 kinase ( pi3k ) , the effect of the pi3k inhibitor wortmannin was addressed . this provides further evidence of the involvement of the mtor route , since the phosphorylation of this translation inhibitor by mtor disrupts its binding to eif4e and activates cap - dependent translation
actinomycin d did not influence the induction of cox-2 protein elicited by mannan and pgn , whereas it fully inhibited the response to lps . actinomycin d treatment induced a further drop of the remaining mrna in vehicle treated cells , whereas this additional drop was hardly observed in pgn - treated pmn . hydroxy-4-trifluoromethylbenzoic acid lacked a significant effect on cox-2 protein expression in response to all of the stimuli tested . unlike pmn , rapamycin did not influence cox-2 protein expression in monocytes nor in macrophages , whereas actinomycin d significantly blocked cox-2 protein induction expression in response to zymosan , mannan , pgn , and the soluble -glucan laminarin . this is particularly relevant to mononuclear phagocytes in view of the different patterns of differentiation they may undergo due to the presence of cytokines and growth factors in the inflammatory milieu . the most likely interpretation of these findings is that the ability of c3bi - ic to interact with complement receptor 3 ( cr3 ) blunts fc / fcr interactions and the attendant aa release associated with fcr cross - linking . treatment of c3bi - ic with anti - c3 igg , but not with an irrelevant rabbit igg , allowed the recovery of aa - releasing ability , thus indicating that masking the c3bi moieties with igg in the c3bi - ic lattice , makes these complexes similar to those formed in the absence of complement . conversely , removal of the fc portion of anti - ova igg , which preserves the ability of the f(ab)2 fragment to bind covalently c3bi on the ser-132 of the ch1 domain , abrogated aa - releasing activity , thus indicating that fc - fcr interaction is essential for ic - induced aa release and that stimulation through c3bi does not elicit productive binding in this system . the mr is mainly expressed in alveolar macrophages , peritoneal macrophages , and macrophages derived from blood monocytes and seems to play a role in the early immune response against invading pathogens . the mr has been found to exert some effect on the induction of effector th17 cells in mixed leukocytes populations and binding of the mannose polymer mannan to the mr induced a mild expression of cox-2 protein above basal levels , whereas treatment with laminarin , zymosan particles , and preformed ic failed to do so . notably , monocyte - derived macrophages obtained after two weeks of culture showed a prominent induction of cox-2 protein with concentrations of mannan as low as 0.1 mg / ml , thus suggesting that recognition of mannose - based molecular patterns by macrophages might play a central role in the induction of the innate immune response . the main function of dendritic cells ( dc ) is the detection of pathogens and the initiation of the host response to microbial invasion . so far , few studies have been dedicated to the analysis of the production of aa metabolites , in spite of the relevant role of eicosanoids in dc function and the prominent changes in lipid metabolism elicited by m - csf and il-4 along the process of monocyte differentiation . as regards 5-lipoxygenase products , deficient extracellular export of ltc4 is associated with a decreased migratory response of dc , whereas cysteinyl - lt increase il-10 production by myeloid dc . recent studies have disclosed lipoxins as a unique class of lipoxygenase interaction metabolites with a strong ability to suppress the production of il-12 and the function of dc . whereas the release of aa elicited by zymosan and other ligands showed no difference between immature and tnf-mature cells , increased expression of cox-2
unlike pmn and monocytes , zymosan particles were the most potent stimulus for aa release , which was observed with concentrations as low as 0.1 mg / ml . in contrast , mannan induced aa release to a lower extent . at first glance
, two mechanisms might explain the different responses : ( i ) expression in some myeloid cell types of an inhibitor , for example , tetraspanin cd37 , that restricts dectin-1-card9 signaling , or ( ii ) gain of function of dc by differentiation - induced expression of a receptor cooperating with dectin-1 . these data would suggest cooperation of both dectin-1 and dc - sign in zymosan - induced aa release and would agree with the aforementioned hypothesis of the selective expression in dc of a receptor not expressed in other myeloid cell types . mannan , laminarin , anti - dc - sign mab , and anti - dectin-1 mab blocked zymosan binding , but combination of these inhibitors enhanced binding blockade . studies in human dc were addressed by examining tyrosine phosphorylation of the kinase ( a measure of syk activation ) and the effect of syk inhibitors . zymosan - induced syk phosphorylation was also inhibited with the addition of laminarin , but not by anti - dc - sign mab . the inhibition of aa release by combinations of laminarin / anti - dectin-1 and anti - dc - sign mab suggested cooperation between dc - sign and dectin-1 . additional experiments in hek293 cells transfected with vectors encoding dc - sign and myc - dectin-1 showed a robust coimmunoprecipitation of both c - lectin receptors when immunoprecipitation was carried out with either anti - dc - sign mab or anti - myc mab . , these data would suggest that the differentiation of human monocytes into dc is accompanied by the induction of dc - sign , a receptor that cooperates with dectin-1 to elicit an active metabolism of aa . fungal pamp acting through dectin-1 and dc - sign induce a cytokine response characterized by a high production of il-10 and il-23 , and a low secretion of il-12 p70 [ 55 , 62 , 63 ] , as compared to the effect on il-12 p70 production of archetypal tlr4 agonists . this fact may have pathophysiological consequences for the persistence of infection and raises the question of the signaling pathways involved in the predominant il-10 response . as regards transcriptional regulation ,
many transcription factors have been considered as master regulators , namely stat3 [ 6467 ] , sp1 and sp3 [ 68 , 69 ] , c - maf , nf - y , nf-b [ 7274 ] , pbx1b ( pre - b cell leukemia transcription factor-1b ) , c / ebp , nfat [ 77 , 78 ] , and creb [ 79 , 80 ] . in addition , posttranscriptional regulation of il-10 message has also been proposed because of the high number of au - rich elements in the 3-utr of il-10 mrna and their binding by the rna - binding protein tristetraprolin , which destabilizes the message . several of the sites detected were previously associated with the transcriptional regulation of il10 , but there have been some discrepancies regarding their functional relevance and studies with fungus - related stimuli have not been reported . no binding activity to stat and c / ebp consensus sequences was observed in the nuclear extracts of cells treated with zymosan , whereas binding activity to stat1 and stat3 was elicited by ifn-. the response to zymosan was dose - dependent and binding was competed by the unlabeled sequence . however , as the sequence involved in nf-b - dependent regulation of il10 expression in the mouse is not conserved in the human il10 promoter , the appearance of b - binding activity in the nuclear extracts upon zymosan challenge is not a proof of the involvement of nf-b in the regulation of il-10 expression in human dc . altogether , the above - mentioned results did not support the involvement of stat1 , stat3 , and c / ebp in the regulation of il-10 induction and further experiments were conducted focusing on the possible involvement of nf-b and creb . since the activity of creb and nf-b can be modulated by pharmacological tools ,
experiments were conducted with 8-br - camp , a cell permeable analogue of cyclic amp , pge2 , and the protein kinase a inhibitor h-89 . elevation of the intracellular levels of cyclic amp by both pge2 and 8-br - camp had on its own a limited effect on il-10 production , whereas stimulation with zymosan in the presence of these compounds produced a synergistic increase of il-10 production . in contrast with the effect of the chemicals producing inhibition of creb activity , blockade of nf-b activity by two structurally unrelated inhibitors was accompanied by an increase of il-10 production . since the regulation of creb activity has been related to calcium / calmodulin kinases and cre coactivators , the activity of which depends on a sensor of both ca and cyclic amp levels , the ca - dependence of il-10 production was addressed . ionomycin induced a limited production of il-10 , thus suggesting that intracellular ca levels are not the only factor determining il-10 production . moreover , low micromolar concentrations of cyclosporin induced a significant decrease of il-10 production , thus pointing to the involvement of calcineurin in the regulation of il-10 production . since e prostanoid receptors type 2 and 4 are involved in the regulation of the intracellular levels of cyclic amp and zymosan
is a strong inducer of cox-2 and pge2 production , the effect of inhibiting cox-1 with the specific inhibitor sc560 and cox-2 with the specific inhibitor ns398 was addressed . notably , combination thereof produced a significant inhibition , thus suggesting that both cox isoforms might be involved in an autocrine production of pge2 that regulates intracellular cyclic amp levels and zymosan - induced il-10 production . taken together , these results suggest that the polarization of dc cytokine response versus il-10 production in response to the fungal surrogate zymosan depends on a fine - tuned balance between nf-b and creb activity , and that pge2 plays a role in this balance . to address directly the involvement of the distinct transcription factors on il-10 regulation ,
chromatin immunoprecipitation ( chip ) assays were conducted using antibodies reactive to p - creb , cbp , c - maf , nf - ya , sp1 , and pbx1 . notably , this was associated with a 64-fold increase of the amount of cbp associated to the il10 promoter , thus suggesting that zymosan induces both binding of p - creb to cre sites and recruitment of the coactivator cbp . binding of p - creb and cbp to the promoters was coincidental with the detection of torc2 ( transducer of regulated creb activity ) , a creb coactivator also known as creb - regulated transcription coactivator ( crtc ) , in the nuclear extracts . expression of the mrna encoding both the long and the short form of c - maf was detected in dc , thus agreeing with the reported induction of this factor by lps and il-4 in monocytes , but binding to the il10 promoter was not detected by chip assays . stat3 has been associated with il10 transcriptional activation , especially in response to ligands of tlr4 , which differ from zymosan because of their capacity to activate the jak / stat pathway by trif ( tir - domain - containing adapter - inducing interferon-)-dependent mechanisms . stat binding activity and tyrosine - phosphorylated stat1 were not detected in nuclear extracts from zymosan - stimulated dc , whereas they were induced upon lps and ifn- treatment . the response of dc to fungal glucans is characterized by a high production of il-23 and the development of a th17 response . paf has been shown to induce the production of il-6 and the development of th17 cells when added at picomolar concentrations to monocyte - derived langerhans cells and to keratinocytes . moreover , when langerhans cells ( lc ) were pretreated with paf and then cocultured with anti - cd3- and anti - cd28-activated t cells , the latter developed a th17 phenotype , with a 3-fold increase in the expression of the transcriptional regulator rort and enhanced production of il-17 , il-21 and il-22 . release of aa and the sequential production of eicosanoid are a blatant outcome of prr binding by their cognate ligands . the amounts of eicosanoids released under these conditions make pamp the most potent and physiologically relevant stimuli of aa metabolism in myeloid cells
. this raises relevant questions regarding the distinct signaling routes coupled to the receptors , the role of the concomitant expression of other receptors recognizing the same or other pamp present on the same ligand , and the effect of positive and negative regulators . of particular interest is the fact that pgn is the most relevant stimulus in pmn , thus underscoring the important role played by these cells in the pyogenic infections produced by gram + bacteria . complement coating of pamp seems essential for the activation of pmn and monocytes by particles mimicking the fungal cell wall , whereas monocyte - derived dc are a cell type specially endowed to respond to fungal patterns , even in the absence of opsonins . the signaling network triggered by the combined binding of dectin-1 and dc - sign in dc
is specially suited for the transcriptional regulation of a pattern of cytokines characterized by a low production of il-12 p70 and a high production of il-10 . this can be explained by the activation of the transcription factor creb through a mechanism involving the coactivators cbp and torc2/crtc2 , and autocrine production of prostaglandin e2 . | [
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breast cancer is one of the most common cancers among women in the united states and in western countries .
an estimated 226,870 cases of invasive breast cancer and 63,300 ductal carcinomas in situ ( dcis ) will be diagnosed among women in the united states in 2012 .
breast cancer is increasingly being diagnosed at an early stage allowing treatment with breast conserving surgery ( bcs ) , in which only the tumor and a small amount of surrounding normal tissue are removed.multiple clinical trials have concluded that patients who undergo bcs with clean margin coupled with radiation have survival rates equivalent to those with mastectomy [ 36 ] .
in addition , it was found that for every four local recurrences avoided in patients treated by bcs , one breast - cancer related death was averted .
furthermore , morbidity and local recurrence rate are higher in patients with positive or close margin ( 16% ) than those with negative margin ( 6% ) [ 8 , 9 ]
. positive and close margins usually refer to margins where cancer cells are present within 2 mm from the surface of the excised tissue .
consequently , it is best to have the tumor removed cleanly with negative margins on the first surgery .
current bcs procedures rely on margin assessment in the pathology department to ensure completeness of tumor removal .
it is only after the pathology report is completed that a final determination of surgical margin adequacy can be made .
if the margin is found to be positive , reexcision is required , which often results in additional cost , let alone the additional pain to the patients . currently , there is no real - time intraoperative method to rapidly and accurately assess the status of lumpectomy margins as a standard of care .
several techniques have been studied including gross examination , touch preparation cytology ( tpc ) [ 11 , 12 ] , frozen section analysis ( fsa ) [ 13 , 14 ] , radio - frequency spectroscopy ( rfs ) , tomography ( tm ) , and raman spectroscopy ( rs ) [ 16 , 17 ] , each of which have various limitations with false negative diagnoses in 2050% of the patients or prolong surgical time .
although rfs , tm , and rs are more sensitive than tpc , they are limited by their dependence on tissue homogeneity . as a result
. it would be desirable to have a method that is not affected by tissue heterogeneity .
on the other hand , molecular imaging has increasingly become more popular as a tool for fluorescence - guided surgery due to its sensitivity and specificity for cancer cells [ 19 , 20 ] .
molecular imaging of cancer margin requires a biomarker that is specific to cancer but not the normal breast tissues .
it is commonly accepted that there is no known unique biomarker for breast cancer due to the dynamic characteristics of the disease . however , for margin assessment purpose , the biomarker does not need to distinguish breast cancer from all other types of cancer but rather to distinguish cancer from the surrounding normal breast tissues .
for this purpose , tumor - associated carbohydrate antigens ( taca ) may be ideal as they are only associated with cancer but not the normal tissues .
one of the most common tacas is tn antigen ( galnac - o - ser / thr ) , a core glycan associated with mucins on the cancer cell surface of more than 90% of human epithelial carcinomas [ 2124 ] .
tn antigen is formed due to the lack of activities of 13 d - galactosytransferase and -2,6-sialyltransferase enzymes leading to incomplete elongation of o - glycan saccharide chains [ 25 , 26 ] .
it is a truncated form of a major type of glycosylation [ 27 , 28 ] .
tn antigen is present in malignant breast lesions , invasive carcinomas[29 , 30 ] , and some types of benign lesions such as ducal hyperplasia or atypical lobular hyperplasia [ 30 , 31 ] .
tn antigen has gained attention in antitumor vaccine applications as it is known to generate immune response in cancer patients . [ 32 , 33 ] . from a study of konska et al . , tn antigen is expressed in 60%80% of cancer cells in ductal carcinomas in situ ( dcis ) and 20%50% of cancer cells in lobular carcinoma in situ . in invasive ductal carcinoma ( idc ) , tn is expressed in 70% of cancer cells of stage i cancer , 90%100% of cancer cells in stage ii cancer , and 40%60% of cancer cells in stage iii cancer . in addition , tn is expressed in 20%70% of cancer cells in invasive lobular carcinoma .
the recent development of nanomaterials has provided considerable improvement in specificity and sensitivity for tumor imaging by using targeted contrasting agents [ 35 , 36 ] .
they exhibit a high fluorescence efficiency , are resistant to photobleaching , and comparable to green fluorescent protein ( gfp ) in size . by changing particle 's size
, the emission spectra can be tunable which allows simultaneously imaging of different markers at the same pathological sites .
bioimaging applications of qds include cell labeling and tracking [ 4042 ] , cell proliferation , sentinel lymph node mapping , brain imaging , molecular beacons for dna detection [ 4648 ] , and in vivo tumor detection [ 49 , 50 ] . for specific target imaging , qds
recent studies have shown that quantum dots can be directly made in an aqueous environment at room temperature ( aqds ) with their capping ligands directly in place [ 51 , 52 ] .
the advantages of such aqds are that they are more stable and easier to conjugate for bio - imaging .
a recent conjugation study of cdse aqds showed that cdse aqds were more than 20 times more efficient in protein conjugation than commercial qds which were made in an organic solvent ( oqds ) and required ligand and solvent exchanges .
furthermore , cdse aqds are very bright with a high quantum yield ( 79% ) .
it also worked well with a 700 nm long - pass emission filter , therefore will have little if any interference from tissue autofluorescence .
the purpose of this study is to demonstrate the use of a molecular probe consisting of a monoclonal antibody of tn antigen coupled with cdse aqds to image margin status of human cancers grown in nude mice .
this approach is molecularly specific , rapid , and not affected by tissue heterogeneity , which sets it apart from all other technologies that are available or being developed .
the ht29 human colon cancer cell line was obtained from the american type culture collection as it is the best characterized tn antigen expressing in solid tumor that is easily available and reproducible .
the ht29 cell line is a colorectal adenocarnioma which secretes carcinoembryonic antigen ( cea ) , transforming growth factor beta binding protein and mucin with high level of tn anigen . under standard growth conditions ,
the cells form a multilayer of non - polarized cells that display an undifferentiated phenotype . therefore , the ht-29 cells are aggressive .
they form solid tumor in a short amount of time ( 2 - 3 weeks ) compared to other carcinomas cell lines .
ht 29 cells were maintained in mccoy 's 5a medium supplemented with 10% fetal bovine serum ( bioexpress , kaysville , ut ) and 1% penicillin and streptomycin ( mediatech inc . , manassas , va ) and cultured at 37c in a 5% co2 incubator .
[ 51 , 55 ] with an optimal mpa : cd : se ratio = 4 : 3 : 1 .
the aqds were conjugated to monoclonal tn antigen antibody ( mab ) ( tn218 igm , abcam , nj ) for direct tumor imaging .
the details of the cdse aqds conjugation will be published in a separate publication .
viability was verified to be greater than 95% using trypan blue ( amresco , solon , oh ) .
the cells were resuspended at 10 cells per 10 l of pbs , mixed 1 : 1 with matrigel ( bd biosciences , san jose , ca ) .
the 10 l of prepared mixtures were injected subcutaneously in eight - week - old female nude mice having an average weight of 20 g. the tumors were allowed to grow for three weeks to reach the suitable size for study . to test the staining capability of the aqd - tn mab conjugate , ht29 cells were grown on cover glass overnight and then fixed with 4% paraformaldehyl for 15 minutes .
cells were blocked with 10% normal goat serum for nonspecific binding for 1 hour at room temperature .
slides were then washed with 0.1% tween / tris buffer saline ( tbs ) for 3 times .
aqd - tn mab complex was added and slides were incubated for 1 hour at room temperature .
slides were washed with tbs for 3 times and mounted with dapi ( mounting medium with fluorescence , vector laboratories , ca , usa ) for nucleus staining .
sharp dissection was used to excise the tumors with a small amount of the surrounding muscles still attached to the tumors .
the fresh tumors were immediately processed ex vivo with the staining procedure as described below , imaged and analyzed with ivis imaging system ( lumina xr , caliper , ca ) .
first , the entire tumor 's surface was washed with tbs then emerged in 1% bovine serum albumin solution ( bsa ) for nonspecific blocking for 10 minutes .
next , the tumor was removed from bsa solution and washed with tbs to remove bsa residue .
the tumor was then immersed in aqd - tn mab solution for tn - antigen staining of cancer cells .
to find the optimal blocking time , fresh livers were washed with tbs for 2 min twice and then immersed in 1% bovine serum albumin ( bsa ) for various amounts of time .
after washing in tbs three times , the livers were stained with aqd - tn mab complex for 1 hour at room temperature .
the livers were suspended in 1% bsa solution by thin wire to maximize surface exposure .
the blocking solution was stirred continuously to help the diffusion of bsa onto the tissue 's surface .
after the optimal blocking time was identified , staining time was evaluated to develop the optimal margin assessing procedure for the whole tumor .
the mouse was euthanized and the entire peritoneum was opened to expose the tumor and internal organs on the ventral side .
on the dorsal side , the tumor was left underneath the skin . however , since the skin around the shoulder blade was removed and exposed abdomen , the aqd - tn mab probe could get to the tumor even if it was covered with skin .
although the cancer cells were injected subcutaneously on the back of the mouse , the tumor invaded through the ventral side .
the whole animal was immersed in blocking solution and then aqd - tn mab probe suspensions .
all the internal organs ( such as lung , liver , kidney , etc . ) and the tumor were exposed to the probe .
this experiment was done to demonstrate the specificity and sensitivity of the probe when the tumor was surrounded by many normal tissues .
the mouse was then washed with tbs and imaged with the ivis system . to examine the potential interference of the method with standard pathological procedures , we studied 10 tumor bearing nude mice .
each mouse had one control tumor and an average of two aqd - treated tumors .
the aqds - treated tumors were stained with the aqds - probe as described in section 2.6 .
both the aqds - treated and the control tumors were submitted to the pathological department for the same regular pathological examination .
both the aqds - treated tumors and the control tumors were fixed in 10% formaline for 12 hours and then embedded in paraffin .
the blocks were cut into 5 m sections at the surfaces of the specimens where the aqds - treated tumors were stained by the aqds - probe . both sets of sections ( control and treated ) were stained with h&e and immunohistochemistry ( ihc ) stained for 8 different markers : tn antigen , vegf , msh6 , mlh1 , pms2 , p27 , p53 , and ki67 .
interpretation was performed using aperio scanscope xt ihc image analysis algorithms ( fda - cleared in - vitro diagnostic ) and light microscopy ( olympus bx50 ) in the carcinoma component .
immuofluorescent staining was performed on the ht29 cell lines to validate the functionality of qd - mab complex .
the expression of tn antigen was evident as can be seen from figure 1(a ) .
these results indicate that qd - mab complexes selectively bind to the tn antigen protein .
furthermore , aqd - tn mab complex was compared with cy3-labeled tn mab . both aqd - tn mab and cy3-tn mab bound to the ht29 cell at similar pattern , indicating aqd - tn mab complexes were functional ( data not shown ) .
autofluorescence has always been a challenge for fluorescent imaging especially in tissue with high adipose content such as breast and liver .
normal tissues are known to emit autofluorescent signal that ranges from 380 nm to 550 nm under uv light excitation ( 350400 nm ) [ 16 , 53 ] . here
, we tried to establish a clear cut - off threshold to separate the background autofluorescence signal and the positive signal .
livers , muscles , and kidneys were used as normal tissue ( negative controls ) to evaluate the background threshold ( figure 2 ) . at emission wavelength 509 nm ,
although other tissues did not show positive signal in the images , there were still background signals when the analysis was performed . with the emission at 610 nm
, the background signals were reduced in all of the tissues especially livers ( 30% reduction ) compared to emission at 509 nm ( figure 2(d ) ) .
a fluorescent intensity threshold of 400 10 could be used as the cut - off to separate normal tissues since all the tissues autofluorescence background was below this level .
the total staining process of aqd - tn mab probe to evaluating excised tumor margin is summarized in figure 3 .
next , the tumor is removed from bsa solution , washed with tbs and immersed in aqd - tn mab probe suspensions for staining .
finally , the tumor is washed again with tbs and imaged on each side with correct orientation .
there are two major steps that affect the sensitivity and specificity of aqd - tn mab probe : blocking time and staining time .
different time periods were examined and it was narrowed down to 15 minutes as the sufficient blocking time .
smaller time intervals were studied to further shorten the blocking time . as shown in figure 4(a ) , no bsa blocking resulted in strong nonspecific binding of the qd - mab probe on liver 's surface both dorsal and ventral sides .
as the blocking time increased , nonspecific binding decreased and reached the saturated point at 10 minutes .
the integrated intensity was similar between 10 minute and 15 minute blocking ( figures 4(c ) and 4(d ) ) .
this was also at the same level as a control liver without qd - tn mab probe exposure ( data not shown ) .
therefore , 10 minutes blocking should be sufficient to prevent nonspecific binding as previously mentioned , although livers and kidneys showed no positive signal , they still had some background intensity around 400 10 .
thus , this was the cut - off level to differentiate between cancer and normal tissue .
the excised tumors were divided into 3 different regions : a tumor region , a muscle region , and an overlap between tumor and muscle or margin region . comparing the fluorescent image with the bright field image
, we could see that bright dark to turquoise blue - region ( region 1 in figures 5(a ) and 5(b ) ) correlated with the tumor and gray region correlated with the muscle ( region 3 ) .
aqd probe was also specific to the tumor and not the muscle as evidenced by the unstained muscle area .
region 2 was more ambiguous based on the image in figures 5(a ) and 5(b ) . in the dorsal view ( figure 5(a ) ) , the color map showed that region 2 was red corresponding to integrated fluorescent intensity of less than 400 10 ( figure 5(c ) ) .
meanwhile , the ventral bright field image looked like muscle area whereas the fluorescent image indicated the presence of cancer cells with quantitative fluorescent intensity value of 503 10 , indicating the method was sensitive and specific to detect small non - palpable lesions .
the red color in the images could be interpreted as negative region ( integrated fluorescent intensity less than 400 10 ) . to further confirm the presence or absence of cancer cells in region 2 of both dorsal and ventral sides ,
the tumor was embedded in paraffin and examined using the h&e - stained sections of these regions .
figure 6 showed the areas in the square boxes of the tumor , with each region separated by the red line .
for the dorsal side , the square box contained all three regions tumor , interface , and muscle .
meanwhile , the square box in the ventral side consisted of only region 2 and region 3 due to larger area of region 2 to be included in the image .
clearly , dorsal region 2 ( figure 6(a ) ) contained only inflammatory and fibroblasts cells , which correlated to the red color in the whole tumor examination indicating negative signal .
the presence of cancer cells were observed in h&e section of ventral region 2 ( figure 6(b ) ) , which confirmed the above positive assessment .
the results suggested that this method was sensitive and specific to identify cancer cells in areas that could have been missed by gross examination during tumor removal process . by applying the quantitative analysis of aqd - tn mab probe signal , normal , and cancer regions
could be distinguished in real - time . to further demonstrate the capability of the method , one tumor was left inside of the mouse body .
the tumor was exposed at the ventral side and underneath the skin at the dorsal side .
we found that aqd - tn mab probe resulted in the ability to visualize areas of tumors that were not apparent with white light because the appearance of the tumor was not easily distinguished from the other tissues with good fluorescence contrast , indicating highly specific tumor targeting of aqd - tn mab probe .
the positive signal was strong enough to identify the outline of the tumor surface ( fluorescence was much greater than 400 10 ) .
all the other organs showed no positive signal confirmed the specificity and sensitivity of the aqd - tn mab probe .
we examined the potential impact of aqd - tn mab probe on the microscopic examination of the operative specimen by submitting the treated tumors to pathology department for standard processing . using multiple routine evaluated markers
, we found that there was no difference between the h&e staining of an aqds - stained tumor and that of a control tumor .
there was also no difference between the ihc staining of the various markers : tn antigen , vegf , msh6 , mlh1 , pms2 , p27 , p53 , and ki67 on an aqds - treated tumor and that of the various markers on a control tumor .
as examples , figure 8 showed the h&e staining and ihc staining of ki67 , p53 , and tn antigen of a control tumor ( figure 8(a ) ) and those of an aqd - treated tumor ( figure 8(b ) ) .
as can be seen , there was no difference between the h&e staining of the control tumor and that of an aqds - treated tumor nor was there a difference between the ihc staining of ki67 , p53 , and tn antigen of a control tumor and of an aqds - strained tumor .
furthermore , quantitative grades of ki67 expression were 38.2 5.2% in the control tumors and 31 6.4% in aqd - treated tumors . for p53 expression ,
the quantitative grades were 38.6 3% and 41.5 3.7% in the control tumors and aqd - treated tumors respectively .
this result clearly indicates that the aqds - based assessment method would not interfere with the standard histological examinations of the surgical specimens .
it is well known that incomplete removal of a tumor is a major factor that compromises the long term survival rate of cancer patients .
this study presents the first demonstration of molecular imaging for intraoperative ex vivo tumor margin assessment . by providing a quantitative threshold level
, aqd - tn mab probe provides surgeons the ability to evaluate margin status in real - time , potentially reducing the number of positive margins found postoperatively , and thus reducing the need for the second operation and risk of local recurrence .
aqd - tn mab effectively identified cancer areas that could be missed by the current gross visual examination .
furthermore , aqd - tn mab bound specifically to the cancer cells and not adipocytes and stromal cells as verified by histopathology .
tissue autofluorescence is a serious background noise issue in any fluorescent imaging and can lead to false positives .
many biomolecules exhibits endogenous fluorescence including amino acids , structural proteins , and lipids . their emission maxima range between 280 nm to 550 nm .
for epithelial tissues such as breast , the concentration of endogenous fluorophores can be substantial between the surface epithelium and the underlying stroma to result in strong autofluorescence in adipose tissue and the stroma . in this study
, the cdse aqds were imaged with a 610 nm emission filter due to the constraint of the imaging system .
however , the cdse aqds probe can be viewed with a 700 nm long - pass emission filter , which will allow the signal of the aqds further separated from tissue autofluorescence , with a higher signal to noise ratio and make the qd - tn mab probe even more sensitive and specific in the future . meanwhile , unlike fluorophores , aqds can undergo constant light exposure with minimal photobleach , which often leads to loss of signal .
aqds allow convenience in handling the probe without the need of a dark room .
current technologies such as wire - guided localization ( wgl ) can perform intraoperative tumor localization with positive margin ranges from 23% to 46% [ 56 , 57 ] and does not provide a clear three - dimensional image of tumor edges .
ultrasound guided resection is limited to ultrasound visible tumors while specimen radiography detects clips or calcifications in a tumor specimen , but both are limited in ability to establish clear margins reliably .
new developing optical - based imaging technologies appear to be applicable for intraoperative imaging due to their portable size and low cost .
for example , the optical spectroscopy method developed by wilke et al . transforms optical images into tissue composition maps with parameters of total hemoglobin concentration , -carotene concentration , and scattering .
the marginprobe method is a near - field radio frequency ( rf ) spectroscopy device that detects differences between dielectric properties of malignant and normal breast tissue .
these methods , however , depend on the intrinsic measurements , such as tissue scattering and autofluorescence of the tissues , leading to unacceptable false - negative rates due to the high heterogeneity of malignant and benign tissues [ 15 , 17 , 61 ]
. the present aqd - tn mab probe does not depend on the physical - mechanical characteristic of the tissue but assessing the differences between normal and cancer at the molecular level .
taca tn antigen has been reported to be expressed exclusively in cancer cells and not normal tissue . using this molecular signature of cancer cells ,
tissue heterogeneity is not an issue as the results presented above clearly showed that the aqd - tn mab probe was capable of displaying very small spots consisting of 100 to 200 cancer cells .
this is a key advantage compared to most of the current developing optical - based imaging technologies which rely on signal average over a large area and thus are unable to image cancer in a heterogeneous background .
total margin evaluation time is one of the most critical requirements for intraoperative margin status determination .
fsa has been reported to have good sensitivity and specificity to cancer cells but has difficulties in performing frozen sections on adipose tissue results in increasing surgery time and cost due to additional pathology evaluation .
the most significant disadvantage of fsa is the inability to evaluate the entire surface area with sampling rate of 1015% surface area . using antibody - antigen binding mechanism ,
the aqd - tn mab probe was able to stain and identify cancer areas quantitatively in less than 30 minutes to prevent the prolonged anesthesia period for patients .
all sides of the tumor are evaluated which give the surgeon the exact location of cancer area .
furthermore , no additional intraoperative pathological evaluation is needed to decide whether an area contains cancer cells or not .
manipulations of the surgical specimen have no impact on the microscopic examination of the operative specimen as shown in the interference study is another advantage of this method
. the specimen can undergo normal histologic examinations for further margin confirmation and other necessary markers evaluations . in this study , we used ht29 colon cancer cell as our tumor model instead of a breast cancer cell line .
ht29 cells expressed tn antigen strongly without any need of transfection to express the protein , as verified by western blot . because this was a proof of concept study , we wanted to ensure that our tumors express the marker strongly so that we could control over the methodology development .
furthermore , we have showed that aqd - tn mab probe was capable of staining the whole tumor inside the mouse body locally once the tumor surface was exposed .
although there was positive signal of tumor underneath the skin at the dorsal side , the skin was relatively thin ( less than 1 mm ) . with the depth of 2 mm ( consider negative margin ) , no signal would be observed due to the penetration depth of 610 nm wavelength .
the tumor imaging inside the mouse further indicated that aqd - tn mab probe was very sensitive and specific to cancer cells only .
this can potentially be developed as a tool to examine the cavity after tumor is removed for additional information about the margin .
in conclusion , we have demonstrated the use of a molecular probe aqd - tn mab to assess the surface of excised human cancers grown in nude mice . the aqd - tn mab molecular probe consisted of the antibody to target cancer - specific tn - antigen on the cancer cell surface
the advantages of the cdse aqds as the fluorescent tag of a cancer molecular probe include brightness , without photo - bleaching , and can be accessible to 700 nm long - pass emission filter that minimizes background tissue autofluorescence .
the results showed that the aqd - tn mab was effective to image tumor margin in less than 30 min .
tissue heterogeneity which was an issue for optical- and electrical - current - based imaging technologies did not have an effect in aqd - tn mab imaging due to its specific binding capability which allows a more precise margin assessment .
the integrity of the surgical specimen was not affected by the aqd treatment and there was no difference in the quality and intensity of standard h&e as well as ihc stains .
the aqd - tn mab molecular probe offers the potential to quantitatively and accurately assess margin during surgery to help reduce reexcision rate . | positive margins have been a critical issue that hinders the success of breast- conserving surgery .
the incidence of positive margins is estimated to range from 20% to as high as 60% . currently , there is no effective intraoperative method for margin assessment
. it would be desirable if there is a rapid and reliable breast cancer margin assessment tool in the operating room so that further surgery can be continued if necessary to reduce re - excision rate . in this study
, we seek to develop a sensitive and specific molecular probe to help surgeons assess if the surgical margin is clean .
the molecular probe consists of the unique aqueous quantum dots developed in our laboratory conjugated with antibodies specific to breast cancer markers such as tn - antigen .
excised tumors from tumor - bearing nude mice were used to demonstrate the method .
aqd - tn mab probe proved to be sensitive and specific to identify cancer area quantitatively without being affected by the heterogeneity of the tissue .
the integrity of the surgical specimen was not affected by the aqd treatment .
furthermore , aqd - tn mab method could determine margin status within 30 minutes of tumor excision , indicating its potential as an accurate intraoperative margin assessment method . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion | breast cancer is one of the most common cancers among women in the united states and in western countries . an estimated 226,870 cases of invasive breast cancer and 63,300 ductal carcinomas in situ ( dcis ) will be diagnosed among women in the united states in 2012 . breast cancer is increasingly being diagnosed at an early stage allowing treatment with breast conserving surgery ( bcs ) , in which only the tumor and a small amount of surrounding normal tissue are removed.multiple clinical trials have concluded that patients who undergo bcs with clean margin coupled with radiation have survival rates equivalent to those with mastectomy [ 36 ] . furthermore , morbidity and local recurrence rate are higher in patients with positive or close margin ( 16% ) than those with negative margin ( 6% ) [ 8 , 9 ]
. positive and close margins usually refer to margins where cancer cells are present within 2 mm from the surface of the excised tissue . consequently , it is best to have the tumor removed cleanly with negative margins on the first surgery . current bcs procedures rely on margin assessment in the pathology department to ensure completeness of tumor removal . it is only after the pathology report is completed that a final determination of surgical margin adequacy can be made . if the margin is found to be positive , reexcision is required , which often results in additional cost , let alone the additional pain to the patients . currently , there is no real - time intraoperative method to rapidly and accurately assess the status of lumpectomy margins as a standard of care . several techniques have been studied including gross examination , touch preparation cytology ( tpc ) [ 11 , 12 ] , frozen section analysis ( fsa ) [ 13 , 14 ] , radio - frequency spectroscopy ( rfs ) , tomography ( tm ) , and raman spectroscopy ( rs ) [ 16 , 17 ] , each of which have various limitations with false negative diagnoses in 2050% of the patients or prolong surgical time . as a result
. it would be desirable to have a method that is not affected by tissue heterogeneity . molecular imaging of cancer margin requires a biomarker that is specific to cancer but not the normal breast tissues . it is commonly accepted that there is no known unique biomarker for breast cancer due to the dynamic characteristics of the disease . however , for margin assessment purpose , the biomarker does not need to distinguish breast cancer from all other types of cancer but rather to distinguish cancer from the surrounding normal breast tissues . for this purpose , tumor - associated carbohydrate antigens ( taca ) may be ideal as they are only associated with cancer but not the normal tissues . one of the most common tacas is tn antigen ( galnac - o - ser / thr ) , a core glycan associated with mucins on the cancer cell surface of more than 90% of human epithelial carcinomas [ 2124 ] . it is a truncated form of a major type of glycosylation [ 27 , 28 ] . tn antigen is present in malignant breast lesions , invasive carcinomas[29 , 30 ] , and some types of benign lesions such as ducal hyperplasia or atypical lobular hyperplasia [ 30 , 31 ] . from a study of konska et al . by changing particle 's size
, the emission spectra can be tunable which allows simultaneously imaging of different markers at the same pathological sites . for specific target imaging , qds
recent studies have shown that quantum dots can be directly made in an aqueous environment at room temperature ( aqds ) with their capping ligands directly in place [ 51 , 52 ] . furthermore , cdse aqds are very bright with a high quantum yield ( 79% ) . the purpose of this study is to demonstrate the use of a molecular probe consisting of a monoclonal antibody of tn antigen coupled with cdse aqds to image margin status of human cancers grown in nude mice . this approach is molecularly specific , rapid , and not affected by tissue heterogeneity , which sets it apart from all other technologies that are available or being developed . the ht29 cell line is a colorectal adenocarnioma which secretes carcinoembryonic antigen ( cea ) , transforming growth factor beta binding protein and mucin with high level of tn anigen . the aqds were conjugated to monoclonal tn antigen antibody ( mab ) ( tn218 igm , abcam , nj ) for direct tumor imaging . the details of the cdse aqds conjugation will be published in a separate publication . viability was verified to be greater than 95% using trypan blue ( amresco , solon , oh ) . the 10 l of prepared mixtures were injected subcutaneously in eight - week - old female nude mice having an average weight of 20 g. the tumors were allowed to grow for three weeks to reach the suitable size for study . to test the staining capability of the aqd - tn mab conjugate , ht29 cells were grown on cover glass overnight and then fixed with 4% paraformaldehyl for 15 minutes . aqd - tn mab complex was added and slides were incubated for 1 hour at room temperature . sharp dissection was used to excise the tumors with a small amount of the surrounding muscles still attached to the tumors . the tumor was then immersed in aqd - tn mab solution for tn - antigen staining of cancer cells . after washing in tbs three times , the livers were stained with aqd - tn mab complex for 1 hour at room temperature . the blocking solution was stirred continuously to help the diffusion of bsa onto the tissue 's surface . after the optimal blocking time was identified , staining time was evaluated to develop the optimal margin assessing procedure for the whole tumor . however , since the skin around the shoulder blade was removed and exposed abdomen , the aqd - tn mab probe could get to the tumor even if it was covered with skin . although the cancer cells were injected subcutaneously on the back of the mouse , the tumor invaded through the ventral side . the whole animal was immersed in blocking solution and then aqd - tn mab probe suspensions . all the internal organs ( such as lung , liver , kidney , etc . ) this experiment was done to demonstrate the specificity and sensitivity of the probe when the tumor was surrounded by many normal tissues . to examine the potential interference of the method with standard pathological procedures , we studied 10 tumor bearing nude mice . each mouse had one control tumor and an average of two aqd - treated tumors . both the aqds - treated and the control tumors were submitted to the pathological department for the same regular pathological examination . the blocks were cut into 5 m sections at the surfaces of the specimens where the aqds - treated tumors were stained by the aqds - probe . interpretation was performed using aperio scanscope xt ihc image analysis algorithms ( fda - cleared in - vitro diagnostic ) and light microscopy ( olympus bx50 ) in the carcinoma component . the expression of tn antigen was evident as can be seen from figure 1(a ) . furthermore , aqd - tn mab complex was compared with cy3-labeled tn mab . both aqd - tn mab and cy3-tn mab bound to the ht29 cell at similar pattern , indicating aqd - tn mab complexes were functional ( data not shown ) . autofluorescence has always been a challenge for fluorescent imaging especially in tissue with high adipose content such as breast and liver . normal tissues are known to emit autofluorescent signal that ranges from 380 nm to 550 nm under uv light excitation ( 350400 nm ) [ 16 , 53 ] . here
, we tried to establish a clear cut - off threshold to separate the background autofluorescence signal and the positive signal . livers , muscles , and kidneys were used as normal tissue ( negative controls ) to evaluate the background threshold ( figure 2 ) . at emission wavelength 509 nm ,
although other tissues did not show positive signal in the images , there were still background signals when the analysis was performed . with the emission at 610 nm
, the background signals were reduced in all of the tissues especially livers ( 30% reduction ) compared to emission at 509 nm ( figure 2(d ) ) . the total staining process of aqd - tn mab probe to evaluating excised tumor margin is summarized in figure 3 . next , the tumor is removed from bsa solution , washed with tbs and immersed in aqd - tn mab probe suspensions for staining . there are two major steps that affect the sensitivity and specificity of aqd - tn mab probe : blocking time and staining time . as shown in figure 4(a ) , no bsa blocking resulted in strong nonspecific binding of the qd - mab probe on liver 's surface both dorsal and ventral sides . as the blocking time increased , nonspecific binding decreased and reached the saturated point at 10 minutes . this was also at the same level as a control liver without qd - tn mab probe exposure ( data not shown ) . therefore , 10 minutes blocking should be sufficient to prevent nonspecific binding as previously mentioned , although livers and kidneys showed no positive signal , they still had some background intensity around 400 10 . the excised tumors were divided into 3 different regions : a tumor region , a muscle region , and an overlap between tumor and muscle or margin region . comparing the fluorescent image with the bright field image
, we could see that bright dark to turquoise blue - region ( region 1 in figures 5(a ) and 5(b ) ) correlated with the tumor and gray region correlated with the muscle ( region 3 ) . aqd probe was also specific to the tumor and not the muscle as evidenced by the unstained muscle area . in the dorsal view ( figure 5(a ) ) , the color map showed that region 2 was red corresponding to integrated fluorescent intensity of less than 400 10 ( figure 5(c ) ) . meanwhile , the ventral bright field image looked like muscle area whereas the fluorescent image indicated the presence of cancer cells with quantitative fluorescent intensity value of 503 10 , indicating the method was sensitive and specific to detect small non - palpable lesions . the red color in the images could be interpreted as negative region ( integrated fluorescent intensity less than 400 10 ) . figure 6 showed the areas in the square boxes of the tumor , with each region separated by the red line . for the dorsal side , the square box contained all three regions tumor , interface , and muscle . meanwhile , the square box in the ventral side consisted of only region 2 and region 3 due to larger area of region 2 to be included in the image . clearly , dorsal region 2 ( figure 6(a ) ) contained only inflammatory and fibroblasts cells , which correlated to the red color in the whole tumor examination indicating negative signal . the results suggested that this method was sensitive and specific to identify cancer cells in areas that could have been missed by gross examination during tumor removal process . by applying the quantitative analysis of aqd - tn mab probe signal , normal , and cancer regions
could be distinguished in real - time . to further demonstrate the capability of the method , one tumor was left inside of the mouse body . we found that aqd - tn mab probe resulted in the ability to visualize areas of tumors that were not apparent with white light because the appearance of the tumor was not easily distinguished from the other tissues with good fluorescence contrast , indicating highly specific tumor targeting of aqd - tn mab probe . the positive signal was strong enough to identify the outline of the tumor surface ( fluorescence was much greater than 400 10 ) . all the other organs showed no positive signal confirmed the specificity and sensitivity of the aqd - tn mab probe . we examined the potential impact of aqd - tn mab probe on the microscopic examination of the operative specimen by submitting the treated tumors to pathology department for standard processing . using multiple routine evaluated markers
, we found that there was no difference between the h&e staining of an aqds - stained tumor and that of a control tumor . there was also no difference between the ihc staining of the various markers : tn antigen , vegf , msh6 , mlh1 , pms2 , p27 , p53 , and ki67 on an aqds - treated tumor and that of the various markers on a control tumor . as examples , figure 8 showed the h&e staining and ihc staining of ki67 , p53 , and tn antigen of a control tumor ( figure 8(a ) ) and those of an aqd - treated tumor ( figure 8(b ) ) . as can be seen , there was no difference between the h&e staining of the control tumor and that of an aqds - treated tumor nor was there a difference between the ihc staining of ki67 , p53 , and tn antigen of a control tumor and of an aqds - strained tumor . furthermore , quantitative grades of ki67 expression were 38.2 5.2% in the control tumors and 31 6.4% in aqd - treated tumors . for p53 expression ,
the quantitative grades were 38.6 3% and 41.5 3.7% in the control tumors and aqd - treated tumors respectively . this result clearly indicates that the aqds - based assessment method would not interfere with the standard histological examinations of the surgical specimens . it is well known that incomplete removal of a tumor is a major factor that compromises the long term survival rate of cancer patients . this study presents the first demonstration of molecular imaging for intraoperative ex vivo tumor margin assessment . by providing a quantitative threshold level
, aqd - tn mab probe provides surgeons the ability to evaluate margin status in real - time , potentially reducing the number of positive margins found postoperatively , and thus reducing the need for the second operation and risk of local recurrence . aqd - tn mab effectively identified cancer areas that could be missed by the current gross visual examination . furthermore , aqd - tn mab bound specifically to the cancer cells and not adipocytes and stromal cells as verified by histopathology . tissue autofluorescence is a serious background noise issue in any fluorescent imaging and can lead to false positives . for epithelial tissues such as breast , the concentration of endogenous fluorophores can be substantial between the surface epithelium and the underlying stroma to result in strong autofluorescence in adipose tissue and the stroma . in this study
, the cdse aqds were imaged with a 610 nm emission filter due to the constraint of the imaging system . however , the cdse aqds probe can be viewed with a 700 nm long - pass emission filter , which will allow the signal of the aqds further separated from tissue autofluorescence , with a higher signal to noise ratio and make the qd - tn mab probe even more sensitive and specific in the future . current technologies such as wire - guided localization ( wgl ) can perform intraoperative tumor localization with positive margin ranges from 23% to 46% [ 56 , 57 ] and does not provide a clear three - dimensional image of tumor edges . new developing optical - based imaging technologies appear to be applicable for intraoperative imaging due to their portable size and low cost . the marginprobe method is a near - field radio frequency ( rf ) spectroscopy device that detects differences between dielectric properties of malignant and normal breast tissue . these methods , however , depend on the intrinsic measurements , such as tissue scattering and autofluorescence of the tissues , leading to unacceptable false - negative rates due to the high heterogeneity of malignant and benign tissues [ 15 , 17 , 61 ]
. the present aqd - tn mab probe does not depend on the physical - mechanical characteristic of the tissue but assessing the differences between normal and cancer at the molecular level . taca tn antigen has been reported to be expressed exclusively in cancer cells and not normal tissue . using this molecular signature of cancer cells ,
tissue heterogeneity is not an issue as the results presented above clearly showed that the aqd - tn mab probe was capable of displaying very small spots consisting of 100 to 200 cancer cells . this is a key advantage compared to most of the current developing optical - based imaging technologies which rely on signal average over a large area and thus are unable to image cancer in a heterogeneous background . total margin evaluation time is one of the most critical requirements for intraoperative margin status determination . the most significant disadvantage of fsa is the inability to evaluate the entire surface area with sampling rate of 1015% surface area . using antibody - antigen binding mechanism ,
the aqd - tn mab probe was able to stain and identify cancer areas quantitatively in less than 30 minutes to prevent the prolonged anesthesia period for patients . all sides of the tumor are evaluated which give the surgeon the exact location of cancer area . furthermore , no additional intraoperative pathological evaluation is needed to decide whether an area contains cancer cells or not . manipulations of the surgical specimen have no impact on the microscopic examination of the operative specimen as shown in the interference study is another advantage of this method
. in this study , we used ht29 colon cancer cell as our tumor model instead of a breast cancer cell line . because this was a proof of concept study , we wanted to ensure that our tumors express the marker strongly so that we could control over the methodology development . furthermore , we have showed that aqd - tn mab probe was capable of staining the whole tumor inside the mouse body locally once the tumor surface was exposed . although there was positive signal of tumor underneath the skin at the dorsal side , the skin was relatively thin ( less than 1 mm ) . with the depth of 2 mm ( consider negative margin ) , no signal would be observed due to the penetration depth of 610 nm wavelength . the tumor imaging inside the mouse further indicated that aqd - tn mab probe was very sensitive and specific to cancer cells only . this can potentially be developed as a tool to examine the cavity after tumor is removed for additional information about the margin . in conclusion , we have demonstrated the use of a molecular probe aqd - tn mab to assess the surface of excised human cancers grown in nude mice . the aqd - tn mab molecular probe consisted of the antibody to target cancer - specific tn - antigen on the cancer cell surface
the advantages of the cdse aqds as the fluorescent tag of a cancer molecular probe include brightness , without photo - bleaching , and can be accessible to 700 nm long - pass emission filter that minimizes background tissue autofluorescence . the results showed that the aqd - tn mab was effective to image tumor margin in less than 30 min . tissue heterogeneity which was an issue for optical- and electrical - current - based imaging technologies did not have an effect in aqd - tn mab imaging due to its specific binding capability which allows a more precise margin assessment . the integrity of the surgical specimen was not affected by the aqd treatment and there was no difference in the quality and intensity of standard h&e as well as ihc stains . the aqd - tn mab molecular probe offers the potential to quantitatively and accurately assess margin during surgery to help reduce reexcision rate . | [
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] |
the study was a randomised double - blind controlled clinical study carried out in private practices , children 's hospitals , university hospitals , or site management organisations ( organisations that provide clinical trial related services ) located in 5 countries in europe and asia : malaysia , the netherlands , poland , portugal , and thailand .
healthy young children ages 11 to 29 months attending a day care centre at least 2 times per week were recruited into the study between september 2008 and may 2009 after informed written consent was obtained from the parents or legal guardians .
day care centres were defined as any private or governmental facility providing care or playgroups for at least 20 children .
exclusion criteria included atopic dermatitis according to the hannifin criteria ; disorders requiring a special diet ; any relevant congenital abnormality , chromosomal disorder , or severe disease ; preexisting pathology of severe respiratory or gi diseases ; use of antiregurgitation , antireflux , or laxative medication at the time of inclusion ; and use of immune modulators or of prescribed prophylactic antibiotics at the time of inclusion .
the study was conducted following the principles of the declaration of helsinki and good clinical practice guidelines .
the protocol was approved by the ethics committees of the different institutes in the countries .
study products were either the active study product , gum with the addition of 1.2 g/100 ml of scgos/ lcfos ( 9:1 ) ( immunofortis ) and 19.2 mg/100 ml of n-3 lcpufas ( epa + dha , 4:6 ) , the control study product , gum without scgos / lcfos / n-3 lcpufas ( danone research ) .
gum is specially formulated milk for young children ages 1 to 3 years , enriched with key nutrients such as vitamins a , c , and d , iron , and calcium .
both study products were packed in identical tins ; they were of the same colour , weight , smell , and taste .
both the research staff and parents and subjects were unaware of the real nature of the product .
the unblinding procedure was performed after the study was completed and after the statistical analyses were finalised .
the study consisted of a 4-week run - in period and a 52-week intervention period .
subjects eligible for participation started with the run - in period , in which control product was consumed .
the run - in period was needed to get the children used to the product and the parents used to the completion of the diaries .
subjects who had successfully completed the run - in period were randomly allocated to receive either the active study product or the control study product in a double - blind period of 52 weeks . to ensure a gradual transition during the first 2 weeks of this period , the run - in product was mixed with the active / control study product following a schedule provided by the centres .
the requested intake of the study product was 400 to 750 ml daily for both the run - in period and the 52-week intervention period .
this wide range was chosen because the different countries involved in the study had a wide variety of dosage recommendations .
every 2 to 4 weeks , there was a contact session with the parents , either by visit or by telephone , in which study diaries were discussed .
in addition , there was a reference group of subjects receiving cow 's milk already before the start of the study and which followed the same procedures as the intervention groups ; however , this group was not randomised and consumed regular cow 's milk for 52 weeks .
background information was collected on the family , their environment , the child 's nutritional habits , and illnesses . during the study , parents daily recorded the intake of study product as well as , in a symptom diary , any respiratory symptoms ( cough , fever , blocked or runny nose , sore throat , wheeze , and/or ear discharge ) and gi symptoms ( vomiting or diarrhoea ) .
they also reported absence from the day care centre , absence of parents from work , contacts with a physician , doctor 's diagnoses , and prescription of medication .
if parents suspected that their child had fever , body temperature of the subject was measured and recorded , with thermometers provided to all of the children .
furthermore , parents completed 3 times a 3-day diary , with questions about gi symptoms and stool characteristics , at the start of the intervention period , at 26 weeks , and at 52 weeks .
the diary covered stool frequency and stool consistency and severity of gi tract characteristics ( diarrhoea , constipation , and flatulence ) with a 4-point scale ( none , mild , moderate , severe ) .
based on a previous study on the incidence of respiratory tract infections ( 17,18 ) , the study was designed to have sufficient power ( 80% ) to detect a 20% difference between the groups with a 0.05 significance level based on a 2-tailed test .
the number of children necessary in each group was 300 subjects , including an estimated dropout rate of 25% .
each child , except for the children in the reference group , was randomly allocated to either the active study product or the control study product .
the randomised subjects were distributed to the 2 study groups based on a randomisation code , developed using a computer random number generator .
a block size of 6 was used , and stratification was applied for investigator site .
the primary outcome was the number of episodes of urti or giis based on a combination of subject 's illness symptoms reported by the parents during the intervention period .
urti was defined as any combination of at least 2 of the following symptoms : cough , fever , blocked or runny nose , sore throat , wheeze , and/or ear discharge .
gii was defined as the occurrence of vomiting or diarrhoea or both for at least 1 day , with or without fever .
an episode of urti and/or gii was defined as having 1 symptoms for at least 1 day .
a child was considered as recovered from an episode when the subject showed no symptoms of the reported infection and had a normal level of activity .
a new episode was considered to have occurred if the symptom(s ) occurred at least 3 days after the stated time of recovery from a previous episode .
he or she could indicate whether he or she found the symptoms to be an infection .
secondary outcomes were total number and duration of episodes of urti and/or gii ; parents absence from work because of illness of the child ; number , duration , and season of subjects absence from day care centre ; and number and type of all infections diagnosed by a physician or investigator .
descriptive statistics were used to describe the basic features according to the age , sex , food intake , and family characteristics .
results on the primary parameter were obtained from both a logistic regression model and a zero - inflated negative binomial regression model .
the latter model combines 2 separate parts : a ( binomial ) part , whether or not a subject would get an infection ; and a ( negative - binomial ) part fitting the counts for infections for the subjects .
all analyses were based on the all - subjects randomised group : all of the subjects who had been randomised and to whose parents the study product had been dispensed . for safety analysis , such as growth , height , and tolerance , the safety analysis population was used consisting of all children except the screen failure subjects for the intervention period . in an analysis of the normally distributed continuous data ,
a t test was performed , whereas a wilcoxon rank - sum test was used in analysis of non - normality continuous data .
in addition , a test ( fisher exact test in the case of < 5 expected cell counts ) was performed in analysis of nominal data .
furthermore , kaplan - meier ( km ) curves together with corresponding log - rank tests were conducted in analysis of time to event type of data .
all statistical analyses were performed with sas ( sas enterprise guide 4.1 or higher ) for windows ( sas institute inc , cary , nc ) .
the study was a randomised double - blind controlled clinical study carried out in private practices , children 's hospitals , university hospitals , or site management organisations ( organisations that provide clinical trial related services ) located in 5 countries in europe and asia : malaysia , the netherlands , poland , portugal , and thailand .
healthy young children ages 11 to 29 months attending a day care centre at least 2 times per week were recruited into the study between september 2008 and may 2009 after informed written consent was obtained from the parents or legal guardians .
day care centres were defined as any private or governmental facility providing care or playgroups for at least 20 children .
exclusion criteria included atopic dermatitis according to the hannifin criteria ; disorders requiring a special diet ; any relevant congenital abnormality , chromosomal disorder , or severe disease ; preexisting pathology of severe respiratory or gi diseases ; use of antiregurgitation , antireflux , or laxative medication at the time of inclusion ; and use of immune modulators or of prescribed prophylactic antibiotics at the time of inclusion .
the study was conducted following the principles of the declaration of helsinki and good clinical practice guidelines .
the protocol was approved by the ethics committees of the different institutes in the countries .
study products were either the active study product , gum with the addition of 1.2 g/100 ml of scgos/ lcfos ( 9:1 ) ( immunofortis ) and 19.2 mg/100 ml of n-3 lcpufas ( epa + dha , 4:6 ) , the control study product , gum without scgos / lcfos / n-3 lcpufas ( danone research ) .
gum is specially formulated milk for young children ages 1 to 3 years , enriched with key nutrients such as vitamins a , c , and d , iron , and calcium .
both study products were packed in identical tins ; they were of the same colour , weight , smell , and taste .
both the research staff and parents and subjects were unaware of the real nature of the product .
the unblinding procedure was performed after the study was completed and after the statistical analyses were finalised .
the study consisted of a 4-week run - in period and a 52-week intervention period .
subjects eligible for participation started with the run - in period , in which control product was consumed .
the run - in period was needed to get the children used to the product and the parents used to the completion of the diaries .
subjects who had successfully completed the run - in period were randomly allocated to receive either the active study product or the control study product in a double - blind period of 52 weeks . to ensure a gradual transition during the first 2 weeks of this period , the run - in product was mixed with the active / control study product following a schedule provided by the centres .
the requested intake of the study product was 400 to 750 ml daily for both the run - in period and the 52-week intervention period .
this wide range was chosen because the different countries involved in the study had a wide variety of dosage recommendations .
every 2 to 4 weeks , there was a contact session with the parents , either by visit or by telephone , in which study diaries were discussed .
in addition , there was a reference group of subjects receiving cow 's milk already before the start of the study and which followed the same procedures as the intervention groups ; however , this group was not randomised and consumed regular cow 's milk for 52 weeks .
background information was collected on the family , their environment , the child 's nutritional habits , and illnesses . during the study , parents daily recorded the intake of study product as well as , in a symptom diary , any respiratory symptoms ( cough , fever , blocked or runny nose , sore throat , wheeze , and/or ear discharge ) and gi symptoms ( vomiting or diarrhoea ) .
they also reported absence from the day care centre , absence of parents from work , contacts with a physician , doctor 's diagnoses , and prescription of medication .
if parents suspected that their child had fever , body temperature of the subject was measured and recorded , with thermometers provided to all of the children .
furthermore , parents completed 3 times a 3-day diary , with questions about gi symptoms and stool characteristics , at the start of the intervention period , at 26 weeks , and at 52 weeks . the diary covered stool frequency and stool consistency and severity of gi tract characteristics ( diarrhoea , constipation , and flatulence ) with a 4-point scale ( none , mild , moderate , severe ) .
sample size was estimated based on the incidence of respiratory tract infections . based on a previous study on the incidence of respiratory tract infections ( 17,18 )
, the study was designed to have sufficient power ( 80% ) to detect a 20% difference between the groups with a 0.05 significance level based on a 2-tailed test .
the number of children necessary in each group was 300 subjects , including an estimated dropout rate of 25% .
each child , except for the children in the reference group , was randomly allocated to either the active study product or the control study product .
the randomised subjects were distributed to the 2 study groups based on a randomisation code , developed using a computer random number generator .
a block size of 6 was used , and stratification was applied for investigator site .
the primary outcome was the number of episodes of urti or giis based on a combination of subject 's illness symptoms reported by the parents during the intervention period .
urti was defined as any combination of at least 2 of the following symptoms : cough , fever , blocked or runny nose , sore throat , wheeze , and/or ear discharge .
gii was defined as the occurrence of vomiting or diarrhoea or both for at least 1 day , with or without fever .
an episode of urti and/or gii was defined as having 1 symptoms for at least 1 day .
a child was considered as recovered from an episode when the subject showed no symptoms of the reported infection and had a normal level of activity .
a new episode was considered to have occurred if the symptom(s ) occurred at least 3 days after the stated time of recovery from a previous episode .
he or she could indicate whether he or she found the symptoms to be an infection .
secondary outcomes were total number and duration of episodes of urti and/or gii ; parents absence from work because of illness of the child ; number , duration , and season of subjects absence from day care centre ; and number and type of all infections diagnosed by a physician or investigator .
descriptive statistics were used to describe the basic features according to the age , sex , food intake , and family characteristics .
results on the primary parameter were obtained from both a logistic regression model and a zero - inflated negative binomial regression model .
the latter model combines 2 separate parts : a ( binomial ) part , whether or not a subject would get an infection ; and a ( negative - binomial ) part fitting the counts for infections for the subjects .
all analyses were based on the all - subjects randomised group : all of the subjects who had been randomised and to whose parents the study product had been dispensed . for safety analysis , such as growth , height , and tolerance , the safety analysis population was used consisting of all children except the screen failure subjects for the intervention period . in an analysis of the normally distributed continuous data ,
a t test was performed , whereas a wilcoxon rank - sum test was used in analysis of non - normality continuous data .
in addition , a test ( fisher exact test in the case of < 5 expected cell counts ) was performed in analysis of nominal data .
furthermore , kaplan - meier ( km ) curves together with corresponding log - rank tests were conducted in analysis of time to event type of data .
all statistical analyses were performed with sas ( sas enterprise guide 4.1 or higher ) for windows ( sas institute inc , cary , nc ) .
of the 907 subjects screened and enrolled in the run - in period , 767 were randomised : 388 received the active study product , and 379 received the control study product . in total
, there were 697 completers : malaysia 135 , the netherlands 199 , poland 126 , portugal 70 , and thailand 167 .
thirty - seven subjects were included in the reference group , which was not randomised ( fig .
this was because of logistical reasons , timing differences of start of inclusion periods at different locations and the run - in period , and the fact that it was uncertain how many subjects would remain in the intervention period .
no statistically significant differences were observed between the 2 groups in regard to age , sex , length , and weight at birth and any of the other baseline characteristics analysed ( table 2 ) .
also , no differences were seen in the mother 's and father 's characteristics , with regard to the highest level of education or the professional status at baseline ( tables 3 and 4 ) .
logistic regression analysis was performed in which the chance of having 1 episodes was modelled against the chance of having 0 episodes ; this yielded a p value of 0.03 ( 299/388 [ 77% ] children active group vs 313/379 [ 83% ] children control group ) and a relative risk of 0.93 with a 95% confidence interval 0.871.00 and number needed to treat ( nnt ) 17 .
the logistic regression model was based on a distinction between the 2 categories : did infections occur , yes or no ; however , the data contained information on the number of reported infection episodes as well ( fig .
a poisson regression model is needed that can address the fact that a considerable proportion of children had the value 0 ( ie , no infections , fig .
the zero - inflated negative binomial regression model is such a model and showed a p value of 0.07 for the overall effect on occurrence of infections .
the binomial part of the model analyses the occurrence of zero infections and showed a significant difference ( 89/388 children active group vs 66/379 children control group ; p = 0.03 ; fig .
3 ) , whereas the negative binomial part of the model did not show a significant difference .
number of subjects with number of infections for active and control group , based on the statistical model .
there are fewer children with small numbers of reported episodes in active group ( 15 number of reported infectious episodes ) ( b ) .
no difference between groups in percentages of children with high number of reported episodes ( 6 number of reported infectious episodes ) ( c ) .
a post hoc analysis that focused on only those parent - reported symptoms that were confirmed by an investigator ( based on their experience ) showed a significant effect ( p = 0.004 and relative risk 0.89 with a 95% confidence interval 0.820.97 , nnt 12 , table 5 ) on the number of infectious episodes .
also , here this was mostly driven by the higher statistically significant effect on having no infectious episodes ( p = 0.003 ) . furthermore , post hoc analyses were performed to check for differences when urti and gii were analysed separately with or without fever .
secondary outcomes such as any type of medication use ( which consisted of oral antibiotics , paracetamol - acetaminophen , cough mixture medication , nose drops / spray , ear drops , oral rehydration solution , homeopathic medication , and any other medication ) and duration of absence from day care showed no significant differences between the 2 study groups ( table 7 ) . in total , 2217 adverse events , including 78 serious adverse events , were reported .
the majority of adverse events ( 49% ) were related to the respiratory system ( eg , cough , fever , blocked or runny nose , sore throat , wheeze , ear discharge ) .
a total of 29 adverse events were assessed by the investigator as possibly or probably related to the study product during the run - in or intervention period .
the related adverse events were mild gi symptoms and occurred equally between the 2 groups .
none of the reported serious adverse events were assessed as related to study product and none were unexpected . in the study , the small nonrandomised cow 's milk reference group , coming from the netherlands only , was compared with the total active and control groups .
both study groups had fewer parent - reported episodes than the cow 's milk group ( cow 's milk vs active : p = 0.008 ; and cow 's milk vs control : p = 0.004 ; table 8) .
time to occurrence of first reported episode was significantly different ( p = 0.04 ) between active and cow 's milk group in favour of the active group with a survival comparison with log - rank test . a kaplan - meier curve ( fig .
4 ) showed the same result when looking at deviations between the active versus the cow 's milk group . the kaplan - meier curve .
active , control , and cow 's milk group : time to occurrence of first reported episode .
the mean average intake of gum products was 528 ml / day , and no significant differences were found between the 2 study groups regarding daily intake of the study products ( active : 529 128 vs control : 527 139 ml / day ; table 9 ) .
the daily intake of scgos / lcfos ( 9:1 ) was at least 3.6 g / day , and for dha + epa , this was at least 57.6 mg / day .
subjects in the active arm weighed statistically significantly less at baseline and were smaller when compared with the control arm ( table 10 ) ; however , these differences are seen as normal variability of weight and height .
the difference in weight was maintained throughout the intervention period ; however , for body mass index and for the change in weight and height , no statistically significant difference between the 2 study arms was observed .
the weight change from baseline at week 52 of children from the reference group compared with the active or control group did not differ significantly ; however , the length change and bmi change did differ significantly ( except for length change when reference was compared with control ) .
logistic regression analysis was performed in which the chance of having 1 episodes was modelled against the chance of having 0 episodes ; this yielded a p value of 0.03 ( 299/388 [ 77% ] children active group vs 313/379 [ 83% ] children control group ) and a relative risk of 0.93 with a 95% confidence interval 0.871.00 and number needed to treat ( nnt ) 17 .
the logistic regression model was based on a distinction between the 2 categories : did infections occur , yes or no ; however , the data contained information on the number of reported infection episodes as well ( fig .
a poisson regression model is needed that can address the fact that a considerable proportion of children had the value 0 ( ie , no infections , fig .
the zero - inflated negative binomial regression model is such a model and showed a p value of 0.07 for the overall effect on occurrence of infections .
the binomial part of the model analyses the occurrence of zero infections and showed a significant difference ( 89/388 children active group vs 66/379 children control group ; p = 0.03 ; fig .
3 ) , whereas the negative binomial part of the model did not show a significant difference .
number of subjects with number of infections for active and control group , based on the statistical model .
there are fewer children with small numbers of reported episodes in active group ( 15 number of reported infectious episodes ) ( b ) .
no difference between groups in percentages of children with high number of reported episodes ( 6 number of reported infectious episodes ) ( c ) .
a post hoc analysis that focused on only those parent - reported symptoms that were confirmed by an investigator ( based on their experience ) showed a significant effect ( p = 0.004 and relative risk 0.89 with a 95% confidence interval 0.820.97 , nnt 12 , table 5 ) on the number of infectious episodes . also , here
this was mostly driven by the higher statistically significant effect on having no infectious episodes ( p = 0.003 ) .
furthermore , post hoc analyses were performed to check for differences when urti and gii were analysed separately with or without fever .
secondary outcomes such as any type of medication use ( which consisted of oral antibiotics , paracetamol - acetaminophen , cough mixture medication , nose drops / spray , ear drops , oral rehydration solution , homeopathic medication , and any other medication ) and duration of absence from day care showed no significant differences between the 2 study groups ( table 7 ) . in total , 2217 adverse events , including 78 serious adverse events , were reported .
the majority of adverse events ( 49% ) were related to the respiratory system ( eg , cough , fever , blocked or runny nose , sore throat , wheeze , ear discharge ) .
a total of 29 adverse events were assessed by the investigator as possibly or probably related to the study product during the run - in or intervention period .
the related adverse events were mild gi symptoms and occurred equally between the 2 groups .
none of the reported serious adverse events were assessed as related to study product and none were unexpected .
in the study , the small nonrandomised cow 's milk reference group , coming from the netherlands only , was compared with the total active and control groups .
both study groups had fewer parent - reported episodes than the cow 's milk group ( cow 's milk vs active : p = 0.008 ; and cow 's milk vs control : p = 0.004 ; table 8) .
time to occurrence of first reported episode was significantly different ( p = 0.04 ) between active and cow 's milk group in favour of the active group with a survival comparison with log - rank test . a kaplan - meier curve ( fig .
4 ) showed the same result when looking at deviations between the active versus the cow 's milk group . the kaplan - meier curve .
active , control , and cow 's milk group : time to occurrence of first reported episode .
the mean average intake of gum products was 528 ml / day , and no significant differences were found between the 2 study groups regarding daily intake of the study products ( active : 529 128 vs control : 527 139 ml / day ; table 9 ) .
the daily intake of scgos / lcfos ( 9:1 ) was at least 3.6 g / day , and for dha + epa , this was at least 57.6 mg / day .
subjects in the active arm weighed statistically significantly less at baseline and were smaller when compared with the control arm ( table 10 ) ; however , these differences are seen as normal variability of weight and height .
the difference in weight was maintained throughout the intervention period ; however , for body mass index and for the change in weight and height , no statistically significant difference between the 2 study arms was observed .
the weight change from baseline at week 52 of children from the reference group compared with the active or control group did not differ significantly ; however , the length change and bmi change did differ significantly ( except for length change when reference was compared with control ) .
this randomised double - blind controlled clinical study is the first to show a reduced risk of infections in young children following consumption of gum supplemented with scgos / lcfos / n-3 lcpufas .
it is remarkable that although the gum is only a relatively small part of a young child 's diet , it can apparently influence the risk of infections .
the borderline significant effect we observed is mainly derived from the decreased risk of developing an infection .
this does not mean that the child does not encounter any viral or bacterial microorganism , but that the child does not experience symptoms of the infection .
this is the first study conducted in young children to demonstrate that the use of a prebiotic mixture can influence the risk of infections .
similar studies have evaluated the effect of consumption of milk with probiotics on incidence of infections , with different results . in a randomised double - blind placebo - controlled study ( n = 281 , age range 17 years ) , administration of lactobacillus gg ( lgg ) resulted in a significantly reduced risk of urti ( 19 ) .
another study in which lgg was administered showed a reduction in the number of children with respiratory infections ( 20 ) .
neither study showed any effect on gii . a more recent study with lgg could not show an effect on respiratory illness in children ( 21 ) , but the daily dose of lgg in this study was lower compared with the other studies .
in contrast , results from another study with other bacteria , bifidobacterium lactis or lactobacillus reuteri , did not show a beneficial probiotic effect on the duration of respiratory illness ( 18 ) .
in contrast to the above reports , in our study , we investigated the effect of gum with prebiotics and lcpufas , not with the addition of probiotics .
our results indicate that the specific scgos / lcfos prebiotic mixture and lcpufas can have an effect only on the risk of infections . moreover , the dropout rate was extremely low ( only 10% ) in this study , and also the compliance of product intake over both groups was better compared with other published studies . in our study , we evaluated the combination of urti and gii together and not separately .
as for the mechanism responsible for the beneficial role of gum supplemented with scgos / lcfos / n-3 lcpufas , studies in infants have documented that the mixture of scgos / lcfos supports immune development by significantly improving the intestinal microbiota .
the positive effects of scgos / lcfos on the immune response have also been observed in experimental animal studies ( 22,23 ) .
furthermore , several lcpufa supplementation studies in preterm infants ( 24 ) , term infants ( 25,26 ) , and school - age children ( 27 ) suggest normalisation and improved immune function following dietary lcpufas supplementation .
supplementation of lcpufas to infant formula resulted in cytokine and t - cell population profiles that were more similar to those of breast - fed infants compared with those of unsupplemented formula - fed infants , and resulted in reduced incidence of respiratory illnesses ( 26 ) .
lcpufa supplementation in children 5 to 7 years of age appeared to result in enhanced initiation of the immune response to b - cell mitogen and a reduced inflammatory response ( 27 ) .
finally , dietary dha enrichment reduced the incidence and severity of illnesses in thai schoolchildren ( 28,29 ) , and these effects were accompanied by a shift of cytokine profile toward a less inflammatory profile .
together , these studies support the concept that a dietary source of n-3 lcpufas can improve immune function in children , and thereby support disease resistance .
few studies have been done in which the effect of prebiotics on infections was examined .
we recently reported the results of a study with healthy young children indicating that the use of a gum with added scgos / lcfos / n-3 lcpufas leads to a higher level of bifidobacteria during the intake period compared with the control group ( 29a ) .
the mechanisms of prebiotics and n-3 lcpufas apparently operated in a complementary way on infections in this study . as for the clinical relevance of the results of this study , there was only a 6% lower risk of infectious episodes when using gum supplemented with scgos / lcfos / n-3 lcpufas and an 8% lower risk when the data of the investigator - confirmed episodes analysis are used .
the nnt to prevent one child from having an infectious episode is 17 , and when the data of the investigator - confirmed episodes analysis are used , this decreases to 12 . knowing that the study product is a food product , which only accounts for a small part of a child 's diet , the effect on nnt is large .
what the exact effect will be on health care cost reduction remains to be investigated .
first , the symptoms were based on parental reporting , but we did not actually determine whether the children with symptoms had infections by measuring immune responses .
second , a low frequency of infections was observed , which could be because of underreporting .
study results were only measured as parent - reported symptoms , to limit the burden for the child .
third , because there are no clear definitions , the definitions for the respiratory and/or gi symptoms used throughout this study are not exactly the same as in other published studies , which makes it sometimes difficult to compare the results .
in addition , there are no validated questionnaires available for the monitoring of respiratory symptoms , which would have made the results more consistent with other studies .
the comparison of the cow 's milk group with the gum groups showed that more infectious episodes were observed in the cow 's milk group .
therefore , the results obtained should be interpreted with caution ; however , the results suggest that gum as such has a beneficial effect on the occurrence of infections compared with cow 's milk
. this could be because of the fact that gum is nutritionally enriched compared with cow 's milk .
gum contains relevant vitamins and minerals required for that age range , which may contribute to a better support of growth and development , and may indirectly positively affect the immune system .
iron and vitamin d , for instance , are 2 nutrients included in gum for which intake is often seen as insufficient in young children ( 30,31 ) ( nutritional surveys in several countries , systematic surveys of nutrition and health status of young children and women of child - bearing age based on literature review and expert opinions conducted in several countries ) .
an explanation for the significant difference in height change between reference group and active group could be that the children from the reference group were from the netherlands only .
it is well known that people from northern europe , and especially the netherlands , are tall compared with people from other european countries and asia .
the fact that there was no significant difference in weight change was to be expected because the ratio of height and weight should not differ .
further studies in which cow 's milk is compared with gum for effects on the occurrence of infections are needed to obtain conclusive results . in conclusion
, this is the first study in children to show a reduced risk of infection following consumption of gum supplemented with scgos / lcfos / n-3 lcpufas .
although the primary outcome was of borderline statistical significance , it is supported by the post hoc significantly decreased risk of infectious symptoms with investigator - confirmed episodes ; therefore , this study supports the use of gum supplemented with scgos / lcfos / n-3 lcpufas to reduce the risk of infection in children attending day care centres . | abstractobjective : the aim of this study was to investigate the effect of growing - up milk ( gum ) with added short - chain galacto - oligosaccharides ( scgos)/long - chain fructo - oligosaccharides ( lcfos ) ( 9:1 ) ( immunofortis ) and n-3 long - chain polyunsaturated fatty acids ( lcpufas ) on the occurrence of infections in healthy children attending day care centres.methods:in a randomised double - blind controlled , parallel , multicountry intervention study , 767 healthy children , ages 11 to 29 months , received gum with scgos / lcfos / lcpufas ( the active group , n = 388 ) , gum without scgos / lcfos / lcpufas ( the control group , n = 379 ) , or cow 's milk ( n = 37 ) for 52 weeks . the primary outcome measure was the number of episodes of upper respiratory tract infections or gastrointestinal infections based on a combination of subject 's illness symptoms reported by the parents during the intervention period.results:children in the active group compared with the control group had a decreased risk of developing at least 1 infection ( 299/388 [ 77% ] vs 313/379 [ 83% ] , respectively , relative risk 0.93 , 95% confidence interval [ ci ] 0.871.00 ; logistic regression p = 0.03 ) .
there was a trend toward a reduction ( p = 0.07 ) in the total number of infections in the active group , which was significant when confirmed by one of the investigators ( 268/388 [ 69% ] vs 293/379 [ 77% ] , respectively , relative risk 0.89 , 95% ci 0.820.97 ; p = 0.004 , post hoc ) .
more infectious episodes were observed in the cow 's milk group , when compared with both gum groups ( 34/37 [ 92% ] vs 612/767 [ 80% ] , respectively , relative risk 1.15 , 95% ci 1.041.28).conclusions : this is the first study in children to show a reduced risk of infection following consumption of gum supplemented with scgos / lcfos / n-3 lcpufas .
the borderline statistical significance justifies a new study to confirm this finding . | METHODS
Participants
Study Design/Intervention
Data Collection
Sample Size and Randomisation
Outcome Measures
Statistical Analysis
RESULTS
Occurrence of Infections
Comparison of GUM Groups With Cow's Milk Group
GUM Intake, Weight, and Height
DISCUSSION | the study was a randomised double - blind controlled clinical study carried out in private practices , children 's hospitals , university hospitals , or site management organisations ( organisations that provide clinical trial related services ) located in 5 countries in europe and asia : malaysia , the netherlands , poland , portugal , and thailand . healthy young children ages 11 to 29 months attending a day care centre at least 2 times per week were recruited into the study between september 2008 and may 2009 after informed written consent was obtained from the parents or legal guardians . study products were either the active study product , gum with the addition of 1.2 g/100 ml of scgos/ lcfos ( 9:1 ) ( immunofortis ) and 19.2 mg/100 ml of n-3 lcpufas ( epa + dha , 4:6 ) , the control study product , gum without scgos / lcfos / n-3 lcpufas ( danone research ) . subjects who had successfully completed the run - in period were randomly allocated to receive either the active study product or the control study product in a double - blind period of 52 weeks . to ensure a gradual transition during the first 2 weeks of this period , the run - in product was mixed with the active / control study product following a schedule provided by the centres . every 2 to 4 weeks , there was a contact session with the parents , either by visit or by telephone , in which study diaries were discussed . in addition , there was a reference group of subjects receiving cow 's milk already before the start of the study and which followed the same procedures as the intervention groups ; however , this group was not randomised and consumed regular cow 's milk for 52 weeks . during the study , parents daily recorded the intake of study product as well as , in a symptom diary , any respiratory symptoms ( cough , fever , blocked or runny nose , sore throat , wheeze , and/or ear discharge ) and gi symptoms ( vomiting or diarrhoea ) . furthermore , parents completed 3 times a 3-day diary , with questions about gi symptoms and stool characteristics , at the start of the intervention period , at 26 weeks , and at 52 weeks . based on a previous study on the incidence of respiratory tract infections ( 17,18 ) , the study was designed to have sufficient power ( 80% ) to detect a 20% difference between the groups with a 0.05 significance level based on a 2-tailed test . each child , except for the children in the reference group , was randomly allocated to either the active study product or the control study product . the primary outcome was the number of episodes of urti or giis based on a combination of subject 's illness symptoms reported by the parents during the intervention period . urti was defined as any combination of at least 2 of the following symptoms : cough , fever , blocked or runny nose , sore throat , wheeze , and/or ear discharge . gii was defined as the occurrence of vomiting or diarrhoea or both for at least 1 day , with or without fever . secondary outcomes were total number and duration of episodes of urti and/or gii ; parents absence from work because of illness of the child ; number , duration , and season of subjects absence from day care centre ; and number and type of all infections diagnosed by a physician or investigator . results on the primary parameter were obtained from both a logistic regression model and a zero - inflated negative binomial regression model . all analyses were based on the all - subjects randomised group : all of the subjects who had been randomised and to whose parents the study product had been dispensed . the study was a randomised double - blind controlled clinical study carried out in private practices , children 's hospitals , university hospitals , or site management organisations ( organisations that provide clinical trial related services ) located in 5 countries in europe and asia : malaysia , the netherlands , poland , portugal , and thailand . healthy young children ages 11 to 29 months attending a day care centre at least 2 times per week were recruited into the study between september 2008 and may 2009 after informed written consent was obtained from the parents or legal guardians . day care centres were defined as any private or governmental facility providing care or playgroups for at least 20 children . the protocol was approved by the ethics committees of the different institutes in the countries . study products were either the active study product , gum with the addition of 1.2 g/100 ml of scgos/ lcfos ( 9:1 ) ( immunofortis ) and 19.2 mg/100 ml of n-3 lcpufas ( epa + dha , 4:6 ) , the control study product , gum without scgos / lcfos / n-3 lcpufas ( danone research ) . subjects who had successfully completed the run - in period were randomly allocated to receive either the active study product or the control study product in a double - blind period of 52 weeks . to ensure a gradual transition during the first 2 weeks of this period , the run - in product was mixed with the active / control study product following a schedule provided by the centres . every 2 to 4 weeks , there was a contact session with the parents , either by visit or by telephone , in which study diaries were discussed . in addition , there was a reference group of subjects receiving cow 's milk already before the start of the study and which followed the same procedures as the intervention groups ; however , this group was not randomised and consumed regular cow 's milk for 52 weeks . during the study , parents daily recorded the intake of study product as well as , in a symptom diary , any respiratory symptoms ( cough , fever , blocked or runny nose , sore throat , wheeze , and/or ear discharge ) and gi symptoms ( vomiting or diarrhoea ) . furthermore , parents completed 3 times a 3-day diary , with questions about gi symptoms and stool characteristics , at the start of the intervention period , at 26 weeks , and at 52 weeks . sample size was estimated based on the incidence of respiratory tract infections . based on a previous study on the incidence of respiratory tract infections ( 17,18 )
, the study was designed to have sufficient power ( 80% ) to detect a 20% difference between the groups with a 0.05 significance level based on a 2-tailed test . each child , except for the children in the reference group , was randomly allocated to either the active study product or the control study product . the primary outcome was the number of episodes of urti or giis based on a combination of subject 's illness symptoms reported by the parents during the intervention period . urti was defined as any combination of at least 2 of the following symptoms : cough , fever , blocked or runny nose , sore throat , wheeze , and/or ear discharge . gii was defined as the occurrence of vomiting or diarrhoea or both for at least 1 day , with or without fever . secondary outcomes were total number and duration of episodes of urti and/or gii ; parents absence from work because of illness of the child ; number , duration , and season of subjects absence from day care centre ; and number and type of all infections diagnosed by a physician or investigator . results on the primary parameter were obtained from both a logistic regression model and a zero - inflated negative binomial regression model . all analyses were based on the all - subjects randomised group : all of the subjects who had been randomised and to whose parents the study product had been dispensed . of the 907 subjects screened and enrolled in the run - in period , 767 were randomised : 388 received the active study product , and 379 received the control study product . thirty - seven subjects were included in the reference group , which was not randomised ( fig . this was because of logistical reasons , timing differences of start of inclusion periods at different locations and the run - in period , and the fact that it was uncertain how many subjects would remain in the intervention period . logistic regression analysis was performed in which the chance of having 1 episodes was modelled against the chance of having 0 episodes ; this yielded a p value of 0.03 ( 299/388 [ 77% ] children active group vs 313/379 [ 83% ] children control group ) and a relative risk of 0.93 with a 95% confidence interval 0.871.00 and number needed to treat ( nnt ) 17 . the logistic regression model was based on a distinction between the 2 categories : did infections occur , yes or no ; however , the data contained information on the number of reported infection episodes as well ( fig . the zero - inflated negative binomial regression model is such a model and showed a p value of 0.07 for the overall effect on occurrence of infections . the binomial part of the model analyses the occurrence of zero infections and showed a significant difference ( 89/388 children active group vs 66/379 children control group ; p = 0.03 ; fig . 3 ) , whereas the negative binomial part of the model did not show a significant difference . number of subjects with number of infections for active and control group , based on the statistical model . there are fewer children with small numbers of reported episodes in active group ( 15 number of reported infectious episodes ) ( b ) . no difference between groups in percentages of children with high number of reported episodes ( 6 number of reported infectious episodes ) ( c ) . a post hoc analysis that focused on only those parent - reported symptoms that were confirmed by an investigator ( based on their experience ) showed a significant effect ( p = 0.004 and relative risk 0.89 with a 95% confidence interval 0.820.97 , nnt 12 , table 5 ) on the number of infectious episodes . also , here this was mostly driven by the higher statistically significant effect on having no infectious episodes ( p = 0.003 ) . secondary outcomes such as any type of medication use ( which consisted of oral antibiotics , paracetamol - acetaminophen , cough mixture medication , nose drops / spray , ear drops , oral rehydration solution , homeopathic medication , and any other medication ) and duration of absence from day care showed no significant differences between the 2 study groups ( table 7 ) . a total of 29 adverse events were assessed by the investigator as possibly or probably related to the study product during the run - in or intervention period . in the study , the small nonrandomised cow 's milk reference group , coming from the netherlands only , was compared with the total active and control groups . both study groups had fewer parent - reported episodes than the cow 's milk group ( cow 's milk vs active : p = 0.008 ; and cow 's milk vs control : p = 0.004 ; table 8) . time to occurrence of first reported episode was significantly different ( p = 0.04 ) between active and cow 's milk group in favour of the active group with a survival comparison with log - rank test . 4 ) showed the same result when looking at deviations between the active versus the cow 's milk group . active , control , and cow 's milk group : time to occurrence of first reported episode . the daily intake of scgos / lcfos ( 9:1 ) was at least 3.6 g / day , and for dha + epa , this was at least 57.6 mg / day . subjects in the active arm weighed statistically significantly less at baseline and were smaller when compared with the control arm ( table 10 ) ; however , these differences are seen as normal variability of weight and height . the weight change from baseline at week 52 of children from the reference group compared with the active or control group did not differ significantly ; however , the length change and bmi change did differ significantly ( except for length change when reference was compared with control ) . logistic regression analysis was performed in which the chance of having 1 episodes was modelled against the chance of having 0 episodes ; this yielded a p value of 0.03 ( 299/388 [ 77% ] children active group vs 313/379 [ 83% ] children control group ) and a relative risk of 0.93 with a 95% confidence interval 0.871.00 and number needed to treat ( nnt ) 17 . the logistic regression model was based on a distinction between the 2 categories : did infections occur , yes or no ; however , the data contained information on the number of reported infection episodes as well ( fig . the zero - inflated negative binomial regression model is such a model and showed a p value of 0.07 for the overall effect on occurrence of infections . the binomial part of the model analyses the occurrence of zero infections and showed a significant difference ( 89/388 children active group vs 66/379 children control group ; p = 0.03 ; fig . 3 ) , whereas the negative binomial part of the model did not show a significant difference . number of subjects with number of infections for active and control group , based on the statistical model . there are fewer children with small numbers of reported episodes in active group ( 15 number of reported infectious episodes ) ( b ) . no difference between groups in percentages of children with high number of reported episodes ( 6 number of reported infectious episodes ) ( c ) . a post hoc analysis that focused on only those parent - reported symptoms that were confirmed by an investigator ( based on their experience ) showed a significant effect ( p = 0.004 and relative risk 0.89 with a 95% confidence interval 0.820.97 , nnt 12 , table 5 ) on the number of infectious episodes . also , here
this was mostly driven by the higher statistically significant effect on having no infectious episodes ( p = 0.003 ) . secondary outcomes such as any type of medication use ( which consisted of oral antibiotics , paracetamol - acetaminophen , cough mixture medication , nose drops / spray , ear drops , oral rehydration solution , homeopathic medication , and any other medication ) and duration of absence from day care showed no significant differences between the 2 study groups ( table 7 ) . a total of 29 adverse events were assessed by the investigator as possibly or probably related to the study product during the run - in or intervention period . in the study , the small nonrandomised cow 's milk reference group , coming from the netherlands only , was compared with the total active and control groups . both study groups had fewer parent - reported episodes than the cow 's milk group ( cow 's milk vs active : p = 0.008 ; and cow 's milk vs control : p = 0.004 ; table 8) . time to occurrence of first reported episode was significantly different ( p = 0.04 ) between active and cow 's milk group in favour of the active group with a survival comparison with log - rank test . 4 ) showed the same result when looking at deviations between the active versus the cow 's milk group . active , control , and cow 's milk group : time to occurrence of first reported episode . the daily intake of scgos / lcfos ( 9:1 ) was at least 3.6 g / day , and for dha + epa , this was at least 57.6 mg / day . subjects in the active arm weighed statistically significantly less at baseline and were smaller when compared with the control arm ( table 10 ) ; however , these differences are seen as normal variability of weight and height . the weight change from baseline at week 52 of children from the reference group compared with the active or control group did not differ significantly ; however , the length change and bmi change did differ significantly ( except for length change when reference was compared with control ) . this randomised double - blind controlled clinical study is the first to show a reduced risk of infections in young children following consumption of gum supplemented with scgos / lcfos / n-3 lcpufas . the borderline significant effect we observed is mainly derived from the decreased risk of developing an infection . this is the first study conducted in young children to demonstrate that the use of a prebiotic mixture can influence the risk of infections . similar studies have evaluated the effect of consumption of milk with probiotics on incidence of infections , with different results . in a randomised double - blind placebo - controlled study ( n = 281 , age range 17 years ) , administration of lactobacillus gg ( lgg ) resulted in a significantly reduced risk of urti ( 19 ) . another study in which lgg was administered showed a reduction in the number of children with respiratory infections ( 20 ) . a more recent study with lgg could not show an effect on respiratory illness in children ( 21 ) , but the daily dose of lgg in this study was lower compared with the other studies . in contrast to the above reports , in our study , we investigated the effect of gum with prebiotics and lcpufas , not with the addition of probiotics . our results indicate that the specific scgos / lcfos prebiotic mixture and lcpufas can have an effect only on the risk of infections . moreover , the dropout rate was extremely low ( only 10% ) in this study , and also the compliance of product intake over both groups was better compared with other published studies . as for the mechanism responsible for the beneficial role of gum supplemented with scgos / lcfos / n-3 lcpufas , studies in infants have documented that the mixture of scgos / lcfos supports immune development by significantly improving the intestinal microbiota . the positive effects of scgos / lcfos on the immune response have also been observed in experimental animal studies ( 22,23 ) . lcpufa supplementation in children 5 to 7 years of age appeared to result in enhanced initiation of the immune response to b - cell mitogen and a reduced inflammatory response ( 27 ) . together , these studies support the concept that a dietary source of n-3 lcpufas can improve immune function in children , and thereby support disease resistance . we recently reported the results of a study with healthy young children indicating that the use of a gum with added scgos / lcfos / n-3 lcpufas leads to a higher level of bifidobacteria during the intake period compared with the control group ( 29a ) . the mechanisms of prebiotics and n-3 lcpufas apparently operated in a complementary way on infections in this study . as for the clinical relevance of the results of this study , there was only a 6% lower risk of infectious episodes when using gum supplemented with scgos / lcfos / n-3 lcpufas and an 8% lower risk when the data of the investigator - confirmed episodes analysis are used . the comparison of the cow 's milk group with the gum groups showed that more infectious episodes were observed in the cow 's milk group . therefore , the results obtained should be interpreted with caution ; however , the results suggest that gum as such has a beneficial effect on the occurrence of infections compared with cow 's milk
. this could be because of the fact that gum is nutritionally enriched compared with cow 's milk . further studies in which cow 's milk is compared with gum for effects on the occurrence of infections are needed to obtain conclusive results . in conclusion
, this is the first study in children to show a reduced risk of infection following consumption of gum supplemented with scgos / lcfos / n-3 lcpufas . although the primary outcome was of borderline statistical significance , it is supported by the post hoc significantly decreased risk of infectious symptoms with investigator - confirmed episodes ; therefore , this study supports the use of gum supplemented with scgos / lcfos / n-3 lcpufas to reduce the risk of infection in children attending day care centres . | [
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these fast and brief eye movements can not rely on online ( visual ) feedback since visual delays are longer than the movement itself .
this means that , in order to maintain accurate eye movements , the motor commands for future saccades must be adjusted after each eye movement is completed .
these plastic mechanisms are present to reduce or compensate motor errors due to either physiological or pathological behavior [ 1 , 2 ] . since mclaughlin ( 1967 ) described the
parametric adjustment , known today as short - term saccade adaptation , his paradigm has been used as a way to assess learning and plasticity in the oculomotor system .
this is done by asking a subject to make a saccade to a new position and while the saccade is in flight , the target moves ( intrasaccadic step ) causing a postsaccadic visual error [ 35 ] . when the subject is repeatedly exposed to the same error
, the oculomotor system will gradually drive a change in the metrics of the eye movement over time , making the error smaller [ 615 ] .
the error can induce saccade shortening ( gain - down ) , when the intrasaccade step of the target is in the direction of the starting point of the saccade ( inward adaptation ) , or saccade lengthening ( gain - up ) when the step is away from the starting point ( outward adaptation ) .
human subjects adapt faster in response to inward adaptation than in response to outward adaptation stimuli , which poses the hypothesis that these two types of adaptation involve different neural mechanisms [ 16 , 17 ] .
the cerebellum plays a crucial role in saccadic error detection [ 1820 ] and thus in saccade adaptation .
evidence of the cerebellar involvement and its necessary integrity to oculomotor learning has been demonstrated as large lesions , focal inactivation , or pathological conditions of different areas of the cerebellum impair the ability to adapt saccades [ 2125 ] .
in addition , various loci in the cerebellum relate to inward and outward errors differently .
for instance , patients with vermal damage are partially capable of inward adaptation but lack outward adaptation . also , mri - guided tms on lateral hemispheres potentiates the postadaptation effects of outward adaptation and , in contrast , depresses gain - down adaptation .
cerebellar output can be modulated with transcranial direct current stimulation ( tdcs ) with great specificity as shown by excitability changes after stimulation ranging from cognitive to motor skills [ 2730 ] . in this study
, we used anodal tdcs as a tool to noninvasively modulate cerebellar output and provide functional insight into the learning aspects during saccade adaptation .
13 healthy subjects ( one author : e. avila , 12 naive subjects to tdcs , mean age of 22.4 , range 1929 years , 6 females ) , right - handed volunteers with no known history of neurological or psychiatric conditions , not taking chronic or acute medications or using drugs , with normal vision , were recruited .
they all gave informed consent to participate in the experiment , which was approved by the local medical ethics committee and adhered to the declaration of helsinki .
ten subjects participated in the inward saccade adaptation experiment and ten in the outward saccade adaptation experiment .
subjects were seated in a completely darkened room at 84 cm in front of a 21 in .
the screen was covered with a red filter to eliminate light reflections of the monitor and after images .
eye movements were recorded binocularly at 250 hz by means of video - oculography ( sr research eyelink ii , ontario , canada ) .
head movements were restrained by a chin rest and monitored throughout the measurements to ensure head stability .
the inward and outward adaptation experiments were created using experiment builder ( sr research , ontario , canada ) . in both experiments ,
the subject was instructed to look at a red dot ( 0.5 degrees of visual angle ) displayed on a black background . at the beginning of the trial ,
the dot was shown at 10 degrees to the left of the center of the screen ( fixation position ) .
after a random delay between 1.5 s and 2 s , the fixation point was switched off and the dot appeared at a position on the right of the center ( target position ) , evoking a visually guided saccade . in the inward adaptation experiment ,
this target position was 10 degrees to the right of the center and in the outward adaptation experiment the target position was 5 degrees to the right of the center .
in other words , in the inward adaptation experiment the target jump was 20 degrees and in the outward adaptation experiment it was 15 degrees .
both experiments consisted of three phases with 250 trials in total ( figures 1(a ) and 1(b)):50 baseline trials , where the dot remained on the rightward position for 1.5 seconds until the end of the trial;150 adaptation trials , in which the initial target position was the same ; at saccade detection , however , the target jumped toward the fixation point in the inward adaptation experiment ( i.e. , backward target jump ) and away from it ( i.e. , forward target jump ) in the outward adaptation experiment during the saccade towards it ; the size of the intrasaccadic step was 5 in both experiments ; the saccade was detected online using a velocity threshold of 50/sec and a boundary threshold of 7.5 to the right of the fixation position , to ensure that saccades were in the right direction ; if no proper saccade was detected , the screen was blanked for 500 ms and the trial was presented again;50 postadaptation
trials , identical to baseline trials . 50 baseline trials , where the dot remained on the rightward position for 1.5 seconds until the end of the trial ; 150 adaptation trials , in which the initial target position was the same ;
at saccade detection , however , the target jumped toward the fixation point in the inward adaptation experiment ( i.e. , backward target jump ) and away from it ( i.e. , forward target jump ) in the outward adaptation experiment during the saccade towards it ; the size of the intrasaccadic step was 5 in both experiments ; the saccade was detected online using a velocity threshold of 50/sec and a boundary threshold of 7.5 to the right of the fixation position , to ensure that saccades were in the right direction ; if no proper saccade was detected , the screen was blanked for 500 ms and the trial was presented again ; 50 postadaptation trials , identical to baseline trials .
anodal tdcs was delivered to the cerebellum through a constant current stimulator ( neuroconn , ilmenau , germany ) through two annular sintered ag / agcl 12 mm diameter electrodes ( medcat , erica , netherlands ) with highly conductive gel ( signa gel , parker laboratories , new jersey , usa ) .
the anodal electrode was placed over the right cerebellum 3 cm to the right of the inion and the reference electrode ( cathode ) was placed over left buccinator muscle .
the total current density was 1.3 ma / cm , ramped up in 30 s to a constant 1.5 ma .
stimulation commenced 3 min before an experiment started and lasted for 15 minutes ( i.e. , during all baseline trials and adaptation trials ) .
a subject participated twice in an experiment , once in a sham tdcs condition and once in an anodal cerebellar tdcs condition .
the order of the tdcs conditions was pseudorandomized and counterbalanced across subjects , with three to seven days between recordings . in the anodal cerebellar condition ,
real stimulation was applied , while , in the sham condition , the current was turned off after 30 s .
subjects and experimenter were blind to the tdcs condition ( double - blind design ) . at the end of each paradigm
, subjects were asked to report perceived pain and fatigue using a verbal analog scale ( 0no fatigue / pain to 5maximal fatigue / pain ) , as well as the presence of headache , balance , nausea , and discomfort .
recordings in subjects who participated in both the inward and outward adaptation experiments were separated by at least seven days to avoid carry - over effects . for each trial , the primary saccade from the left ( fixation ) to the right ( target ) was analyzed .
saccades were marked automatically using a velocity threshold of 50/s and a duration threshold of 20 ms .
trials were excluded if ( 1 ) there was no fixation inside a 1.7 window around the fixation point or ( 2 ) there was no saccadic movement from left to right .
the amplitudes of the primary saccades were transformed into gain values , with gain being defined as the ratio between saccade amplitude and the distance between fixation and target position .
a gain of 1 indicates a saccadic amplitude of 20 in the inward and 15 in the outward paradigm .
median , mean , and sd of the gains were calculated for individual subjects and pooled by paradigm and condition .
saccades that fell outside 1.96 sd from the mean of a subject were excluded separately for every phase . from the inward adaptation experiment 4.76% of trials were excluded and 3.56% from the outward adaptation experiment .
baseline gain was defined as the median gain in all baseline trials , adaptation gain as the median gain of the last 10 saccades made in the adaptation phase , and postadaptation gain as the median gain of the last 10 saccades in the postadaptation phase .
adaptation gain - change was calculated as the difference between adaptation gain and baseline gain .
retention was calculated as the difference between the postadaptation gain and baseline gain , giving a measure of how much learning was retained after the adaptation phase .
statistical analyses were performed using a custom script written in matlab ( mathworks , natick , ma , usa ) , and spss ( v. 20.0 , ibm corp . , armonk , ny , usa ) .
we assessed the presence of adaptation for each subject by testing the difference between baseline and adaptation gain with a student 's t - test .
for both inward and outward experiments , gains and saccade kinematics ( duration and peak velocity ) were analyzed using repeated measures ' manova with two intrasubject factors : tdcs condition ( two levels : sham versus cerebellar tdcs ) and phase ( three levels : baseline , adaptation , and postadaptation ) .
post hoc planned comparisons between the two stimulation conditions for each of the three phases were performed using paired t - tests on the saccadic gains .
the effects of tdcs on adaptation gain - change and on retention were assessed using paired t - tests . for each experiment ,
the difference in adaptation gain - change and the difference in retention between tdcs and sham stimulation were calculated .
these differences were statistically compared between the inward and outward experiments using a wilcoxon signed rank test using the 7 subjects that participated in both experiments .
pain and fatigue were statistically assessed using a one way anova with tdcs condition as intrasubject factor .
all participants successfully completed the experiments and showed a significant change in gain during the adaptation phase .
example data of one subject and group data are shown in figures 1(c ) and 1(d ) , respectively .
table 1 summarizes the results obtained in each phase for inward and outward adaptation for the two tdcs conditions .
pain and fatigue scores were not different between the tdcs or sham conditions ( p > 0.5 ) .
a manova on the gains for the inward adaptation experiment with sham and cerebellar tdcs and phase as factors revealed an effect of tdcs condition ( f(1,9 ) = 6.755 , p = 0.02 , = 0.429 ) and phase ( f(2,8 ) = 49.801 , p 0.0001 , = 0.926 ) as well as the interaction between tdcs condition and phase ( f(2,8 ) = 6.439 , p = 0.02 , = 0.617 ; table 1 ) .
saccades during baseline trials tended to be slightly hypometric for both the sham and tdcs conditions ( table 1 ) , which is normal for saccades above 10 degrees [ 37 , 38 ] .
the adaptation phase showed a gradual decrease in gain throughout the trials in the two tdcs conditions , in which smaller gains are present for the tdcs condition ( table 1 ) . in the postadaptation phase
, we found that subjects in both groups did not present full recovery to baseline gains ( table 1 ) .
planned comparisons between the two conditions ( sham and tdcs ) showed no significant differences between the two stimulation conditions in baseline gains ( 0.95 0.01 versus 0.96 0.02 , t(9 ) = 0.88 , p = 0.39 , figure 1(e ) ) .
the gain at the end of the adaptation phase was significantly smaller under cerebellar tdcs compared to sham stimulation ( sham 0.83 0.04 , tdcs 0.81 0.03 , t(9 ) = 2.71 , p = 0.02 , figure 1(e ) ) .
postadaptation phase did not exhibit differences between the two conditions ( sham 0.92 0.03 , tdcs 0.90 0.03 , t(9 ) = 1.75 , p = 0.11 , figure 1(e ) ) . the optimal adaptation gain - change ( difference between baseline and the last 10 adaptation trials ) is of 25% ( gain of 1 to 0.75 ) .
the observed adaptation gain - change was lower in the cerebellar tdcs condition than in the sham condition ( 0.12 0.04 versus 0.15 0.03 , t(9 ) = 2.26 , p = 0.04 , figure 2 ) .
difference in retention , which reveals learning residual between the two conditions , was just not significant ( t(9 ) = 2.09 , p = 0.06 , figure 2 ) .
repeated measures ' manova analyses revealed an effect of phase on peak velocities ( baseline : 518 21 deg / s , adaptation : 452 22 deg / s , and postadaptation : 499 29 deg / s , f(2,8 ) = 17.45 , p = 0.001 , = 0.814 ) , but the effects of tdcs condition ( f(1,9 ) = 1.00 , p = 0.34 , = 0.101 ) or the interaction between tdcs condition and phase were not significant ( f(2,8 ) = 1.24 , p = 0.33 , = 0.23 ; figure 3(a ) ) .
no significant effects were found for saccade durations ( figure 3(b ) ) . here , participants were subjected to an outward intrasaccadic jump of the target in the adaptation phase . as in inward adaptation
, subjects received anodal stimulation during baseline and adaptation phases ( figures 1(a ) and 1(b ) ) .
figure 1(d ) shows an example subject during the outward adaptation experiment in the two conditions .
the resulting data from all subjects was approached in the same way as the previous experiment .
the manova analyses presented a main effect of phase on saccadic gains ( f(2,8 ) = 51.10 , p 0.0001 , = 0.927 ) and on the tdcs condition ( f(1,9 ) = 8.36 , p = 0.01 , = 0.482 ) , whereas the tdcs condition and phase interaction was not significant ( f(2,8 ) = 0.658 , p = 0.544 , = 0.141 ; table 1 ) .
planned comparisons did not show any statistical difference between the two tdcs conditions . when observing group data during baseline , subjects present relatively smaller gains in sham condition compared to tdcs ( sham 0.98 0.03 , tdcs 1 0.02 , t(9 ) = 1.79 , p = 0.10 ) .
subjects also presented a normal course of adaptation throughout the trials increasing their gains ( sham 1.08 0.04 , tdcs 1.12 0.07 , t(9 ) = 1.97 , p = 0.08 ) , to thereafter decrease them in the postadaptation phase ( sham 1 0.06 , tdcs 1.05 0.07 , t(9 ) = 2.23 , p = 0.05 ) , not reaching baseline again ( figure 1(f ) ) . here
we also assessed the amount of learning ( gain - change ) of each individual by comparing the baseline and the last 10 trials of the adaptation phase .
no significant differences were found between sham and tdcs conditions ( t(9 ) = 0.79 , p = 0.44 ) or for retention ( t(9 ) = 1.21 , p = 0.25 , figure 2 ) .
kinematic differences were assessed in the same way as gains : no effects of tdcs condition or the interaction between phase and tdcs condition , except for an effect of phase on saccade durations as a result of gain increase ( baseline 58 1 ms , adaptation 67 4 ms , and postadaptation 63 1 ms , f(2,8 ) = 21.89 , p = 0.001 , = 0.84 ; figure 3 ) .
inward and outward adaptation did not differ from each other in the seven subjects that participated in both experiments with respect to adaptation gain - change ( 0.16 0.03 versus 0.11 0.07 , wilcoxon z = 1.35 , p = 0.17 ) or retention ( 0.05 0.03 versus 0.06 0.07 , wilcoxon z = 0.33 , p = 0.73 ) .
we observed that applying tdcs with a small contact electrode at 1.5 ma in an inward saccade adaptation experiment , with a 25% backward intrasaccadic step , induces a greater gain reduction when compared to sham condition .
the effect of tdcs on gain - change is just not significant for outward adaptation , probably due to the low number of subjects .
saccade adaptation is a widely used model for motor learning . when the eyes land on a location where a target was displayed , the oculomotor system detects that an error has been made and updates its motor commands to adjust its amplitude on a trial - by - trial basis .
we have explored the use of tdcs in a different type of motor learning in which previous results from other research groups have demonstrated the effect of this type of stimulation on cerebellar output .
. showed that anodal tdcs enhanced acquisition in a visuomotor transformation task by stimulation over the cerebellum and other experiments have also shown the effects of cerebellar tdcs in learning , attention , or working memory [ 28 , 29 ] .
the results of this study also show that tdcs exerts modulatory effects in behavior when applied to the cerebellum .
the affirmation for the confined effects of the stimulation over the cerebellum is demonstrated mostly by previous reports of similar configurations which did not find any effects on brainstem or visual cortex [ 27 , 41 , 42 ] and the use of modeling techniques , [ 43 , 44 ] in which the current flow has been proven uniform with good sensitivity and response by purkinje cells ( pc ) . during the postadaptation the subjects must deadapt and
any difference in this phase could indicate an effect on retention or a continuous effect of tdcs on ( de)adaptation . while we did not see any significant difference the groups here
, our sense is that this does not necessarily reflect a real lack of effect .
our sample size and the degree of noise here make strong conclusions difficult . in any case , this is not the main issue that this research sought to address .
, jayaram et al . , and zuchowski et al . who observed differences in the speed of adaptation but found no poststimulation effects in the extinction rate of the learned response in their tdcs group .
there are some possible explanations for the lack of differences between the two conditions in outward adaptation .
the mechanisms for these two types of adaptation are not completely understood and are thought to involve different neural substrates .
diverse theories explore why this could be happening , such as a natural tendency of the system to be hypometric , and this way reducing gains will develop in a faster way than increasing them .
using functional mri showed that forward and backward error target shifts elicited different cerebellar activation patterns .
also , different behavioral mechanisms might be in place for the two types of adaptation , namely , a target remapping for outward adaptation .
on saccade adaptation showed that anodal stimulation tended to slow down adaptation in both directions , while cathodal stimulation enhanced outward adaptation .
differences could arise on account of different electrode size , position , current , and time of stimulation as with other studies where apparent opposite effects might be present .
on inward adaptation , significant differences were found in peak velocities due to the gain - decrease adaptation and on outward adaptation we only observed a significant increase in the duration of the saccades as a result of adaptation .
this suggests that tdcs is exerting an effect in the stages or at a level where saccade kinematics are not coded yet .
this supports the notion that tdcs actually affects adaptation and not the saccade generation per se .
direct comparison between the two paradigms yielded no significant results on the tdcs effects . despite the fact that the effect sizes are almost similar for the two experiments , outward adaption presents larger noise in the resulting data .
we presume that this increased noise does not prevent tdcs from having an effect on performance or learning , but it may still cause that the effect of tdcs on outward adaptation failed to reach significance . the current inability to stimulate specific areas in the cerebellar cortex could also account for the apparent lack of response in outward adaptation .
another probable source is a difference in the mechanisms needed to elicit either type of adaptation . in other words
, we think a preliminary hypothesis that the effect exists in both inward and outward adaptation is a good starting point for further exploration .
total cerebellectomies abolish complete means of adaptation ; oculomotor vermis inactivation impairs adaptation without affecting the production of saccades . results from kojima et al .
inactivated the same area with total incapacity for outward adaptation and a partial effect for inward adaptation .
an mri - guided tms study on crus i had a dual effect on saccade adaptation , potentiating gain - up adaptation after effects and depressing gain - down adaptation .
we suggest that tdcs might have enhanced the cerebellar plastic mechanisms needed for a more prominent participation of the cerebellum in inward adaptation .
being able to modulate cerebellar output earns particular interest as pc change their firing pattern in response to saccade adaptation .
as observed by catz et al . while recording pc activity in primates performing an inward and outward adaptation task , they observed a change in the population burst throughout the course of adaptation .
the population signal may have a modulatory role throughout the saccade , which could in turn be modulated or broadened by applying tdcs .
consequently , pc activity may be enhanced and more sensitive to error at the individual level ; and at a regional level , tdcs might engage faster areas that are available for adaptation .
assumptions of local and regional cerebellar stimulation are further supported by modeling studies where somatic polarization together with axon terminal polarization seems to be key to the direct current response .
another possible mechanism that tdcs could be possibly influencing is by affecting short - term plasticity through brain - derived neurotrophic factor ( bdnf ) .
trkb , the receptor for bdnf , is located at the parallel fiber to pc synapse , where plasticity in the cerebellum takes place and might be regulating pc / parallel fiber mechanisms underlying short - term synaptic plasticity [ 55 , 56 ] .
tests have shown that direct current stimulation plays a critical role in long - lasting synaptic potentiation in mouse slices . at this moment
, only inferences can be made of how tdcs might be working at a cellular level and more studies are needed in this area to elucidate what the actual effects of tdcs at the pc level are . in conclusion , we showed an effect of tdcs over the cerebellum in an inward saccade adaptation task displayed by a greater gain - reduction compared to sham stimulation .
we could not demonstrate a similar effect in the outward adaptation task , although we also could not rule one out .
moreover , we contribute to the evidence that cerebellar tdcs may be used to enhance cerebellar ( oculomotor ) function .
tdcs could help lead the way to a better understanding of motor learning and how the cerebellum is contributing to each of these processes ; therefore , more studies are needed to clarify the extent and the mechanisms through which tdcs can modulate cerebellar functions . | saccade adaptation is a cerebellar - mediated type of motor learning in which the oculomotor system is exposed to repetitive errors .
different types of saccade adaptations are thought to involve distinct underlying cerebellar mechanisms .
transcranial direct current stimulation ( tdcs ) induces changes in neuronal excitability in a polarity - specific manner and offers a modulatory , noninvasive , functional insight into the learning aspects of different brain regions .
we aimed to modulate the cerebellar influence on saccade gains during adaptation using tdcs .
subjects performed an inward ( n = 10 ) or outward ( n = 10 ) saccade adaptation experiment ( 25% intrasaccadic target step ) while receiving 1.5 ma of anodal cerebellar tdcs delivered by a small contact electrode .
compared to sham stimulation , tdcs increased learning of saccadic inward adaptation but did not affect learning of outward adaptation .
this may imply that plasticity mechanisms in the cerebellum are different between inward and outward adaptation .
tdcs could have influenced specific cerebellar areas that contribute to inward but not outward adaptation .
we conclude that tdcs can be used as a neuromodulatory technique to alter cerebellar oculomotor output , arguably by engaging wider cerebellar areas and increasing the available resources for learning . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion | since mclaughlin ( 1967 ) described the
parametric adjustment , known today as short - term saccade adaptation , his paradigm has been used as a way to assess learning and plasticity in the oculomotor system . this is done by asking a subject to make a saccade to a new position and while the saccade is in flight , the target moves ( intrasaccadic step ) causing a postsaccadic visual error [ 35 ] . when the subject is repeatedly exposed to the same error
, the oculomotor system will gradually drive a change in the metrics of the eye movement over time , making the error smaller [ 615 ] . the error can induce saccade shortening ( gain - down ) , when the intrasaccade step of the target is in the direction of the starting point of the saccade ( inward adaptation ) , or saccade lengthening ( gain - up ) when the step is away from the starting point ( outward adaptation ) . human subjects adapt faster in response to inward adaptation than in response to outward adaptation stimuli , which poses the hypothesis that these two types of adaptation involve different neural mechanisms [ 16 , 17 ] . the cerebellum plays a crucial role in saccadic error detection [ 1820 ] and thus in saccade adaptation . evidence of the cerebellar involvement and its necessary integrity to oculomotor learning has been demonstrated as large lesions , focal inactivation , or pathological conditions of different areas of the cerebellum impair the ability to adapt saccades [ 2125 ] . in addition , various loci in the cerebellum relate to inward and outward errors differently . for instance , patients with vermal damage are partially capable of inward adaptation but lack outward adaptation . also , mri - guided tms on lateral hemispheres potentiates the postadaptation effects of outward adaptation and , in contrast , depresses gain - down adaptation . cerebellar output can be modulated with transcranial direct current stimulation ( tdcs ) with great specificity as shown by excitability changes after stimulation ranging from cognitive to motor skills [ 2730 ] . in this study
, we used anodal tdcs as a tool to noninvasively modulate cerebellar output and provide functional insight into the learning aspects during saccade adaptation . they all gave informed consent to participate in the experiment , which was approved by the local medical ethics committee and adhered to the declaration of helsinki . ten subjects participated in the inward saccade adaptation experiment and ten in the outward saccade adaptation experiment . subjects were seated in a completely darkened room at 84 cm in front of a 21 in . head movements were restrained by a chin rest and monitored throughout the measurements to ensure head stability . the inward and outward adaptation experiments were created using experiment builder ( sr research , ontario , canada ) . in both experiments ,
the subject was instructed to look at a red dot ( 0.5 degrees of visual angle ) displayed on a black background . after a random delay between 1.5 s and 2 s , the fixation point was switched off and the dot appeared at a position on the right of the center ( target position ) , evoking a visually guided saccade . in the inward adaptation experiment ,
this target position was 10 degrees to the right of the center and in the outward adaptation experiment the target position was 5 degrees to the right of the center . in other words , in the inward adaptation experiment the target jump was 20 degrees and in the outward adaptation experiment it was 15 degrees . both experiments consisted of three phases with 250 trials in total ( figures 1(a ) and 1(b)):50 baseline trials , where the dot remained on the rightward position for 1.5 seconds until the end of the trial;150 adaptation trials , in which the initial target position was the same ; at saccade detection , however , the target jumped toward the fixation point in the inward adaptation experiment ( i.e. , forward target jump ) in the outward adaptation experiment during the saccade towards it ; the size of the intrasaccadic step was 5 in both experiments ; the saccade was detected online using a velocity threshold of 50/sec and a boundary threshold of 7.5 to the right of the fixation position , to ensure that saccades were in the right direction ; if no proper saccade was detected , the screen was blanked for 500 ms and the trial was presented again;50 postadaptation
trials , identical to baseline trials . 50 baseline trials , where the dot remained on the rightward position for 1.5 seconds until the end of the trial ; 150 adaptation trials , in which the initial target position was the same ;
at saccade detection , however , the target jumped toward the fixation point in the inward adaptation experiment ( i.e. , forward target jump ) in the outward adaptation experiment during the saccade towards it ; the size of the intrasaccadic step was 5 in both experiments ; the saccade was detected online using a velocity threshold of 50/sec and a boundary threshold of 7.5 to the right of the fixation position , to ensure that saccades were in the right direction ; if no proper saccade was detected , the screen was blanked for 500 ms and the trial was presented again ; 50 postadaptation trials , identical to baseline trials . anodal tdcs was delivered to the cerebellum through a constant current stimulator ( neuroconn , ilmenau , germany ) through two annular sintered ag / agcl 12 mm diameter electrodes ( medcat , erica , netherlands ) with highly conductive gel ( signa gel , parker laboratories , new jersey , usa ) . the anodal electrode was placed over the right cerebellum 3 cm to the right of the inion and the reference electrode ( cathode ) was placed over left buccinator muscle . the total current density was 1.3 ma / cm , ramped up in 30 s to a constant 1.5 ma . stimulation commenced 3 min before an experiment started and lasted for 15 minutes ( i.e. a subject participated twice in an experiment , once in a sham tdcs condition and once in an anodal cerebellar tdcs condition . in the anodal cerebellar condition ,
real stimulation was applied , while , in the sham condition , the current was turned off after 30 s . recordings in subjects who participated in both the inward and outward adaptation experiments were separated by at least seven days to avoid carry - over effects . a gain of 1 indicates a saccadic amplitude of 20 in the inward and 15 in the outward paradigm . from the inward adaptation experiment 4.76% of trials were excluded and 3.56% from the outward adaptation experiment . baseline gain was defined as the median gain in all baseline trials , adaptation gain as the median gain of the last 10 saccades made in the adaptation phase , and postadaptation gain as the median gain of the last 10 saccades in the postadaptation phase . we assessed the presence of adaptation for each subject by testing the difference between baseline and adaptation gain with a student 's t - test . for both inward and outward experiments , gains and saccade kinematics ( duration and peak velocity ) were analyzed using repeated measures ' manova with two intrasubject factors : tdcs condition ( two levels : sham versus cerebellar tdcs ) and phase ( three levels : baseline , adaptation , and postadaptation ) . for each experiment ,
the difference in adaptation gain - change and the difference in retention between tdcs and sham stimulation were calculated . these differences were statistically compared between the inward and outward experiments using a wilcoxon signed rank test using the 7 subjects that participated in both experiments . pain and fatigue were statistically assessed using a one way anova with tdcs condition as intrasubject factor . all participants successfully completed the experiments and showed a significant change in gain during the adaptation phase . table 1 summarizes the results obtained in each phase for inward and outward adaptation for the two tdcs conditions . pain and fatigue scores were not different between the tdcs or sham conditions ( p > 0.5 ) . a manova on the gains for the inward adaptation experiment with sham and cerebellar tdcs and phase as factors revealed an effect of tdcs condition ( f(1,9 ) = 6.755 , p = 0.02 , = 0.429 ) and phase ( f(2,8 ) = 49.801 , p 0.0001 , = 0.926 ) as well as the interaction between tdcs condition and phase ( f(2,8 ) = 6.439 , p = 0.02 , = 0.617 ; table 1 ) . the adaptation phase showed a gradual decrease in gain throughout the trials in the two tdcs conditions , in which smaller gains are present for the tdcs condition ( table 1 ) . in the postadaptation phase
, we found that subjects in both groups did not present full recovery to baseline gains ( table 1 ) . planned comparisons between the two conditions ( sham and tdcs ) showed no significant differences between the two stimulation conditions in baseline gains ( 0.95 0.01 versus 0.96 0.02 , t(9 ) = 0.88 , p = 0.39 , figure 1(e ) ) . the gain at the end of the adaptation phase was significantly smaller under cerebellar tdcs compared to sham stimulation ( sham 0.83 0.04 , tdcs 0.81 0.03 , t(9 ) = 2.71 , p = 0.02 , figure 1(e ) ) . postadaptation phase did not exhibit differences between the two conditions ( sham 0.92 0.03 , tdcs 0.90 0.03 , t(9 ) = 1.75 , p = 0.11 , figure 1(e ) ) . the observed adaptation gain - change was lower in the cerebellar tdcs condition than in the sham condition ( 0.12 0.04 versus 0.15 0.03 , t(9 ) = 2.26 , p = 0.04 , figure 2 ) . repeated measures ' manova analyses revealed an effect of phase on peak velocities ( baseline : 518 21 deg / s , adaptation : 452 22 deg / s , and postadaptation : 499 29 deg / s , f(2,8 ) = 17.45 , p = 0.001 , = 0.814 ) , but the effects of tdcs condition ( f(1,9 ) = 1.00 , p = 0.34 , = 0.101 ) or the interaction between tdcs condition and phase were not significant ( f(2,8 ) = 1.24 , p = 0.33 , = 0.23 ; figure 3(a ) ) . here , participants were subjected to an outward intrasaccadic jump of the target in the adaptation phase . as in inward adaptation
, subjects received anodal stimulation during baseline and adaptation phases ( figures 1(a ) and 1(b ) ) . figure 1(d ) shows an example subject during the outward adaptation experiment in the two conditions . the resulting data from all subjects was approached in the same way as the previous experiment . planned comparisons did not show any statistical difference between the two tdcs conditions . when observing group data during baseline , subjects present relatively smaller gains in sham condition compared to tdcs ( sham 0.98 0.03 , tdcs 1 0.02 , t(9 ) = 1.79 , p = 0.10 ) . subjects also presented a normal course of adaptation throughout the trials increasing their gains ( sham 1.08 0.04 , tdcs 1.12 0.07 , t(9 ) = 1.97 , p = 0.08 ) , to thereafter decrease them in the postadaptation phase ( sham 1 0.06 , tdcs 1.05 0.07 , t(9 ) = 2.23 , p = 0.05 ) , not reaching baseline again ( figure 1(f ) ) . no significant differences were found between sham and tdcs conditions ( t(9 ) = 0.79 , p = 0.44 ) or for retention ( t(9 ) = 1.21 , p = 0.25 , figure 2 ) . kinematic differences were assessed in the same way as gains : no effects of tdcs condition or the interaction between phase and tdcs condition , except for an effect of phase on saccade durations as a result of gain increase ( baseline 58 1 ms , adaptation 67 4 ms , and postadaptation 63 1 ms , f(2,8 ) = 21.89 , p = 0.001 , = 0.84 ; figure 3 ) . inward and outward adaptation did not differ from each other in the seven subjects that participated in both experiments with respect to adaptation gain - change ( 0.16 0.03 versus 0.11 0.07 , wilcoxon z = 1.35 , p = 0.17 ) or retention ( 0.05 0.03 versus 0.06 0.07 , wilcoxon z = 0.33 , p = 0.73 ) . we observed that applying tdcs with a small contact electrode at 1.5 ma in an inward saccade adaptation experiment , with a 25% backward intrasaccadic step , induces a greater gain reduction when compared to sham condition . the effect of tdcs on gain - change is just not significant for outward adaptation , probably due to the low number of subjects . saccade adaptation is a widely used model for motor learning . when the eyes land on a location where a target was displayed , the oculomotor system detects that an error has been made and updates its motor commands to adjust its amplitude on a trial - by - trial basis . we have explored the use of tdcs in a different type of motor learning in which previous results from other research groups have demonstrated the effect of this type of stimulation on cerebellar output . showed that anodal tdcs enhanced acquisition in a visuomotor transformation task by stimulation over the cerebellum and other experiments have also shown the effects of cerebellar tdcs in learning , attention , or working memory [ 28 , 29 ] . the results of this study also show that tdcs exerts modulatory effects in behavior when applied to the cerebellum . the affirmation for the confined effects of the stimulation over the cerebellum is demonstrated mostly by previous reports of similar configurations which did not find any effects on brainstem or visual cortex [ 27 , 41 , 42 ] and the use of modeling techniques , [ 43 , 44 ] in which the current flow has been proven uniform with good sensitivity and response by purkinje cells ( pc ) . while we did not see any significant difference the groups here
, our sense is that this does not necessarily reflect a real lack of effect . who observed differences in the speed of adaptation but found no poststimulation effects in the extinction rate of the learned response in their tdcs group . there are some possible explanations for the lack of differences between the two conditions in outward adaptation . the mechanisms for these two types of adaptation are not completely understood and are thought to involve different neural substrates . diverse theories explore why this could be happening , such as a natural tendency of the system to be hypometric , and this way reducing gains will develop in a faster way than increasing them . also , different behavioral mechanisms might be in place for the two types of adaptation , namely , a target remapping for outward adaptation . on saccade adaptation showed that anodal stimulation tended to slow down adaptation in both directions , while cathodal stimulation enhanced outward adaptation . differences could arise on account of different electrode size , position , current , and time of stimulation as with other studies where apparent opposite effects might be present . on inward adaptation , significant differences were found in peak velocities due to the gain - decrease adaptation and on outward adaptation we only observed a significant increase in the duration of the saccades as a result of adaptation . this suggests that tdcs is exerting an effect in the stages or at a level where saccade kinematics are not coded yet . this supports the notion that tdcs actually affects adaptation and not the saccade generation per se . despite the fact that the effect sizes are almost similar for the two experiments , outward adaption presents larger noise in the resulting data . we presume that this increased noise does not prevent tdcs from having an effect on performance or learning , but it may still cause that the effect of tdcs on outward adaptation failed to reach significance . the current inability to stimulate specific areas in the cerebellar cortex could also account for the apparent lack of response in outward adaptation . another probable source is a difference in the mechanisms needed to elicit either type of adaptation . in other words
, we think a preliminary hypothesis that the effect exists in both inward and outward adaptation is a good starting point for further exploration . inactivated the same area with total incapacity for outward adaptation and a partial effect for inward adaptation . an mri - guided tms study on crus i had a dual effect on saccade adaptation , potentiating gain - up adaptation after effects and depressing gain - down adaptation . we suggest that tdcs might have enhanced the cerebellar plastic mechanisms needed for a more prominent participation of the cerebellum in inward adaptation . being able to modulate cerebellar output earns particular interest as pc change their firing pattern in response to saccade adaptation . while recording pc activity in primates performing an inward and outward adaptation task , they observed a change in the population burst throughout the course of adaptation . the population signal may have a modulatory role throughout the saccade , which could in turn be modulated or broadened by applying tdcs . consequently , pc activity may be enhanced and more sensitive to error at the individual level ; and at a regional level , tdcs might engage faster areas that are available for adaptation . assumptions of local and regional cerebellar stimulation are further supported by modeling studies where somatic polarization together with axon terminal polarization seems to be key to the direct current response . another possible mechanism that tdcs could be possibly influencing is by affecting short - term plasticity through brain - derived neurotrophic factor ( bdnf ) . trkb , the receptor for bdnf , is located at the parallel fiber to pc synapse , where plasticity in the cerebellum takes place and might be regulating pc / parallel fiber mechanisms underlying short - term synaptic plasticity [ 55 , 56 ] . tests have shown that direct current stimulation plays a critical role in long - lasting synaptic potentiation in mouse slices . at this moment
, only inferences can be made of how tdcs might be working at a cellular level and more studies are needed in this area to elucidate what the actual effects of tdcs at the pc level are . in conclusion , we showed an effect of tdcs over the cerebellum in an inward saccade adaptation task displayed by a greater gain - reduction compared to sham stimulation . we could not demonstrate a similar effect in the outward adaptation task , although we also could not rule one out . moreover , we contribute to the evidence that cerebellar tdcs may be used to enhance cerebellar ( oculomotor ) function . tdcs could help lead the way to a better understanding of motor learning and how the cerebellum is contributing to each of these processes ; therefore , more studies are needed to clarify the extent and the mechanisms through which tdcs can modulate cerebellar functions . | [
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patients with absence epilepsy or other idiopathic generalized epilepsy ( ige ) syndromes manifest 2.5 hz to 3.5 hz ( 3 hz on average ) bisynchronous typical spike - and - wave activity ( tswa ) in the interictal electroencephalogram ( eeg ) and during typical absence seizures.1
nonconvulsive status epilepticus ( ncse ) consists of sustained rhythmic / periodic epileptiforma activity in the eeg , along with alterations in mental status and/or behavior , in the absence of convulsive movements .
there are other definitions of ncse , but no definition ( including ours ) is universally accepted .
a wide range of cognitive - behavioral changes occurs in ncse , including mild inattention , confusion , stupor , and coma .
an even wider range of eeg patterns is seen in ncse , but many of these patterns are not conclusively epileptiform.2 ncse is traditionally divided into complex partial status epilepticus ( cpse)3 and absence status epilepticus ( ase).4 diagnosing ncse can be difficult in the presence of coma.5,6 first , the eeg in coma may show a typical cpse / ase pattern even though the clinical course and response to treatment are not compatible with cpse / ase .
second , the eeg in coma may show epileptiform abnormalities that defy classification into cpse or ase .
third , the eeg in coma may show abnormalities that can not be easily labeled as epileptiform .
ige - related ncse usually takes the form of absence status epilepticus.4 some adults with no history of seizure / epilepsy develop ase
de novo in the context of an acute brain disorder.4 the acute brain disorder in de novo ase is often an encephalopathy from drug overdose / withdrawal , fluid / electrolyte imbalance , or systemic infections.7 however , de novo ase is not routinely reported in hypoxic - ischemic encephalopathyb ; we are aware of only one previous case of de novo ase with tswa in hypoxic - ischemic brain injury.8 we present an unusual case of hypoxic - ischemic brain injury in which the patient s eeg showed benzodiazepine - resistant tswa even though the patient had no past or family history of seizure or epilepsy . after presenting the case ,
we examine the pathophysiology of post - hypoxic tswac and its implications for classifying and managing ncse .
the patient , a 65-year - old man with congestive heart failure ( left ventricular ejection fraction of 20% ) suffered cardiorespiratory arrest resulting in hypoxic - ischemic brain injury .
the history is also significant for hypertension , kidney disease , hepatitis c , and substance abuse with cocaine and marijuana .
home medications include digoxin , furosemide , spironolactone , carvedilol , lisinopril , aspirin , potassium chloride , and folic acid .
the patient was found unconscious , apneic , asystolic , and pulseless on a street in his neighborhood .
emergency responders arrived within 15 minutes and performed cardiopulmonary resuscitation ( cpr ) , including defibrillation , intubation , and artificial ventilation .
the patient arrived in the emergency room with a glascow coma scale of 3 ; dopamine was started .
on admission to the intensive care unit ( icu ) , the patient was in coma with intact brainstem function and stable vital signs ( rr = 14 , hr = 73 , bp = 100/70 , t = 36 c ) .
brain ct scan showed mild diffuse atrophy , chronic microvascular changes , and no acute lesions .
blood tests showed normal white cell count , low hemoglobin ( 11.5 g / dl ) , elevated serum glucose ( 255 mg / dl ) , normal blood urea , nitrogen and creatinine , and normal serum sodium , potassium , and calcium .
ventricular fibrillation occurred 21 hours after the first hypoxic - ischemic event ; cpr was performed for 15 minutes .
post - resuscitation , the patient manifested eyelid myoclonus occurring spontaneously every 3 to 12 seconds without any clear relationship to external stimulation ; this is most likely a form of acute posthypoxic myoclonus .
intravenous lorazepam was given : 7-mg over 7 hours ( first 4-mg , then 2-mg , then 1-mg ) resulting in cessation of eyelid myoclonus .
more lorazepam ( 10-mg ) was infused over the next 7 hours ( 0.01 mg / kg / hour over 4 hours , then 0.02 mg / kg / hour over 3 hours ) .
serum phenytoin was 18.6 g / ml 10 hours after the loading dose and 18.0 g / ml 38 hours later even though fosphenytoin was discontinued before the patient received any maintenance dose .
eeg monitoringd was initiated 23 hours after administering fosphenytoin and discontinuing lorazepam ; at that time the patient was in deep coma with no involuntary movements .
the eeg showed sustained typical spike - and - wave activity ( tswa ) confirming ncse and indicating typical absence status epilepticus ( fig .
intravenous midazolam was started : 0.2 mg / kg bolus and 0.05 mg / kg / hour infusion .
tswa persisted so another 0.2 mg / kg bolus was given 2.5 hours after the first bolus .
the eeg continued to show tswa , albeit with somewhat lower discharge frequency ( fig .
2 ) . the infusion rate was increased and at 0.5 mg / kg / hour the eeg showed intermittent suppression of spike - and - wave discharges ( fig .
3 ) . the midazolam infusion rate was further increased to achieve eeg burst - suppression . at a rate of 1.2 mg / kg / hour , tswa disappeared and
stable burst - suppression ( 612 bursts / minute ) was achieved with an infusion rate of 1.4 to 1.6 mg / kg / hour .
the patient s family requested withdrawal of life support 72 hours after admission and the patient died the following day .
eeg showed typical spike - and - wave activity ( tswa ) even though he had no history of seizure or epilepsy .
non - convulsive status epilepticus ( ncse ) was diagnosed and intravenous lorazepam and midazolam was administered .
the tswa proved to be relatively resistant to benzodiazepines , raising some important issues regarding the pathophysiology of post - hypoxic tswa and providing some insight into how ncse should be classified and managed .
typical spike - and - wave activity ( tswa ) is the eeg hallmark of absence seizure .
it is also a routine interictal eeg finding in absence epilepsy and other ige syndromes.1 for twsa to occur , the thalamocortical - corticothalamic network must be functionally intact and in a state where intrinsically bursting cortical pyramidal and thalamic neurons are mildly to moderately hyperpolarized and susceptible to producing high - frequency bursts of action potentials.9 meeren and colleagues published a review of the theories on the pathophysiology of tswa.9 this can be summarized as follows : ( 1 ) the centrencephalic theory posits a diffusely projecting pacemaker in the midline thalamus as the origin of tswa . ( 2 )
the thalamic clock theory is a refinement of the centrencephalic theory ; it localizes the abnormal pacemaker to the reticular thalamic nucleus .
( 3 ) the corticoreticular theory claims that tswa is generated by the abnormal interaction of the cortex and the reticular system of the thalamus and brainstem . ( 4 ) the cortical theory considers cortical hyperexcitability as the primary defect ; a hyperexcitable cortex transforms the normal rhythms of the thalamus into tswa .
( 5 ) the cortical focus theory is a synthesis of the cortical and corticoreticular theories ; it asserts that a cortical focus ( the perioral region of the somatosensory cortex is a prime suspect ) initiates the epileptic activity , spreads to other cortical areas , and entrains the thalamus producing a resonant circuitry for expressing tswa .
to these five theories , we add blumenfeld s comprehensive theory which argues that tswa can be provoked by different insults acting on different excitatory / inhibitory circuits.10 it is likely that absence status epilepticus ( ase ) shares the basic mechanisms of absence seizure . whether ictogenesis results in a self - limited absence seizure or in ase depends on the brain s seizure terminating mechanisms ;
such mechanisms are likely impaired in ase.11 patients with no history of ige can develop ase
de novo in the context of an acute brain disorder.4 pro and colleagues reported a patient with late - onset de novo ase whose eeg consistently showed interictal tswa over a 14-year period.12 their case indicates that , in some individuals with latent ige , exposure of the brain to toxic or metabolic factors can trigger a seizure later in life .
the fact that post - hypoxic tswa can look exactly like ige - related tswa implies that different insults acting on different brain circuits can turn on the basic mechanisms that lead to tswa ( see blumenfeld s theory above).10 this case also implies that extensive hypoxic - ischemic brain injury can spare the thalamocortical - corticothalamic network responsible for generating tswa / absence seizure.9 as stated above , a genetic predisposition to develop tswa / ase may be the reason our patient manifested tswa.12 it is also possible that all individuals have an inherent ability to produce tswa and that , in our patient , the pattern of cerebral hypoxic - ischemic injury has released
such ability.10 the patient s eye - lid movements occurred repeatedly at irregular intervals within a 7-hour period and stopped after a total of 7-mg of lorazepam was given .
these movements are consistent with post - hypoxic myoclonus and not with the more periodic eye - blink automatism seen in absence seizure .
it should also be emphasized that eyelid movements were not present when tswa was detected and that tswa is not a known eeg correlate of post - hypoxic myoclonic status epilepticus ; hence myoclonic status epilepticus would not be an appropriate diagnosis for our patient .
the patient has a history of substance abuse and he tested positive for cocaine and marijuana .
however , most drug - related de novo ase cases in the literature were linked to withdrawal of benzodiazepine ( bdz ) or to intoxication with alcohol or psychotropic drugs.4,7 alcohol was not detected in the patient s blood and we are not aware of any reports in which cocaine / marijuana was linked to de novo ase . still , this explanation can not be ruled out .
the patient received a 1440-mg loading dose of fosphenytoin ( a phenytoin prodrug ) 23 hours prior to the onset of eeg monitoring . in the setting of ige , phenytoin ( and other antiepileptic drugs )
can aggravate seizures13 and can even result in refractory ase.14 however , we are not aware of any report of phenytoin / fosphenytoin causing ase in patients with no ige .
the patient also received 17-mg of lorazepam ( a bdz and gaba - a receptor agonist ) 23 to 37 hours before the onset of eeg recording .
however , the absence of a history of chronic bdz use makes it unlikely that the patient went through a bdz withdrawal state .
enhanced gaba - a inhibition in the thalamus can trigger absence seizures in genetic and pharmacological models of absence epilepsy.15 however , we are not aware of any report of lorazepam causing ase in patients with no ige
. what is the significance of benzodiazepine - resistance in tswa / ase ? as a rule , ige - related ase responds rapidly and completely to bdz therapy.4,11 this is in line with the ability of gaba - a receptors to mediate the inhibition of the thalamic
pacemaker of tswa.15 a few cases of refractory ase were described in the literature but all of these patients had ige and were receiving phenytoin or carbamazepine.13 the rarity of post - hypoxic tswa makes it much more difficult to find any report of bdz - resistant post - hypoxic tswa .
we initially hypothesized that resistance indicates a difference in the mechanisms of post - hypoxic tswa and ige - related tswa.16 this is not necessarily the case and the basic mechanism of tswa genesis could still be the same , only that cerebral hypoxia - ischemia resulted in impairment of neural circuits where bdz act to suppress tswa .
for example , impairment of gaba inhibitory circuits in the cerebral cortex can increase the chance of cortical ictogenesis , thalamic entrainment , and tswa generation ( see cortical focus theory).9 in classifying seizures , the international league against epilepsy ( ilae ) made no formal reference to non - convulsive status epilepticus ( ncse).1719 the 1981 ilae seizure classification contains three diagnostic entities that qualify as ncse aura continua , complex partial status epilepticus , and absence status epilepticus ( ase).17 subtle status epilepticus was added in the 2006 report of the ilae classification core group bringing the number of ncse seizures to four.18 in our patient , the finding of tswa indicated ase ( more specifically typical ase ) but treatment response ( bdz - resistance ) and clinical outcome ( the patient died ) are diametrically opposite to that of ase ( especially typical ase ) .
such an electroclinical paradox is not uncommon in critically ill patients with ncse .
unfortunately , the 1981 and 2006 seizure classifications ignore this issue by omitting the many variations of ncse , especially ncse in coma.5,6 the 2010 ilae seizure classification also fails to address this issue.19 what s more , the ilae has completely excluded status epilepticus and focal seizures from the 2010 classification.20 the fact that ncse is more common than previously thought underscores the urgency to create a better classification system for status epilepticus and ncse .
recent studies have shown that ncse constitutes 25%50% of all cases of status epilepticus and the incidence of ncse in the critically ill can be as high as 50%.6 excluding some ncse subtypes from the seizure classification system on philosophical grounds is disadvantageous to the patient , the physician , and the researcher.19 while a seizure classification system can not be perfect at the outset , it should be heuristic and include all phenomena considered as ncse , including the ncse variations in comatose patients .
only a heuristic seizure classification system will result in better understanding and rational treatment of ncse . according to bauer and trinka , the confusion vis - - vis ncse in coma
can be overcome by having a separate ncse categorycomatose ncse or coma - ncse.5 the authors classified cpse and ase as ncse proper .
ncse proper has a good prognosis and , in most cases , the treatment of choice is turning off the seizure without aggressively anesthetizing the patient .
coma - ncse is more ominous and its prognosis depends almost entirely on the underlying brain disorder.5 while there is no firm evidence that suppressing the seizure improves prognosis in coma - ncse , anesthetizing the patient with midazolam or propofol may not be unreasonable if the patient is already on mechanical ventilation and is being monitored in the icu .
maganti and colleagues proposed an etiology - based classification for ncse , including ncse in coma , ncse in acute cerebral lesions , ncse in metabolic disorders , and ncse in those with pre - existing epilepsy.6 this more detailed approach may lend itself to better treatment recommendation , but most importantly better evaluation and testing of treatment recommendations to determine their value based on outcome .
we agree with bauer and trinka that it is important to differentiate coma - ncse from ncse proper5 and with maganti and colleagues that it is critical to account for etiology in classifying ncse.6 by combining the bauer / trinka and maganti proposals and adding some minor refinements , we were able to create a practical decision tree for ncse ( fig .
4 ) . as shown in this decision tree , as soon as ncse is diagnosed ( based on electroclinical data ) , the clinician should separate
ncse with significant irreversible brain injury ( sibi ) and ncse with no evidence of sibi .
most coma ncse with sibi is due to hypoxic - ischemic , traumatic , ischemic , infectious , or inflammatory insults to the brain .
most coma ncse with no sibi is due to epileptic , toxic , or metabolic factors , often with the confounding effects of anesthetics or sedative drugs that are regularly employed in the icu .
most non - coma ncse is due to purely epileptic , toxic , or metabolic factors , the majority of which are reversible .
occasionally , sibi of lesser severity may give rise to non - coma ncse .
such a heuristic approach can facilitate our understanding of ncse , especially ncse in coma . while the incidence depends on how one defines ncse , even more conservative estimates of the incidence of ncse ( eg , 25% )
warrant a detailed analysis of ncse cases with a goal toward increased understanding and better classification of ncse .
further work is needed to better define ncse , to determine which eeg patterns indicate ncse , and to establish treatment paradigms for the different types of ncse .
a more collaborative approach across hospitals / universities is warranted given the sample size that would be needed to determine treatment efficacy in so many ncse categories .
typical spike - and - wave activity ( tswa ) is the eeg hallmark of absence seizure .
it is also a routine interictal eeg finding in absence epilepsy and other ige syndromes.1 for twsa to occur , the thalamocortical - corticothalamic network must be functionally intact and in a state where intrinsically bursting cortical pyramidal and thalamic neurons are mildly to moderately hyperpolarized and susceptible to producing high - frequency bursts of action potentials.9 meeren and colleagues published a review of the theories on the pathophysiology of tswa.9 this can be summarized as follows : ( 1 ) the centrencephalic theory posits a diffusely projecting pacemaker in the midline thalamus as the origin of tswa . ( 2 )
the thalamic clock theory is a refinement of the centrencephalic theory ; it localizes the abnormal pacemaker to the reticular thalamic nucleus .
( 3 ) the corticoreticular theory claims that tswa is generated by the abnormal interaction of the cortex and the reticular system of the thalamus and brainstem . ( 4 ) the cortical theory considers cortical hyperexcitability as the primary defect ; a hyperexcitable cortex transforms the normal rhythms of the thalamus into tswa .
( 5 ) the cortical focus theory is a synthesis of the cortical and corticoreticular theories ; it asserts that a cortical focus ( the perioral region of the somatosensory cortex is a prime suspect ) initiates the epileptic activity , spreads to other cortical areas , and entrains the thalamus producing a resonant circuitry for expressing tswa .
to these five theories , we add blumenfeld s comprehensive theory which argues that tswa can be provoked by different insults acting on different excitatory / inhibitory circuits.10 it is likely that absence status epilepticus ( ase ) shares the basic mechanisms of absence seizure . whether ictogenesis results in a self - limited absence seizure or in ase depends on the brain s seizure terminating mechanisms ;
such mechanisms are likely impaired in ase.11 patients with no history of ige can develop ase
de novo in the context of an acute brain disorder.4 pro and colleagues reported a patient with late - onset de novo ase whose eeg consistently showed interictal tswa over a 14-year period.12 their case indicates that , in some individuals with latent ige , exposure of the brain to toxic or metabolic factors can trigger a seizure later in life .
the fact that post - hypoxic tswa can look exactly like ige - related tswa implies that different insults acting on different brain circuits can turn on the basic mechanisms that lead to tswa ( see blumenfeld s theory above).10 this case also implies that extensive hypoxic - ischemic brain injury can spare the thalamocortical - corticothalamic network responsible for generating tswa / absence seizure.9 as stated above , a genetic predisposition to develop tswa / ase may be the reason our patient manifested tswa.12 it is also possible that all individuals have an inherent ability to produce tswa and that , in our patient , the pattern of cerebral hypoxic - ischemic injury has released
such ability.10 the patient s eye - lid movements occurred repeatedly at irregular intervals within a 7-hour period and stopped after a total of 7-mg of lorazepam was given .
these movements are consistent with post - hypoxic myoclonus and not with the more periodic eye - blink automatism seen in absence seizure .
it should also be emphasized that eyelid movements were not present when tswa was detected and that tswa is not a known eeg correlate of post - hypoxic myoclonic status epilepticus ; hence myoclonic status epilepticus would not be an appropriate diagnosis for our patient .
the patient has a history of substance abuse and he tested positive for cocaine and marijuana .
however , most drug - related de novo ase cases in the literature were linked to withdrawal of benzodiazepine ( bdz ) or to intoxication with alcohol or psychotropic drugs.4,7 alcohol was not detected in the patient s blood and we are not aware of any reports in which cocaine / marijuana was linked to de novo ase . still , this explanation can not be ruled out .
the patient received a 1440-mg loading dose of fosphenytoin ( a phenytoin prodrug ) 23 hours prior to the onset of eeg monitoring . in the setting of ige , phenytoin ( and other antiepileptic drugs ) can aggravate seizures13 and can even result in refractory ase.14 however , we are not aware of any report of phenytoin / fosphenytoin causing ase in patients with no ige .
the patient also received 17-mg of lorazepam ( a bdz and gaba - a receptor agonist ) 23 to 37 hours before the onset of eeg recording .
however , the absence of a history of chronic bdz use makes it unlikely that the patient went through a bdz withdrawal state .
enhanced gaba - a inhibition in the thalamus can trigger absence seizures in genetic and pharmacological models of absence epilepsy.15 however , we are not aware of any report of lorazepam causing ase in patients with no ige
. what is the significance of benzodiazepine - resistance in tswa / ase ? as a rule , ige - related ase responds rapidly and completely to bdz therapy.4,11 this is in line with the ability of gaba - a receptors to mediate the inhibition of the thalamic
pacemaker of tswa.15 a few cases of refractory ase were described in the literature but all of these patients had ige and were receiving phenytoin or carbamazepine.13 the rarity of post - hypoxic tswa makes it much more difficult to find any report of bdz - resistant post - hypoxic tswa .
we initially hypothesized that resistance indicates a difference in the mechanisms of post - hypoxic tswa and ige - related tswa.16 this is not necessarily the case and the basic mechanism of tswa genesis could still be the same , only that cerebral hypoxia - ischemia resulted in impairment of neural circuits where bdz act to suppress tswa .
for example , impairment of gaba inhibitory circuits in the cerebral cortex can increase the chance of cortical ictogenesis , thalamic entrainment , and tswa generation ( see cortical focus theory).9
in classifying seizures , the international league against epilepsy ( ilae ) made no formal reference to non - convulsive status epilepticus ( ncse).1719 the 1981 ilae seizure classification contains three diagnostic entities that qualify as ncse aura continua , complex partial status epilepticus , and absence status epilepticus ( ase).17 subtle status epilepticus was added in the 2006 report of the ilae classification core group bringing the number of ncse seizures to four.18 in our patient , the finding of tswa indicated ase ( more specifically typical ase ) but treatment response ( bdz - resistance ) and clinical outcome ( the patient died ) are diametrically opposite to that of ase ( especially typical ase ) .
such an electroclinical paradox is not uncommon in critically ill patients with ncse .
unfortunately , the 1981 and 2006 seizure classifications ignore this issue by omitting the many variations of ncse , especially ncse in coma.5,6 the 2010 ilae seizure classification also fails to address this issue.19 what s more , the ilae has completely excluded status epilepticus and focal seizures from the 2010 classification.20 the fact that ncse is more common than previously thought underscores the urgency to create a better classification system for status epilepticus and ncse .
recent studies have shown that ncse constitutes 25%50% of all cases of status epilepticus and the incidence of ncse in the critically ill can be as high as 50%.6 excluding some ncse subtypes from the seizure classification system on philosophical grounds is disadvantageous to the patient , the physician , and the researcher.19 while a seizure classification system can not be perfect at the outset , it should be heuristic and include all phenomena considered as ncse , including the ncse variations in comatose patients .
only a heuristic seizure classification system will result in better understanding and rational treatment of ncse . according to bauer and trinka , the confusion vis - - vis ncse in coma
can be overcome by having a separate ncse categorycomatose ncse or coma - ncse.5 the authors classified cpse and ase as ncse proper .
ncse proper has a good prognosis and , in most cases , the treatment of choice is turning off the seizure without aggressively anesthetizing the patient .
coma - ncse is more ominous and its prognosis depends almost entirely on the underlying brain disorder.5 while there is no firm evidence that suppressing the seizure improves prognosis in coma - ncse , anesthetizing the patient with midazolam or propofol may not be unreasonable if the patient is already on mechanical ventilation and is being monitored in the icu .
maganti and colleagues proposed an etiology - based classification for ncse , including ncse in coma , ncse in acute cerebral lesions , ncse in metabolic disorders , and ncse in those with pre - existing epilepsy.6 this more detailed approach may lend itself to better treatment recommendation , but most importantly better evaluation and testing of treatment recommendations to determine their value based on outcome .
we agree with bauer and trinka that it is important to differentiate coma - ncse from ncse proper5 and with maganti and colleagues that it is critical to account for etiology in classifying ncse.6 by combining the bauer / trinka and maganti proposals and adding some minor refinements , we were able to create a practical decision tree for ncse ( fig .
4 ) . as shown in this decision tree , as soon as ncse is diagnosed ( based on electroclinical data ) , the clinician should separate
the next step is to differentiate between ncse with significant irreversible brain injury ( sibi ) and ncse with no evidence of sibi
. most coma ncse with sibi is due to hypoxic - ischemic , traumatic , ischemic , infectious , or inflammatory insults to the brain .
most coma ncse with no sibi is due to epileptic , toxic , or metabolic factors , often with the confounding effects of anesthetics or sedative drugs that are regularly employed in the icu .
most non - coma ncse is due to purely epileptic , toxic , or metabolic factors , the majority of which are reversible .
occasionally , sibi of lesser severity may give rise to non - coma ncse .
such a heuristic approach can facilitate our understanding of ncse , especially ncse in coma .
while the incidence depends on how one defines ncse , even more conservative estimates of the incidence of ncse ( eg , 25% ) warrant a detailed analysis of ncse cases with a goal toward increased understanding and better classification of ncse .
further work is needed to better define ncse , to determine which eeg patterns indicate ncse , and to establish treatment paradigms for the different types of ncse .
a more collaborative approach across hospitals / universities is warranted given the sample size that would be needed to determine treatment efficacy in so many ncse categories .
this is an unusual presentation of hypoxic - ischemic brain injury in that typical spike - and - wave activity ( tswa ) appeared in the eeg even though the patient never had seizure / epilepsy .
the tswa was relatively resistant to benzodiazepines suggesting that cerebral hypoxia - ischemia spared the thalamocortical apparatus generating tswa , but destroyed the inhibitory circuits where benzodiazepines act to suppress tswa .
post - hypoxic tswa is rare ; nevertheless , it highlights two important principles : first , the eeg allows us to diagnose ncse but does not always tell us how to treat , or whether we should even treat , a particular ncse pattern .
second , it is only by separating ncse proper from comatose ncse and taking into account the overall picture , including the presence of structural lesions , reversible causes , history of epilepsy , and prognosis that we can tailor ncse treatment to the best interest of the patient . | introductiontypical spike - and - wave activity ( tswa ) in the electroencephalogram ( eeg ) indicates idiopathic generalized epilepsy ( ige ) .
ige - related nonconvulsive status epilepticus ( ncse ) is typically an absence status epilepticus ( ase ) .
ase and tswa respond dramatically to benzodiazepines .
patients with no history of seizure / epilepsy may develop ase
de novo in the context of an acute brain disorder . however , we are aware of only one previous case of de novo ase with tswa in hypoxic - ischemic brain injury.case presentationa 65-year - old man , with congestive heart failure and history of substance abuse , survived cardiorespiratory arrest after 18 minutes of cardiopulmonary resuscitation .
post - resuscitation , the patient was in coma with intact brainstem function .
toxicology was positive for cocaine and marijuana .
eyelid myoclonus suggested ncse , which was initially treated with lorazepam and fosphenytoin .
eeg monitoring showed sustained tswa confirming ncse and demonstrating de novo ase ( the patient and his family never had seizure / epilepsy ) .
the tswa was resistant to lorazepam , levetiracetam , and low - dose midazolam ; it was eliminated only with midazolam at a dose that resulted in burst - suppression ( 1.2 mg / kg / hour).conclusionthis is an unusual case of tswa and hypoxic - ischemic brain injury in a patient with no history of seizure / epilepsy .
the tswa was relatively resistant to benzodiazepines suggesting that cerebral hypoxia - ischemia spared the thalamocortical apparatus generating tswa but impaired the cortical / thalamic inhibitory circuits where benzodiazepines act to suppress tswa .
albeit rare , post - hypoxic tswa offers us some valuable insights for classifying and managing nonconvulsive status epilepticus . | Introduction
Case Presentation
Discussion
Pathophysiology of TSWA
Classification of NCSE
Conclusion | patients with absence epilepsy or other idiopathic generalized epilepsy ( ige ) syndromes manifest 2.5 hz to 3.5 hz ( 3 hz on average ) bisynchronous typical spike - and - wave activity ( tswa ) in the interictal electroencephalogram ( eeg ) and during typical absence seizures.1
nonconvulsive status epilepticus ( ncse ) consists of sustained rhythmic / periodic epileptiforma activity in the eeg , along with alterations in mental status and/or behavior , in the absence of convulsive movements . an even wider range of eeg patterns is seen in ncse , but many of these patterns are not conclusively epileptiform.2 ncse is traditionally divided into complex partial status epilepticus ( cpse)3 and absence status epilepticus ( ase).4 diagnosing ncse can be difficult in the presence of coma.5,6 first , the eeg in coma may show a typical cpse / ase pattern even though the clinical course and response to treatment are not compatible with cpse / ase . ige - related ncse usually takes the form of absence status epilepticus.4 some adults with no history of seizure / epilepsy develop ase
de novo in the context of an acute brain disorder.4 the acute brain disorder in de novo ase is often an encephalopathy from drug overdose / withdrawal , fluid / electrolyte imbalance , or systemic infections.7 however , de novo ase is not routinely reported in hypoxic - ischemic encephalopathyb ; we are aware of only one previous case of de novo ase with tswa in hypoxic - ischemic brain injury.8 we present an unusual case of hypoxic - ischemic brain injury in which the patient s eeg showed benzodiazepine - resistant tswa even though the patient had no past or family history of seizure or epilepsy . after presenting the case ,
we examine the pathophysiology of post - hypoxic tswac and its implications for classifying and managing ncse . the patient , a 65-year - old man with congestive heart failure ( left ventricular ejection fraction of 20% ) suffered cardiorespiratory arrest resulting in hypoxic - ischemic brain injury . on admission to the intensive care unit ( icu ) , the patient was in coma with intact brainstem function and stable vital signs ( rr = 14 , hr = 73 , bp = 100/70 , t = 36 c ) . post - resuscitation , the patient manifested eyelid myoclonus occurring spontaneously every 3 to 12 seconds without any clear relationship to external stimulation ; this is most likely a form of acute posthypoxic myoclonus . eeg monitoringd was initiated 23 hours after administering fosphenytoin and discontinuing lorazepam ; at that time the patient was in deep coma with no involuntary movements . the eeg showed sustained typical spike - and - wave activity ( tswa ) confirming ncse and indicating typical absence status epilepticus ( fig . the infusion rate was increased and at 0.5 mg / kg / hour the eeg showed intermittent suppression of spike - and - wave discharges ( fig . at a rate of 1.2 mg / kg / hour , tswa disappeared and
stable burst - suppression ( 612 bursts / minute ) was achieved with an infusion rate of 1.4 to 1.6 mg / kg / hour . eeg showed typical spike - and - wave activity ( tswa ) even though he had no history of seizure or epilepsy . non - convulsive status epilepticus ( ncse ) was diagnosed and intravenous lorazepam and midazolam was administered . the tswa proved to be relatively resistant to benzodiazepines , raising some important issues regarding the pathophysiology of post - hypoxic tswa and providing some insight into how ncse should be classified and managed . typical spike - and - wave activity ( tswa ) is the eeg hallmark of absence seizure . it is also a routine interictal eeg finding in absence epilepsy and other ige syndromes.1 for twsa to occur , the thalamocortical - corticothalamic network must be functionally intact and in a state where intrinsically bursting cortical pyramidal and thalamic neurons are mildly to moderately hyperpolarized and susceptible to producing high - frequency bursts of action potentials.9 meeren and colleagues published a review of the theories on the pathophysiology of tswa.9 this can be summarized as follows : ( 1 ) the centrencephalic theory posits a diffusely projecting pacemaker in the midline thalamus as the origin of tswa . ( 5 ) the cortical focus theory is a synthesis of the cortical and corticoreticular theories ; it asserts that a cortical focus ( the perioral region of the somatosensory cortex is a prime suspect ) initiates the epileptic activity , spreads to other cortical areas , and entrains the thalamus producing a resonant circuitry for expressing tswa . to these five theories , we add blumenfeld s comprehensive theory which argues that tswa can be provoked by different insults acting on different excitatory / inhibitory circuits.10 it is likely that absence status epilepticus ( ase ) shares the basic mechanisms of absence seizure . whether ictogenesis results in a self - limited absence seizure or in ase depends on the brain s seizure terminating mechanisms ;
such mechanisms are likely impaired in ase.11 patients with no history of ige can develop ase
de novo in the context of an acute brain disorder.4 pro and colleagues reported a patient with late - onset de novo ase whose eeg consistently showed interictal tswa over a 14-year period.12 their case indicates that , in some individuals with latent ige , exposure of the brain to toxic or metabolic factors can trigger a seizure later in life . the fact that post - hypoxic tswa can look exactly like ige - related tswa implies that different insults acting on different brain circuits can turn on the basic mechanisms that lead to tswa ( see blumenfeld s theory above).10 this case also implies that extensive hypoxic - ischemic brain injury can spare the thalamocortical - corticothalamic network responsible for generating tswa / absence seizure.9 as stated above , a genetic predisposition to develop tswa / ase may be the reason our patient manifested tswa.12 it is also possible that all individuals have an inherent ability to produce tswa and that , in our patient , the pattern of cerebral hypoxic - ischemic injury has released
such ability.10 the patient s eye - lid movements occurred repeatedly at irregular intervals within a 7-hour period and stopped after a total of 7-mg of lorazepam was given . the patient has a history of substance abuse and he tested positive for cocaine and marijuana . however , most drug - related de novo ase cases in the literature were linked to withdrawal of benzodiazepine ( bdz ) or to intoxication with alcohol or psychotropic drugs.4,7 alcohol was not detected in the patient s blood and we are not aware of any reports in which cocaine / marijuana was linked to de novo ase . in the setting of ige , phenytoin ( and other antiepileptic drugs )
can aggravate seizures13 and can even result in refractory ase.14 however , we are not aware of any report of phenytoin / fosphenytoin causing ase in patients with no ige . however , the absence of a history of chronic bdz use makes it unlikely that the patient went through a bdz withdrawal state . enhanced gaba - a inhibition in the thalamus can trigger absence seizures in genetic and pharmacological models of absence epilepsy.15 however , we are not aware of any report of lorazepam causing ase in patients with no ige
. as a rule , ige - related ase responds rapidly and completely to bdz therapy.4,11 this is in line with the ability of gaba - a receptors to mediate the inhibition of the thalamic
pacemaker of tswa.15 a few cases of refractory ase were described in the literature but all of these patients had ige and were receiving phenytoin or carbamazepine.13 the rarity of post - hypoxic tswa makes it much more difficult to find any report of bdz - resistant post - hypoxic tswa . we initially hypothesized that resistance indicates a difference in the mechanisms of post - hypoxic tswa and ige - related tswa.16 this is not necessarily the case and the basic mechanism of tswa genesis could still be the same , only that cerebral hypoxia - ischemia resulted in impairment of neural circuits where bdz act to suppress tswa . for example , impairment of gaba inhibitory circuits in the cerebral cortex can increase the chance of cortical ictogenesis , thalamic entrainment , and tswa generation ( see cortical focus theory).9 in classifying seizures , the international league against epilepsy ( ilae ) made no formal reference to non - convulsive status epilepticus ( ncse).1719 the 1981 ilae seizure classification contains three diagnostic entities that qualify as ncse aura continua , complex partial status epilepticus , and absence status epilepticus ( ase).17 subtle status epilepticus was added in the 2006 report of the ilae classification core group bringing the number of ncse seizures to four.18 in our patient , the finding of tswa indicated ase ( more specifically typical ase ) but treatment response ( bdz - resistance ) and clinical outcome ( the patient died ) are diametrically opposite to that of ase ( especially typical ase ) . unfortunately , the 1981 and 2006 seizure classifications ignore this issue by omitting the many variations of ncse , especially ncse in coma.5,6 the 2010 ilae seizure classification also fails to address this issue.19 what s more , the ilae has completely excluded status epilepticus and focal seizures from the 2010 classification.20 the fact that ncse is more common than previously thought underscores the urgency to create a better classification system for status epilepticus and ncse . recent studies have shown that ncse constitutes 25%50% of all cases of status epilepticus and the incidence of ncse in the critically ill can be as high as 50%.6 excluding some ncse subtypes from the seizure classification system on philosophical grounds is disadvantageous to the patient , the physician , and the researcher.19 while a seizure classification system can not be perfect at the outset , it should be heuristic and include all phenomena considered as ncse , including the ncse variations in comatose patients . coma - ncse is more ominous and its prognosis depends almost entirely on the underlying brain disorder.5 while there is no firm evidence that suppressing the seizure improves prognosis in coma - ncse , anesthetizing the patient with midazolam or propofol may not be unreasonable if the patient is already on mechanical ventilation and is being monitored in the icu . typical spike - and - wave activity ( tswa ) is the eeg hallmark of absence seizure . it is also a routine interictal eeg finding in absence epilepsy and other ige syndromes.1 for twsa to occur , the thalamocortical - corticothalamic network must be functionally intact and in a state where intrinsically bursting cortical pyramidal and thalamic neurons are mildly to moderately hyperpolarized and susceptible to producing high - frequency bursts of action potentials.9 meeren and colleagues published a review of the theories on the pathophysiology of tswa.9 this can be summarized as follows : ( 1 ) the centrencephalic theory posits a diffusely projecting pacemaker in the midline thalamus as the origin of tswa . ( 5 ) the cortical focus theory is a synthesis of the cortical and corticoreticular theories ; it asserts that a cortical focus ( the perioral region of the somatosensory cortex is a prime suspect ) initiates the epileptic activity , spreads to other cortical areas , and entrains the thalamus producing a resonant circuitry for expressing tswa . to these five theories , we add blumenfeld s comprehensive theory which argues that tswa can be provoked by different insults acting on different excitatory / inhibitory circuits.10 it is likely that absence status epilepticus ( ase ) shares the basic mechanisms of absence seizure . whether ictogenesis results in a self - limited absence seizure or in ase depends on the brain s seizure terminating mechanisms ;
such mechanisms are likely impaired in ase.11 patients with no history of ige can develop ase
de novo in the context of an acute brain disorder.4 pro and colleagues reported a patient with late - onset de novo ase whose eeg consistently showed interictal tswa over a 14-year period.12 their case indicates that , in some individuals with latent ige , exposure of the brain to toxic or metabolic factors can trigger a seizure later in life . the fact that post - hypoxic tswa can look exactly like ige - related tswa implies that different insults acting on different brain circuits can turn on the basic mechanisms that lead to tswa ( see blumenfeld s theory above).10 this case also implies that extensive hypoxic - ischemic brain injury can spare the thalamocortical - corticothalamic network responsible for generating tswa / absence seizure.9 as stated above , a genetic predisposition to develop tswa / ase may be the reason our patient manifested tswa.12 it is also possible that all individuals have an inherent ability to produce tswa and that , in our patient , the pattern of cerebral hypoxic - ischemic injury has released
such ability.10 the patient s eye - lid movements occurred repeatedly at irregular intervals within a 7-hour period and stopped after a total of 7-mg of lorazepam was given . it should also be emphasized that eyelid movements were not present when tswa was detected and that tswa is not a known eeg correlate of post - hypoxic myoclonic status epilepticus ; hence myoclonic status epilepticus would not be an appropriate diagnosis for our patient . the patient has a history of substance abuse and he tested positive for cocaine and marijuana . however , most drug - related de novo ase cases in the literature were linked to withdrawal of benzodiazepine ( bdz ) or to intoxication with alcohol or psychotropic drugs.4,7 alcohol was not detected in the patient s blood and we are not aware of any reports in which cocaine / marijuana was linked to de novo ase . in the setting of ige , phenytoin ( and other antiepileptic drugs ) can aggravate seizures13 and can even result in refractory ase.14 however , we are not aware of any report of phenytoin / fosphenytoin causing ase in patients with no ige . enhanced gaba - a inhibition in the thalamus can trigger absence seizures in genetic and pharmacological models of absence epilepsy.15 however , we are not aware of any report of lorazepam causing ase in patients with no ige
. as a rule , ige - related ase responds rapidly and completely to bdz therapy.4,11 this is in line with the ability of gaba - a receptors to mediate the inhibition of the thalamic
pacemaker of tswa.15 a few cases of refractory ase were described in the literature but all of these patients had ige and were receiving phenytoin or carbamazepine.13 the rarity of post - hypoxic tswa makes it much more difficult to find any report of bdz - resistant post - hypoxic tswa . we initially hypothesized that resistance indicates a difference in the mechanisms of post - hypoxic tswa and ige - related tswa.16 this is not necessarily the case and the basic mechanism of tswa genesis could still be the same , only that cerebral hypoxia - ischemia resulted in impairment of neural circuits where bdz act to suppress tswa . for example , impairment of gaba inhibitory circuits in the cerebral cortex can increase the chance of cortical ictogenesis , thalamic entrainment , and tswa generation ( see cortical focus theory).9
in classifying seizures , the international league against epilepsy ( ilae ) made no formal reference to non - convulsive status epilepticus ( ncse).1719 the 1981 ilae seizure classification contains three diagnostic entities that qualify as ncse aura continua , complex partial status epilepticus , and absence status epilepticus ( ase).17 subtle status epilepticus was added in the 2006 report of the ilae classification core group bringing the number of ncse seizures to four.18 in our patient , the finding of tswa indicated ase ( more specifically typical ase ) but treatment response ( bdz - resistance ) and clinical outcome ( the patient died ) are diametrically opposite to that of ase ( especially typical ase ) . recent studies have shown that ncse constitutes 25%50% of all cases of status epilepticus and the incidence of ncse in the critically ill can be as high as 50%.6 excluding some ncse subtypes from the seizure classification system on philosophical grounds is disadvantageous to the patient , the physician , and the researcher.19 while a seizure classification system can not be perfect at the outset , it should be heuristic and include all phenomena considered as ncse , including the ncse variations in comatose patients . coma - ncse is more ominous and its prognosis depends almost entirely on the underlying brain disorder.5 while there is no firm evidence that suppressing the seizure improves prognosis in coma - ncse , anesthetizing the patient with midazolam or propofol may not be unreasonable if the patient is already on mechanical ventilation and is being monitored in the icu . this is an unusual presentation of hypoxic - ischemic brain injury in that typical spike - and - wave activity ( tswa ) appeared in the eeg even though the patient never had seizure / epilepsy . the tswa was relatively resistant to benzodiazepines suggesting that cerebral hypoxia - ischemia spared the thalamocortical apparatus generating tswa , but destroyed the inhibitory circuits where benzodiazepines act to suppress tswa . post - hypoxic tswa is rare ; nevertheless , it highlights two important principles : first , the eeg allows us to diagnose ncse but does not always tell us how to treat , or whether we should even treat , a particular ncse pattern . second , it is only by separating ncse proper from comatose ncse and taking into account the overall picture , including the presence of structural lesions , reversible causes , history of epilepsy , and prognosis that we can tailor ncse treatment to the best interest of the patient . | [
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successful management of moderate - to - severe copd can be accomplished with combination therapy that delivers a concurrent dose of inhaled corticosteroid and a long - acting 2-agonist ( ics / laba ) or with tiotropium ( tio ) , an inhaled long - acting anticholinergic bronchodilator.1 both twice - daily ics / laba and once - daily tio treatment for copd have been demonstrated to improve lung function and health status as well as reduce copd exacerbations.16 head - to - head comparisons between ics / laba drugs and tio , however , are limited and the efficacy results inconclusive,7,8 leading researchers to call for further large , long - term randomized controlled trials to establish relative efficacy and safety of these treatments , as well as compare newly developed alternative ics / labas with tio.8 a newly introduced ics / laba treatment for copd in a novel inhaler device combines fluticasone furoate ( ff ) , an ics with enhanced selectivity , retention , and affinity for the glucocorticoid receptor,9 and vilanterol ( vi ) , a laba with prolonged bronchodilating properties.10 ff / vi is the only once - daily ics / laba treatment and is well tolerated , improves lung function , and sustains bronchodilation after 24 weeks and 52 weeks of treatment for copd.1113 when once - daily treatment with ff / vi for 12 weeks was compared with twice - daily fluticasone propionate / salmeterol 250/50 ( fp / salm ) for copd , there was evidence of numerically greater effect on the forced expiratory volume in 1 second ( fev1 ) and faster onset for ff / vi compared with fp / salm with similar safety profiles for the two drugs.14 copd is not only a disease of the airways and lung parenchyma , but there are several extrapulmonary comorbidities associated with copd,15 of which cardiovascular disease ( cvd ) is arguably the most significant .
cvd is a comorbidity of copd,1618 and the risk of cardiovascular ( cv ) events rises in subjects with copd with a history of myocardial infarction.19 furthermore , concerns regarding cv safety and copd medications have been raised for both tio5 and laba drugs.20 therefore , we conducted the study in a population of subjects with or at risk for cvd .
the objective of this study was to compare the efficacy and safety of ff / vi and tio , both delivered once daily , for copd treatment over a 12-week period in a randomized , controlled trial .
an additional objective was to investigate whether the efficacy and/or safety profile of ff / vi or tio would differ in subjects with cvd or in subjects at risk for cvd .
the study ( protocol hzc115805 , nct01627327 ) was approved by an institutional review board or independent ethics committee at each site and conducted in accordance with good clinical practice guidelines and the declaration of helsinki .
the study was conducted between april and december 2012 at 56 centers in six countries ( canada , czech republic , germany , poland , romania , and the usa ) .
all subjects were current or former smokers ( 10 pack years ) and had a postbronchodilator fev1 30% to 70% of predicted normal ( nhanes iii ) and an fev1/forced vital capacity ( fvc ) ratio 70% at screening .
subjects also had a history of cvd / a cvd event or , in addition to being a current / former smoker , had at least one current cv risk factor ( hypertension , hypercholesterolemia , or treated diabetes ) .
subjects were excluded if they had asthma or respiratory disorders other than copd ; recent ( 12 months ) lung resection ; clinically significant abnormal chest x - ray , laboratory , holter , or electrocardiogram ( ecg ) finding at screening ; recent ( 12 weeks ) hospitalization for copd ; recent ( 6 weeks ) acute worsening of copd requiring treatment with corticosteroids or antibiotics or lower respiratory tract infection requiring antibiotics ; noncompliance , copd exacerbation , or lower respiratory tract infection during the run - in period ; cancer not in complete remission for 5 years ; long - term or nocturnal oxygen therapy ( > 12 h / day ) ; or other diseases or contraindications that would put the subject at risk .
this study was a 12-week , randomized ( 1:1 ) , blinded , stratified , double - dummy , parallel - group , active comparator study . after providing written informed consent and prior to screening ,
subjects discontinued copd medications ( including maintenance medications ; excluded medications included systemic / oral / parenteral corticosteroids [ within 6 weeks prior to screening and thereafter ] , ics or ics / laba combinations [ within 4 weeks prior to screening and thereafter ] , anticholinergics [ long acting , 1 week prior to screening and thereafter ; short acting , 4 hours prior to screening and thereafter ] , oral phosphodiesterase type 4 inhibitors [ pde-4 ] inhibitors [ 1 week prior to screening and thereafter ] , and labas [ within 48 hours prior to screening and thereafter ] ) ; exceptions were study - supplied rescue medication ( albuterol ) and mucolytics at a constant dosage .
after the screening visit , eligible subjects entered a 2-week single - blind placebo run - in period to obtain baseline rescue medication use and copd symptom ( scores recorded on diary cards ) and to evaluate subjects compliance and disease stability . at the end of the run - in period ,
eligible subjects were randomly assigned to once - daily morning treatment with either ff / vi 100/25 mcg ( breo [ relvar , revinty ] , glaxosmithkline plc [ gsk ] , london , uk ) delivered via the ellipta inhaler ( gsk , london , uk ) or tio 18 mcg ( spiriva , boehringer ingelheim , ingelheim , germany ) delivered from blister capsules by the handihaler ( spiriva , boehringer ingelheim ) . when used , the novel ellipta dry powder inhaler simultaneously delivers one breath - actuated dose of both ff 100 mcg and vi 25 mcg from their individual blister strips .
a central randomization schedule was generated using a validated computerized system ( randall ; gsk ) and communicated with a validated computerized voice response system , the registration and medication ordering system ( ramos ; gsk ) , when the investigative site called to randomize a subject . to provide a double - dummy design ,
subjects were given two inhalers , one containing active drug and the other placebo , and they and their investigators were blinded to assigned treatment .
tio and placebo capsules were closely matched in color , but tio capsules had trade markings that were not present on the placebo capsules . whether subjects would notice and correctly or incorrectly interpret this difference is unclear .
both the tio and placebo blister packages were covered with opaque overlabels that hide the information on the tio packaging .
staff involved with safety and efficacy assessments were not present during dosing in the clinic so that they would not identify the capsules removed from the blisters .
every morning , subjects in the ff / vi group inhaled once from the ellipta inhaler containing active drug followed by two inhalations from the handihaler containing placebo . subjects in the tio group inhaled once from the ellipta inhaler containing placebo followed by two inhalations from the handihaler containing tio .
compliance with the study drug was assessed at each visit by reviewing the dose counter on the ellipta or counting capsules from the handihaler .
subjects were also provided with albuterol ( metered dose inhaler [ mdi ] and/or nebules ) to use as needed for symptomatic relief of copd during the run - in and treatment periods , except for the 4-hour period prior to spirometry and during each study visit .
subjects were stratified at randomization for copd exacerbation history ( either did or did not have 1 copd exacerbation requiring oral corticosteroid and/or antibiotic treatment and/or hospitalization in the 3 years prior to screening ) and for reversibility status ( either reversible [ defined as an increase in fev1 of 12% and 200 ml ] or nonreversible [ defined as an increase in fev1 < 200 ml or 200 ml and < 12% after albuterol administration at screening ] ) .
a follow - up safety telephone contact was performed 7 days after the last treatment visit .
subjects who experienced a copd exacerbation or pneumonia were withdrawn ; however , only exacerbations that resulted in hospitalization ( ie , severe exacerbations ) were captured as an adverse event ( ae ) .
standardized equipment ( spirometry : microquark , 12-lead ecg : universal ecg , and 24-hour holter monitor : mortara instrument ) , study - customized software , and training were provided , and data were transmitted and overread by independent centralized certified staff at compleware . at the screening visit , pre- and postbronchodilator spirometry , medical history , physical examination , 12-lead ecg , 24-hour holter monitoring ( subset of subjects ) , chest x - ray , vital signs , serum pregnancy tests , clinical laboratory tests , oropharyngeal examination , and 24-hour urine cortisol collection ( subset of subjects ) were conducted , and diary cards were dispensed for recording of medical problems , concomitant medications , rescue medication use , and symptoms ( dyspnea , cough , sputum production ) . at all subsequent visits , eligible randomized subjects completed the st george s respiratory questionnaire - copd ( sgrq - c ) and copd assessment tests ( cat ) prior to other assessments , and safety monitoring was conducted , including oropharyngeal examination , ae reporting , exacerbation and pneumonia assessment , and diary card review . at the randomization visit 2 , subjects also underwent vital sign measurement and blood draws for biomarkers ( subset of subjects ) . at visit 5 , which was an overnight stay , 24-hour spirometry , ecgs , 24-hour holter monitoring ( subset of subjects ) , 24-hour urinary cortisol collection ( subset of subjects ) , physical examinations , vital sign measurement , blood draws for clinical laboratory tests and biomarkers , and pregnancy tests were conducted .
serum was collected for seven known inflammatory biomarkers at baseline and week 12 and analyzed as exploratory endpoints : high - sensitivity c - reactive protein ( hscrp ) , pulmonary and activation - regulated chemokine ( more currently referred to as chemokine [ c - c motif ] ligand 18 [ ccl-18 ] ) , fibrinogen , interleukin-6 ( il-6 ) , surfactant protein d ( sp - d ) , clara cell secretory protein-16 ( cc-16 ) , and brain natriuretic peptide ( bnp ) .
spirometry was performed after withholding rescue medication , study medication , and caffeinated drinks and after abstinence from smoking and physical exertion .
inspiratory capacity ( ic ) assessments were performed prior to fev1 assessments . at the randomization visit 2 , after predose spirometry and the first study dose , subjects underwent postdose serial spirometry at 5 minutes , 15 minutes , and 30 minutes and at 1 hour , 2 hours , and 4 hours . at visit 5 on day 84 after predose spirometry and after the last study dose , subjects underwent postdose serial spirometry at 5 minutes , 15 minutes , and 30 minutes and at 1 hour , 2 hours , 4 hours , 6 hours , 8 hours , 12 hours , 13 hours , 14 hours , 16 hours , 20 hours , and 24 hours . assuming a standard deviation of 215 ml and significance at the two - sided 5% level , a sample size of 271 subjects per treatment arm was required for 90% power to detect a 60 ml difference between ff / vi and tio for 24-hour weighted - mean fev1 at week 12 , an estimate based on historical data . to allow for a 10% withdrawal rate , at least 300 subjects were to be randomized per treatment arm .
primary analyses were performed on the intent - to - treat population , which comprised all subjects randomized to treatment and who had received at least one dose of study medication .
the primary endpoint , change from baseline in 24-hour weighted mean fev1 at week 12 , was analyzed using analysis of covariance with covariates of baseline fev1 , randomization stratum ( copd exacerbation history and reversibility status ) , smoking status at screening , and country .
for calculation of 24-hour weighted mean fev1 , missing serial spirometry fev1 values were linearly interpolated between two nonmissing values ; however , the 0-hour and 24-hour values , as well as additional values between 0 hour and 6 hours and between 12 hours and 24 hours , must not have been missing . the secondary endpoints ( time to onset [ increase of 100 ml above baseline in fev1 ] 04 hours postdose on day 1 and change from baseline in trough fev1 on day 84 ) were nested under the primary endpoint . to account for multiplicity across primary and secondary efficacy endpoints ,
a p - value < 0.05 was required for the primary endpoint to allow statistical significance to be inferred for secondary endpoints .
time to onset was analyzed using a log - rank test , stratified by exacerbation history and reversibility stratum .
change from baseline in trough ( predose ) fev1 was analyzed using the same methodology as the primary endpoint .
daily diary measures were averaged weekly and over the entire 12-week treatment period for analysis .
change from baseline in sgrq - c and cat scores was analyzed using mixed model repeated measures with covariates of baseline score , exacerbation history and reversibility stratum , smoking status , country , treatment group , day , day by baseline , and day by treatment group .
subjects were classified as a responder at a given visit if they had a change from baseline at that visit of 4 points improvement , the minimal clinically important difference ( mcid).21 otherwise , the subject was classified as a nonresponder . a logistic regression model with terms for treatment group , exacerbation history and reversibility stratum , and baseline score
other efficacy endpoints , as well as safety measures of pulse rate and log - transformed urinary cortisol , were analyzed using analysis of covariance .
all subjects were current or former smokers ( 10 pack years ) and had a postbronchodilator fev1 30% to 70% of predicted normal ( nhanes iii ) and an fev1/forced vital capacity ( fvc ) ratio 70% at screening .
subjects also had a history of cvd / a cvd event or , in addition to being a current / former smoker , had at least one current cv risk factor ( hypertension , hypercholesterolemia , or treated diabetes ) .
subjects were excluded if they had asthma or respiratory disorders other than copd ; recent ( 12 months ) lung resection ; clinically significant abnormal chest x - ray , laboratory , holter , or electrocardiogram ( ecg ) finding at screening ; recent ( 12 weeks ) hospitalization for copd ; recent ( 6 weeks ) acute worsening of copd requiring treatment with corticosteroids or antibiotics or lower respiratory tract infection requiring antibiotics ; noncompliance , copd exacerbation , or lower respiratory tract infection during the run - in period ; cancer not in complete remission for 5 years ; long - term or nocturnal oxygen therapy ( > 12 h / day ) ; or other diseases or contraindications that would put the subject at risk .
this study was a 12-week , randomized ( 1:1 ) , blinded , stratified , double - dummy , parallel - group , active comparator study . after providing written informed consent and prior to screening , subjects discontinued copd medications ( including maintenance medications ; excluded medications included systemic / oral / parenteral corticosteroids [ within 6 weeks prior to screening and thereafter ] , ics or ics / laba combinations [ within 4 weeks prior to screening and thereafter ] , anticholinergics [ long acting , 1 week prior to screening and thereafter ; short acting , 4 hours prior to screening and thereafter ] , oral phosphodiesterase type 4 inhibitors [ pde-4 ] inhibitors [ 1 week prior to screening and thereafter ] , and labas [ within 48 hours prior to screening and thereafter ] ) ; exceptions were study - supplied rescue medication ( albuterol ) and mucolytics at a constant dosage .
after the screening visit , eligible subjects entered a 2-week single - blind placebo run - in period to obtain baseline rescue medication use and copd symptom ( scores recorded on diary cards ) and to evaluate subjects compliance and disease stability . at the end of the run - in period ,
eligible subjects were randomly assigned to once - daily morning treatment with either ff / vi 100/25 mcg ( breo [ relvar , revinty ] , glaxosmithkline plc [ gsk ] , london , uk ) delivered via the ellipta inhaler ( gsk , london , uk ) or tio 18 mcg ( spiriva , boehringer ingelheim , ingelheim , germany ) delivered from blister capsules by the handihaler ( spiriva , boehringer ingelheim ) . when used , the novel ellipta dry powder inhaler simultaneously delivers one breath - actuated dose of both ff 100 mcg and vi 25 mcg from their individual blister strips .
a central randomization schedule was generated using a validated computerized system ( randall ; gsk ) and communicated with a validated computerized voice response system , the registration and medication ordering system ( ramos ; gsk ) , when the investigative site called to randomize a subject .
to provide a double - dummy design , subjects were given two inhalers , one containing active drug and the other placebo , and they and their investigators were blinded to assigned treatment .
tio and placebo capsules were closely matched in color , but tio capsules had trade markings that were not present on the placebo capsules . whether subjects would notice and correctly or incorrectly interpret this difference is unclear .
both the tio and placebo blister packages were covered with opaque overlabels that hide the information on the tio packaging .
staff involved with safety and efficacy assessments were not present during dosing in the clinic so that they would not identify the capsules removed from the blisters .
every morning , subjects in the ff / vi group inhaled once from the ellipta inhaler containing active drug followed by two inhalations from the handihaler containing placebo . subjects in the tio group inhaled once from the ellipta inhaler containing placebo followed by two inhalations from the handihaler containing tio .
compliance with the study drug was assessed at each visit by reviewing the dose counter on the ellipta or counting capsules from the handihaler .
subjects were also provided with albuterol ( metered dose inhaler [ mdi ] and/or nebules ) to use as needed for symptomatic relief of copd during the run - in and treatment periods , except for the 4-hour period prior to spirometry and during each study visit .
subjects were stratified at randomization for copd exacerbation history ( either did or did not have 1 copd exacerbation requiring oral corticosteroid and/or antibiotic treatment and/or hospitalization in the 3 years prior to screening ) and for reversibility status ( either reversible [ defined as an increase in fev1 of 12% and 200 ml ] or nonreversible [ defined as an increase in fev1 < 200 ml or 200 ml and < 12% after albuterol administration at screening ] ) .
a follow - up safety telephone contact was performed 7 days after the last treatment visit .
subjects who experienced a copd exacerbation or pneumonia were withdrawn ; however , only exacerbations that resulted in hospitalization ( ie , severe exacerbations ) were captured as an adverse event ( ae ) .
standardized equipment ( spirometry : microquark , 12-lead ecg : universal ecg , and 24-hour holter monitor : mortara instrument ) , study - customized software , and training were provided , and data were transmitted and overread by independent centralized certified staff at compleware . at the screening visit , pre- and postbronchodilator spirometry , medical history , physical examination , 12-lead ecg , 24-hour holter monitoring ( subset of subjects ) , chest x - ray , vital signs , serum pregnancy tests , clinical laboratory tests , oropharyngeal examination , and 24-hour urine cortisol collection ( subset of subjects ) were conducted , and diary cards were dispensed for recording of medical problems , concomitant medications , rescue medication use , and symptoms ( dyspnea , cough , sputum production ) . at all subsequent visits , eligible randomized subjects completed the st george s respiratory questionnaire - copd ( sgrq - c ) and copd assessment tests ( cat ) prior to other assessments , and safety monitoring was conducted , including oropharyngeal examination , ae reporting , exacerbation and pneumonia assessment , and diary card review . at the randomization visit 2 , subjects also underwent vital sign measurement and blood draws for biomarkers ( subset of subjects ) . at visit 5 , which was an overnight stay , 24-hour spirometry , ecgs , 24-hour holter monitoring ( subset of subjects ) , 24-hour urinary cortisol collection ( subset of subjects ) , physical examinations , vital sign measurement , blood draws for clinical laboratory tests and biomarkers , and pregnancy tests were conducted .
serum was collected for seven known inflammatory biomarkers at baseline and week 12 and analyzed as exploratory endpoints : high - sensitivity c - reactive protein ( hscrp ) , pulmonary and activation - regulated chemokine ( more currently referred to as chemokine [ c - c motif ] ligand 18 [ ccl-18 ] ) , fibrinogen , interleukin-6 ( il-6 ) , surfactant protein d ( sp - d ) , clara cell secretory protein-16 ( cc-16 ) , and brain natriuretic peptide ( bnp ) .
spirometry was performed after withholding rescue medication , study medication , and caffeinated drinks and after abstinence from smoking and physical exertion .
inspiratory capacity ( ic ) assessments were performed prior to fev1 assessments . at the randomization visit 2 , after predose spirometry and the first study dose , subjects underwent postdose serial spirometry at 5 minutes , 15 minutes , and 30 minutes and at 1 hour , 2 hours , and 4 hours . at visit 5 on day 84 after predose spirometry and after the last study dose , subjects underwent postdose serial spirometry at 5 minutes , 15 minutes , and 30 minutes and at 1 hour , 2 hours , 4 hours , 6 hours , 8 hours , 12 hours , 13 hours , 14 hours , 16 hours , 20 hours , and 24 hours .
assuming a standard deviation of 215 ml and significance at the two - sided 5% level , a sample size of 271 subjects per treatment arm was required for 90% power to detect a 60 ml difference between ff / vi and tio for 24-hour weighted - mean fev1 at week 12 , an estimate based on historical data . to allow for a 10% withdrawal rate , at least 300 subjects were to be randomized per treatment arm .
primary analyses were performed on the intent - to - treat population , which comprised all subjects randomized to treatment and who had received at least one dose of study medication .
the primary endpoint , change from baseline in 24-hour weighted mean fev1 at week 12 , was analyzed using analysis of covariance with covariates of baseline fev1 , randomization stratum ( copd exacerbation history and reversibility status ) , smoking status at screening , and country .
for calculation of 24-hour weighted mean fev1 , missing serial spirometry fev1 values were linearly interpolated between two nonmissing values ; however , the 0-hour and 24-hour values , as well as additional values between 0 hour and 6 hours and between 12 hours and 24 hours , must not have been missing . the secondary endpoints ( time to onset [ increase of 100 ml above baseline in fev1 ] 04 hours postdose on day 1 and change from baseline in trough fev1 on day 84 ) were nested under the primary endpoint . to account for multiplicity across primary and secondary efficacy endpoints ,
a p - value < 0.05 was required for the primary endpoint to allow statistical significance to be inferred for secondary endpoints .
time to onset was analyzed using a log - rank test , stratified by exacerbation history and reversibility stratum .
change from baseline in trough ( predose ) fev1 was analyzed using the same methodology as the primary endpoint .
daily diary measures were averaged weekly and over the entire 12-week treatment period for analysis .
change from baseline in sgrq - c and cat scores was analyzed using mixed model repeated measures with covariates of baseline score , exacerbation history and reversibility stratum , smoking status , country , treatment group , day , day by baseline , and day by treatment group .
subjects were classified as a responder at a given visit if they had a change from baseline at that visit of 4 points improvement , the minimal clinically important difference ( mcid).21 otherwise , the subject was classified as a nonresponder . a logistic regression model with terms for treatment group , exacerbation history and reversibility stratum , and baseline score
other efficacy endpoints , as well as safety measures of pulse rate and log - transformed urinary cortisol , were analyzed using analysis of covariance .
at screening , 890 subjects were evaluated for eligibility ( figure 1 ) . of those ,
267 ( 30% ) subjects were not randomized mainly because they failed the strict enrollment criteria , for example , fev1 30%70% predicted ( n=112 ) , and/or had an abnormal holter ( n=75 ) and/or ecg ( n=55 ) finding .
following the run - in period , 310 and 313 subjects were randomized to treatment with ff / vi and tio , respectively , and constituted the intent - to - treat population . of those , 291 ( 94% ) and 274 ( 88% ) subjects , respectively , completed the study .
more subjects were withdrawn from treatment with tio , mainly due to aes and lack of efficacy / copd exacerbations , than with ff / vi .
three percent of tio - treated and 1% of ff / vi - treated subjects withdrew from the study due to protocol deviations .
subjects demographic and baseline characteristics were similar between treatment groups ( table 1 ) , with the majority of subjects being white and male . in this population of subjects with a history of cvd
/ cv event or risk factors , the most common baseline conditions were hypertension and hypercholesterolemia . slightly under half of the subjects
baseline lung function results indicated that the study population had moderate - to - severe airflow limitation ( table 1 ) , and approximately one - third of the subjects demonstrated acute bronchodilator reversibility .
baseline health - related quality of life ( qol ) and health status , indicated by sgrq - c and cat scores , respectively , suggested impairment and a high level of copd symptoms .
results of the primary efficacy measure , change from baseline trough in 24-hour weighted mean fev1 after 12 weeks treatment , were similar between the treatment groups ( figure 2 ) , with a least squares ( ls ) mean change from baseline of 117 ml and 95 ml in the ff / vi and tio groups , respectively , with a difference of 0.022 l ( 95% confidence interval [ ci ] 0.012 to 0.055 ) ( p=0.201 ) . in the reversible subgroup , however , the mean change was greater in the ff / vi group compared with the tio group ( 181 ml and 110 ml , respectively ) , with a difference of 0.071 l ( 95% ci 0.0120.129 ) . other stratification subgroups ( ie , nonreversible , 1 copd exacerbation in last 3 years , no copd exacerbation in last 3 years ) demonstrated small or no treatment group differences .
because the primary endpoint difference did not reach statistical significance , all differences reported for the secondary efficacy endpoints are presented descriptively only as statistical inference can not be made .
table 2 shows that 79% and 82% of subjects receiving ff / vi or tio , respectively , achieved an increase in fev1 of 100 ml from baseline within 4 hours postdose on treatment day 1 , with a somewhat faster onset in the ff / vi group , with the median time to onset of 17.0 minutes compared with the median time of 20.5 minutes in the tio group .
more subjects in the ff / vi group than the tio group demonstrated an onset of effect within the first 5 minutes of dosing ( table 2 ) .
ff / vi and tio improved trough fev1 on day 84 to a similar degree . both change from baseline in trough ic and change from baseline in trough fvc favored tio treatment .
changes from baseline in the individual symptoms of cough and sputum production were similar between groups ( table 3 ) .
treatment with ff / vi reduced subjects use of rescue medication during weeks 112 , with an ls mean change difference of 0.37 inhalations ( 95% ci 0.55 to 0.19 ) and during each of the week - long treatment periods ( figure 3a ) .
likewise , the percentage of rescue - free 24-hour periods was increased by ff / vi treatment compared with tio during weeks 112 , with an ls mean change difference of 9.1% ( 95% ci 4.014.2 ) and during each week ( figure 3b ) .
while sgrq - c baseline scores were similar between treatment groups ( table 1 ) and both groups improved with treatment ( figure 4a ) , ff / vi demonstrated a consistently greater numerical improvement in total score than tio treatment , and only ff / vi treatment reached the clinically significant mcid change score of 4 ( week 8) ; achievement of the total score mcid was not persistent at week 12 .
with respect to individual sgrq - c components ( symptoms , impacts , activity ) , ff / vi consistently reduced each component score more than tio , and only the ff / vi group achieved the mcid for symptoms across all time points .
a responder analysis for total sgrq - c score revealed that a higher percentage of ff / vi - treated than tio - treated subjects , respectively , achieved the mcid after 4 weeks ( 46% vs 34% ) , 8 weeks ( 49% vs 36% ) , and 12 weeks ( 48% vs 42% ) of treatment .
cat scores at baseline were high ( 10 ) and similar between groups ( table 1 ) .
the ff / vi group consistently demonstrated a numerically greater improvement in cat score over the treatment period ( figure 4b ) .
seven biomarkers ( hscrp , ccl-18 , sp - d , fibrinogen , il-6 , cc-16 , and bnp ) were measured in a subset of subjects ( 165 ff / vi , 169 tio ) at baseline and week 12 .
there were no significant changes from baseline in bnp , fibrinogen , or il-6 with either ff / vi or tio .
for both the treatment groups , there were decreases in ccl-18 and cc-16 and an increase in sp - d ; hscrp only increased at week 12 in the tio group .
however , for all seven biomarkers , the change from baseline was not significantly different between the two treatment groups ( data not shown ) .
over one - third of the subjects experienced an ae during treatment ( table 4 ) , the most frequent of which were headache , nasopharyngitis , back pain , and oral candidiasis . more tio - treated than ff / vi - treated subjects were withdrawn from the study due to aes .
two tio - treated subjects died ( one due to cardiopulmonary arrest and the other due to cardiorespiratory arrest and cardiac failure ) .
more ff / vi than tio - treated subjects experienced an ae considered drug related by the investigator .
pneumonia was reported in three subjects during treatment with ff / vi , all of which were radiologically confirmed and treated with antibiotics .
one ff / vi - treated subject developed pneumonia 19 days after study medication ( ff / vi ) was finished .
aes of special interest based on the known effects of corticosteroid and laba treatment were prespecified .
the most commonly reported were cv effects and local steroid effects ( table 4 ) .
fewer subjects in the ff / vi group ( 7/310 ; 2% ) experienced a copd exacerbation than in the tio group ( 11/313 ; 4% ) , and all were withdrawn from the study . although the protocol required subjects to be withdrawn upon experiencing an exacerbation , one subject in the ff / vi group was not withdrawn until after having a second event . of those withdrawn ,
five ff / vi - treated and nine tio - treated subjects had a history of copd exacerbation .
three of eight exacerbations in the ff / vi group required hospitalization compared with one of eleven in the tio group ; per protocol , only the hospitalized events were captured as adverse events .
compared with the ff / vi group , more subjects in the tio group who had an exacerbation were treated with antibiotics alone or in conjunction with oral corticosteroids . there were no clinically significant differences between groups in mean changes from baseline for pulse rate , heart rate , or qtc intervals . at week 12 , few subjects ( 2% in each group ) had a qtc(f ) > 450 , and few ( 1% in each group ) had a change from baseline in qtc(f ) 60 ms . the percentage of subjects with ecg findings defined a priori as abnormalities of potential clinical importance was similar at baseline ( 9% ff / vi group , 13% tio group ) and at the end of treatment ( 8% ff / vi group , 7% tio group ) .
holter monitoring was conducted in a subset of subjects ( 158 ff / vi , 153 tio ) , with no significant difference between ff / vi and tio groups in abnormalities of potential clinical significance , including mean heart rate , cardiac rhythm abnormalities ( ventricular ectopic events ) , or other prespecified abnormalities of potential clinical importance .
most of the abnormalities were nonsustained ventricular tachycardias ( defined as > 100 beats / min , 330 beats ) or right bundle branch blocks .
there was a higher incidence of nonsustained ventricular tachycardia in the ff / vi group ( 9% ) than the tio group ( 4% ) and of right bundle branch block in the tio group ( 5% ) than the ff / vi group ( 2% ) .
there were no reports of sustained ventricular tachycardia or sustained supraventricular tachycardia ( defined as > 100 beats / min , > 30 beats ) at any time postrandomization in either group .
abnormalities of potential clinical importance were reported in more subjects at the end of treatment ( 14% ff / vi , 12% tio ) than at screening ( 4% ff / vi , 5% tio ) . more subjects in both groups had ventricular ectopics with 1 ventricular run at the end of treatment ( 11% ff / vi , 6% tio ) than at screening ( < 1% ff / vi , 2% tio ) .
clinical chemistry and hematology results were generally similar between groups , and mean changes from baseline were minimal .
cortisol excretion was measured by 24-hour urine collection in a subset of subjects ( 104 on ff / vi , 103 on tio ) .
there were no clinically significant differences in ls mean urinary cortisol ratio of week 12 to baseline between groups ( ff / vi [ 1.06 ] vs tio [ 1.02 ] ratio of 1.038 ; 95% ci 0.861.26 ) .
at screening , 890 subjects were evaluated for eligibility ( figure 1 ) . of those ,
267 ( 30% ) subjects were not randomized mainly because they failed the strict enrollment criteria , for example , fev1 30%70% predicted ( n=112 ) , and/or had an abnormal holter ( n=75 ) and/or ecg ( n=55 ) finding .
following the run - in period , 310 and 313 subjects were randomized to treatment with ff / vi and tio , respectively , and constituted the intent - to - treat population . of those , 291 ( 94% ) and 274 ( 88% ) subjects , respectively , completed the study .
more subjects were withdrawn from treatment with tio , mainly due to aes and lack of efficacy / copd exacerbations , than with ff / vi .
three percent of tio - treated and 1% of ff / vi - treated subjects withdrew from the study due to protocol deviations .
subjects demographic and baseline characteristics were similar between treatment groups ( table 1 ) , with the majority of subjects being white and male . in this population of subjects with a history of cvd
/ cv event or risk factors , the most common baseline conditions were hypertension and hypercholesterolemia . slightly under half of the subjects
baseline lung function results indicated that the study population had moderate - to - severe airflow limitation ( table 1 ) , and approximately one - third of the subjects demonstrated acute bronchodilator reversibility .
baseline health - related quality of life ( qol ) and health status , indicated by sgrq - c and cat scores , respectively , suggested impairment and a high level of copd symptoms .
results of the primary efficacy measure , change from baseline trough in 24-hour weighted mean fev1 after 12 weeks treatment , were similar between the treatment groups ( figure 2 ) , with a least squares ( ls ) mean change from baseline of 117 ml and 95 ml in the ff / vi and tio groups , respectively , with a difference of 0.022 l ( 95% confidence interval [ ci ] 0.012 to 0.055 ) ( p=0.201 ) .
in the reversible subgroup , however , the mean change was greater in the ff / vi group compared with the tio group ( 181 ml and 110 ml , respectively ) , with a difference of 0.071 l ( 95% ci 0.0120.129 ) . other stratification subgroups ( ie , nonreversible , 1 copd exacerbation in last 3 years , no copd exacerbation in last 3 years ) demonstrated small or no treatment group differences .
because the primary endpoint difference did not reach statistical significance , all differences reported for the secondary efficacy endpoints are presented descriptively only as statistical inference can not be made .
table 2 shows that 79% and 82% of subjects receiving ff / vi or tio , respectively , achieved an increase in fev1 of 100 ml from baseline within 4 hours postdose on treatment day 1 , with a somewhat faster onset in the ff / vi group , with the median time to onset of 17.0 minutes compared with the median time of 20.5 minutes in the tio group .
more subjects in the ff / vi group than the tio group demonstrated an onset of effect within the first 5 minutes of dosing ( table 2 ) .
ff / vi and tio improved trough fev1 on day 84 to a similar degree .
both change from baseline in trough ic and change from baseline in trough fvc favored tio treatment .
changes from baseline in the individual symptoms of cough and sputum production were similar between groups ( table 3 ) .
treatment with ff / vi reduced subjects use of rescue medication during weeks 112 , with an ls mean change difference of 0.37 inhalations ( 95% ci 0.55 to 0.19 ) and during each of the week - long treatment periods ( figure 3a ) .
likewise , the percentage of rescue - free 24-hour periods was increased by ff / vi treatment compared with tio during weeks 112 , with an ls mean change difference of 9.1% ( 95% ci 4.014.2 ) and during each week ( figure 3b ) .
while sgrq - c baseline scores were similar between treatment groups ( table 1 ) and both groups improved with treatment ( figure 4a ) , ff / vi demonstrated a consistently greater numerical improvement in total score than tio treatment , and only ff / vi treatment reached the clinically significant mcid change score of 4 ( week 8) ; achievement of the total score mcid was not persistent at week 12 .
with respect to individual sgrq - c components ( symptoms , impacts , activity ) , ff / vi consistently reduced each component score more than tio , and only the ff / vi group achieved the mcid for symptoms across all time points .
a responder analysis for total sgrq - c score revealed that a higher percentage of ff / vi - treated than tio - treated subjects , respectively , achieved the mcid after 4 weeks ( 46% vs 34% ) , 8 weeks ( 49% vs 36% ) , and 12 weeks ( 48% vs 42% ) of treatment .
cat scores at baseline were high ( 10 ) and similar between groups ( table 1 ) .
the ff / vi group consistently demonstrated a numerically greater improvement in cat score over the treatment period ( figure 4b ) .
seven biomarkers ( hscrp , ccl-18 , sp - d , fibrinogen , il-6 , cc-16 , and bnp ) were measured in a subset of subjects ( 165 ff / vi , 169 tio ) at baseline and week 12 .
there were no significant changes from baseline in bnp , fibrinogen , or il-6 with either ff / vi or tio .
for both the treatment groups , there were decreases in ccl-18 and cc-16 and an increase in sp - d ; hscrp only increased at week 12 in the tio group .
however , for all seven biomarkers , the change from baseline was not significantly different between the two treatment groups ( data not shown ) .
over one - third of the subjects experienced an ae during treatment ( table 4 ) , the most frequent of which were headache , nasopharyngitis , back pain , and oral candidiasis . more tio - treated than ff / vi - treated subjects were withdrawn from the study due to aes .
two tio - treated subjects died ( one due to cardiopulmonary arrest and the other due to cardiorespiratory arrest and cardiac failure ) .
more ff / vi than tio - treated subjects experienced an ae considered drug related by the investigator .
pneumonia was reported in three subjects during treatment with ff / vi , all of which were radiologically confirmed and treated with antibiotics .
one ff / vi - treated subject developed pneumonia 19 days after study medication ( ff / vi ) was finished .
aes of special interest based on the known effects of corticosteroid and laba treatment were prespecified .
the most commonly reported were cv effects and local steroid effects ( table 4 ) .
fewer subjects in the ff / vi group ( 7/310 ; 2% ) experienced a copd exacerbation than in the tio group ( 11/313 ; 4% ) , and all were withdrawn from the study . although the protocol required subjects to be withdrawn upon experiencing an exacerbation , one subject in the ff / vi group was not withdrawn until after having a second event . of those withdrawn ,
five ff / vi - treated and nine tio - treated subjects had a history of copd exacerbation .
three of eight exacerbations in the ff / vi group required hospitalization compared with one of eleven in the tio group ; per protocol , only the hospitalized events were captured as adverse events .
compared with the ff / vi group , more subjects in the tio group who had an exacerbation were treated with antibiotics alone or in conjunction with oral corticosteroids . there were no clinically significant differences between groups in mean changes from baseline for pulse rate , heart rate , or qtc intervals . at week 12 , few subjects ( 2% in each group ) had a qtc(f ) > 450 , and few ( 1% in each group ) had a change from baseline in qtc(f ) 60 ms .
the percentage of subjects with ecg findings defined a priori as abnormalities of potential clinical importance was similar at baseline ( 9% ff / vi group , 13% tio group ) and at the end of treatment ( 8% ff / vi group , 7% tio group ) .
holter monitoring was conducted in a subset of subjects ( 158 ff / vi , 153 tio ) , with no significant difference between ff / vi and tio groups in abnormalities of potential clinical significance , including mean heart rate , cardiac rhythm abnormalities ( ventricular ectopic events ) , or other prespecified abnormalities of potential clinical importance .
most of the abnormalities were nonsustained ventricular tachycardias ( defined as > 100 beats / min , 330 beats ) or right bundle branch blocks .
there was a higher incidence of nonsustained ventricular tachycardia in the ff / vi group ( 9% ) than the tio group ( 4% ) and of right bundle branch block in the tio group ( 5% ) than the ff / vi group ( 2% ) .
there were no reports of sustained ventricular tachycardia or sustained supraventricular tachycardia ( defined as > 100 beats / min , > 30 beats ) at any time postrandomization in either group
. abnormalities of potential clinical importance were reported in more subjects at the end of treatment ( 14% ff / vi , 12% tio ) than at screening ( 4% ff / vi , 5% tio ) .
more subjects in both groups had ventricular ectopics with 1 ventricular run at the end of treatment ( 11% ff / vi , 6% tio ) than at screening ( < 1% ff / vi , 2% tio ) .
clinical chemistry and hematology results were generally similar between groups , and mean changes from baseline were minimal .
cortisol excretion was measured by 24-hour urine collection in a subset of subjects ( 104 on ff / vi , 103 on tio ) .
there were no clinically significant differences in ls mean urinary cortisol ratio of week 12 to baseline between groups ( ff / vi [ 1.06 ] vs tio [ 1.02 ] ratio of 1.038 ; 95% ci 0.861.26 ) .
our results in this head - to - head comparator trial between ff / vi and tio in subjects with copd who had or were at risk of cvd showed that both treatments resulted in lung function improvement > 12 weeks and were consistent with previous reports of ff / vi treatment effects from randomized controlled trials.1113,2224 the change from baseline in 24-hour weighted mean fev1 after 12-week treatment ( 117 ml for ff / vi and 95 ml for tio ) exceeded a proposed mcid for weighted mean fev125 and was within the putative mcid range of 100140 ml for trough
ff / vi;26 however , the treatment difference between the two therapies was not statistically significant . in our study ,
secondary endpoints were nested under the primary endpoint ; since the primary endpoint difference did not reach statistical significance , all differences reported for the secondary efficacy endpoints must be regarded as descriptive only .
nonetheless , numerical differences were observed between the treatment groups in some other lung function endpoints , symptoms , use of rescue medication , and qol .
rescue medication use and dyspnea were consistently reduced over the 12 weeks of treatment with ff / vi compared with tio .
qol ( measured by mean sgrq - c total and component scores and by the percentage of sgrq - c responders ) and health status ( measured by mean cat scores ) improved with both treatments , but the improvement was greater with ff / vi treatment compared with tio . the total sgrq - c improvement with ff / vi was 5.0 at 8 weeks and 3.9 at 12 weeks , similar to the result of 4.3 at 12 weeks in another study.25 a previous comparison of another ics / laba with tio provided similar findings of a superior effect of ics / laba on qol.7 in our study , mean sgrq - c improvements reached or approached the mcid after treatment with ff / vi , but not tio .
in contrast , cat improvement scores were more modest after ff / vi treatment and did not approach the mcid .
although the most reliable estimate of mcid may be two points,27 the cat only allows integer scores , and the bar may be set too high for population - based comparisons.28 as copd progresses , patients experience increasing air trapping and hyperinflation reflected by ic declines.29 in our study , 12 weeks tio treatment compared with ff / vi provided improvements in trough ic and fvc .
improvements in ic have been associated with decreased exertional but not resting dyspnea.30,31 in this regard , it is noteworthy that although treatment with tio was associated with a greater improvement in ic , dyspnea improved more so with ff / vi .
the safety profiles of ff / vi and tio in this study were similar , with a couple of exceptions .
pneumonia was reported more frequently in ff / vi - treated subjects , all radiographically confirmed , consistent with previous reports of a higher incidence with ff / vi treatment13 or other ics treatment alone or in combination with a laba.32,33 more subjects failed to complete the study while receiving tio ( 12% vs 6% for ff / vi ) , including those who experienced copd exacerbations ( 4% vs 2% , respectively ) . in 1-year studies of ff / vi versus vi,13 treatment with ff / vi reduced exacerbation rates compared with treatment with vi alone .
likewise , in a 2-year study comparing tio with fp / salm , more tio - treated subjects withdrew , but the rates of copd exacerbation did not differ between groups.7 in our population of patients with comorbid cvd or increased risk of cvd , there were no apparent effects of ff / vi or tio treatment on the cv safety profile or differences between treatments , with the exception of two deaths from cv events in the tio - treated subjects .
we paid particular attention to measuring cv safety , employing 24-hour holter monitoring in a subset of subjects , performing frequent ecgs , and monitoring cv aes because this was an at - risk population and due to previous reports of tio and laba cv safety .
while some earlier analyses suggested that the risk of cvd or cv mortality was increased in copd patients using the tio respimat ( boehringer ingelheim ) inhaler,5,34,35 this was not confirmed in a subsequent trial,35 and the tio handihaler has not been associated with an increased risk compared with placebo in recent studies.6,3639 in our study , the rate of ventricular tachycardias was somewhat higher than anticipated .
however , the definition can vary from that used here ( > 100 beats / min ) to that of > 120 beats / min.4041 also , subjects in both groups experienced a greater incidence of ventricular ectopic beats at the end of the study than at screening , but the circumstances of those holter measurements differed , as the end - of - study visit was conducted with a 24-hour in - patient stay .
the cv risk profile of ff / vi versus tio has also been examined in a recent clinical study of ff / vi effects on arterial stiffness , a marker that is elevated in copd and is a predictor of cv events and mortality.42,43 arterial stiffness , as measured by aortic pulse wave velocity , was lowered by both ff / vi and tio in subjects with moderate - to - very severe copd and baseline aortic pulse wave velocity 11.0 m / s.44 these subjects also had a high incidence of baseline cvd , but the number of cv events over the 12-week treatment period was low and similar between treatments.44 results of the ongoing summit trial in 16,000 subjects with comorbid cvd risk and copd will further determine the impact of ff / vi on cvd and mortality.45 the major limitations of our study were its lack of placebo arm , relatively small sample size , and 12-week duration , making the study incapable of establishing effects on pneumonia incidence , cvd , or mortality . however , this randomized study had several strengths .
second , it applied rigorous cv monitoring and compared ics / laba and tio treatment in a population with comorbid cvd .
most importantly , the study is the first to compare the efficacy of a once - daily ics / laba with tio .
patients with copd demonstrate low adherence to study medication,46 which is significantly associated with increased risk of death and copd exacerbations.47 once - daily medications such as tio have been shown to improve adherence in medication use over medications with more frequent dosing regimens,4850 and it is expected that ff / vi will have similar benefits , especially in certain patient populations who prefer once - daily medications.51 adherence to study medication in the current study was high , with no difference between the ff / vi and tio groups .
in conclusion , treatment with ff / vi or tio for 12 weeks improved lung function in copd subjects who have or are at risk for comorbid cvd , but there was no significant difference between the treatment groups .
reduction in rescue medication use and dyspnea and improving sgrq - c - indicated qol favored ff / vi , whereas improvements in fvc and ic favored tio .
other than more reports of pneumonia in ff / vi - treated subjects and cv aes in tio - treated subjects , the safety profiles of the treatments were similar .
the risk of pneumonia should be weighed against the benefits of ff / vi treatment , such as the reduction in copd exacerbations , increased qol , and medication adherence , when clinicians consider the potential use of ff / vi for patients with copd .
the results of an ongoing larger ff / vi trial in subjects with copd with cvd ( summit)45 are anticipated to provide additional evidence regarding efficacy and safety in this population . | backgroundfluticasone furoate / vilanterol ( ff / vi ) is a novel , once - daily , inhaled corticosteroid / long - acting 2-agonist combination approved for the treatment of copd and asthma .
we compared the safety and efficacy of ff / vi and tiotropium ( tio ) in subjects with moderate - to - severe copd with greater risk for comorbid cardiovascular disease ( cvd).methodsthis randomized , blinded , double - dummy , parallel - group study compared a once - daily morning dose of ff / vi 100/25 mcg delivered via ellipta with tio 18 mcg via handihaler for 12 weeks in subjects with diagnosed copd , forced expiratory volume in 1 second ( fev1 ) 30%70% predicted , and cvd or cvd risk .
the primary endpoint was change from baseline in 24-hour weighted mean fev1 on day 84 .
other efficacy endpoints included time to onset of bronchodilation , trough fev1 , other spirometry measures , rescue medication use , symptoms , quality of life ( st george s respiratory questionnaire - copd [ sgrq - c ] ) , and health status ( copd assessment tests [ cat ] ) measures .
safety endpoints included cardiovascular monitoring , cortisol excretion , copd exacerbations , and adverse events , including prespecified drug effects.resultsboth ff / vi and tio improved the 24-hour weighted mean fev1 from baseline after 12 weeks with no significant difference between treatments .
other endpoints favored ff / vi for time to onset of bronchodilation , rescue medication use , dyspnea , sgrq - c and cat scores , or favored tio for change from baseline in forced vital capacity and inspiratory capacity .
pneumonia occurred more frequently in the ff / vi group , and two tio - treated subjects died following cardiovascular events .
other safety measures were similar between groups , and cardiovascular monitoring did not reveal increased cvd risk.conclusionboth ff / vi and tio were efficacious in improving lung function in subjects with copd and comorbid cvd or cvd risk factors , with minor differences in efficacy and safety profiles . | Introduction
Methods
Subjects
Study design and treatment
Study assessments
Statistical analysis
Results
Subject characteristics
Efficacy
Biomarkers
Safety
Discussion
Conclusion | successful management of moderate - to - severe copd can be accomplished with combination therapy that delivers a concurrent dose of inhaled corticosteroid and a long - acting 2-agonist ( ics / laba ) or with tiotropium ( tio ) , an inhaled long - acting anticholinergic bronchodilator.1 both twice - daily ics / laba and once - daily tio treatment for copd have been demonstrated to improve lung function and health status as well as reduce copd exacerbations.16 head - to - head comparisons between ics / laba drugs and tio , however , are limited and the efficacy results inconclusive,7,8 leading researchers to call for further large , long - term randomized controlled trials to establish relative efficacy and safety of these treatments , as well as compare newly developed alternative ics / labas with tio.8 a newly introduced ics / laba treatment for copd in a novel inhaler device combines fluticasone furoate ( ff ) , an ics with enhanced selectivity , retention , and affinity for the glucocorticoid receptor,9 and vilanterol ( vi ) , a laba with prolonged bronchodilating properties.10 ff / vi is the only once - daily ics / laba treatment and is well tolerated , improves lung function , and sustains bronchodilation after 24 weeks and 52 weeks of treatment for copd.1113 when once - daily treatment with ff / vi for 12 weeks was compared with twice - daily fluticasone propionate / salmeterol 250/50 ( fp / salm ) for copd , there was evidence of numerically greater effect on the forced expiratory volume in 1 second ( fev1 ) and faster onset for ff / vi compared with fp / salm with similar safety profiles for the two drugs.14 copd is not only a disease of the airways and lung parenchyma , but there are several extrapulmonary comorbidities associated with copd,15 of which cardiovascular disease ( cvd ) is arguably the most significant . cvd is a comorbidity of copd,1618 and the risk of cardiovascular ( cv ) events rises in subjects with copd with a history of myocardial infarction.19 furthermore , concerns regarding cv safety and copd medications have been raised for both tio5 and laba drugs.20 therefore , we conducted the study in a population of subjects with or at risk for cvd . the objective of this study was to compare the efficacy and safety of ff / vi and tio , both delivered once daily , for copd treatment over a 12-week period in a randomized , controlled trial . an additional objective was to investigate whether the efficacy and/or safety profile of ff / vi or tio would differ in subjects with cvd or in subjects at risk for cvd . this study was a 12-week , randomized ( 1:1 ) , blinded , stratified , double - dummy , parallel - group , active comparator study . at the end of the run - in period ,
eligible subjects were randomly assigned to once - daily morning treatment with either ff / vi 100/25 mcg ( breo [ relvar , revinty ] , glaxosmithkline plc [ gsk ] , london , uk ) delivered via the ellipta inhaler ( gsk , london , uk ) or tio 18 mcg ( spiriva , boehringer ingelheim , ingelheim , germany ) delivered from blister capsules by the handihaler ( spiriva , boehringer ingelheim ) . at the screening visit , pre- and postbronchodilator spirometry , medical history , physical examination , 12-lead ecg , 24-hour holter monitoring ( subset of subjects ) , chest x - ray , vital signs , serum pregnancy tests , clinical laboratory tests , oropharyngeal examination , and 24-hour urine cortisol collection ( subset of subjects ) were conducted , and diary cards were dispensed for recording of medical problems , concomitant medications , rescue medication use , and symptoms ( dyspnea , cough , sputum production ) . at all subsequent visits , eligible randomized subjects completed the st george s respiratory questionnaire - copd ( sgrq - c ) and copd assessment tests ( cat ) prior to other assessments , and safety monitoring was conducted , including oropharyngeal examination , ae reporting , exacerbation and pneumonia assessment , and diary card review . assuming a standard deviation of 215 ml and significance at the two - sided 5% level , a sample size of 271 subjects per treatment arm was required for 90% power to detect a 60 ml difference between ff / vi and tio for 24-hour weighted - mean fev1 at week 12 , an estimate based on historical data . the primary endpoint , change from baseline in 24-hour weighted mean fev1 at week 12 , was analyzed using analysis of covariance with covariates of baseline fev1 , randomization stratum ( copd exacerbation history and reversibility status ) , smoking status at screening , and country . the secondary endpoints ( time to onset [ increase of 100 ml above baseline in fev1 ] 04 hours postdose on day 1 and change from baseline in trough fev1 on day 84 ) were nested under the primary endpoint . change from baseline in sgrq - c and cat scores was analyzed using mixed model repeated measures with covariates of baseline score , exacerbation history and reversibility stratum , smoking status , country , treatment group , day , day by baseline , and day by treatment group . this study was a 12-week , randomized ( 1:1 ) , blinded , stratified , double - dummy , parallel - group , active comparator study . at the end of the run - in period ,
eligible subjects were randomly assigned to once - daily morning treatment with either ff / vi 100/25 mcg ( breo [ relvar , revinty ] , glaxosmithkline plc [ gsk ] , london , uk ) delivered via the ellipta inhaler ( gsk , london , uk ) or tio 18 mcg ( spiriva , boehringer ingelheim , ingelheim , germany ) delivered from blister capsules by the handihaler ( spiriva , boehringer ingelheim ) . at the screening visit , pre- and postbronchodilator spirometry , medical history , physical examination , 12-lead ecg , 24-hour holter monitoring ( subset of subjects ) , chest x - ray , vital signs , serum pregnancy tests , clinical laboratory tests , oropharyngeal examination , and 24-hour urine cortisol collection ( subset of subjects ) were conducted , and diary cards were dispensed for recording of medical problems , concomitant medications , rescue medication use , and symptoms ( dyspnea , cough , sputum production ) . at all subsequent visits , eligible randomized subjects completed the st george s respiratory questionnaire - copd ( sgrq - c ) and copd assessment tests ( cat ) prior to other assessments , and safety monitoring was conducted , including oropharyngeal examination , ae reporting , exacerbation and pneumonia assessment , and diary card review . assuming a standard deviation of 215 ml and significance at the two - sided 5% level , a sample size of 271 subjects per treatment arm was required for 90% power to detect a 60 ml difference between ff / vi and tio for 24-hour weighted - mean fev1 at week 12 , an estimate based on historical data . the primary endpoint , change from baseline in 24-hour weighted mean fev1 at week 12 , was analyzed using analysis of covariance with covariates of baseline fev1 , randomization stratum ( copd exacerbation history and reversibility status ) , smoking status at screening , and country . the secondary endpoints ( time to onset [ increase of 100 ml above baseline in fev1 ] 04 hours postdose on day 1 and change from baseline in trough fev1 on day 84 ) were nested under the primary endpoint . change from baseline in sgrq - c and cat scores was analyzed using mixed model repeated measures with covariates of baseline score , exacerbation history and reversibility stratum , smoking status , country , treatment group , day , day by baseline , and day by treatment group . following the run - in period , 310 and 313 subjects were randomized to treatment with ff / vi and tio , respectively , and constituted the intent - to - treat population . baseline health - related quality of life ( qol ) and health status , indicated by sgrq - c and cat scores , respectively , suggested impairment and a high level of copd symptoms . results of the primary efficacy measure , change from baseline trough in 24-hour weighted mean fev1 after 12 weeks treatment , were similar between the treatment groups ( figure 2 ) , with a least squares ( ls ) mean change from baseline of 117 ml and 95 ml in the ff / vi and tio groups , respectively , with a difference of 0.022 l ( 95% confidence interval [ ci ] 0.012 to 0.055 ) ( p=0.201 ) . in the reversible subgroup , however , the mean change was greater in the ff / vi group compared with the tio group ( 181 ml and 110 ml , respectively ) , with a difference of 0.071 l ( 95% ci 0.0120.129 ) . table 2 shows that 79% and 82% of subjects receiving ff / vi or tio , respectively , achieved an increase in fev1 of 100 ml from baseline within 4 hours postdose on treatment day 1 , with a somewhat faster onset in the ff / vi group , with the median time to onset of 17.0 minutes compared with the median time of 20.5 minutes in the tio group . ff / vi and tio improved trough fev1 on day 84 to a similar degree . while sgrq - c baseline scores were similar between treatment groups ( table 1 ) and both groups improved with treatment ( figure 4a ) , ff / vi demonstrated a consistently greater numerical improvement in total score than tio treatment , and only ff / vi treatment reached the clinically significant mcid change score of 4 ( week 8) ; achievement of the total score mcid was not persistent at week 12 . with respect to individual sgrq - c components ( symptoms , impacts , activity ) , ff / vi consistently reduced each component score more than tio , and only the ff / vi group achieved the mcid for symptoms across all time points . a responder analysis for total sgrq - c score revealed that a higher percentage of ff / vi - treated than tio - treated subjects , respectively , achieved the mcid after 4 weeks ( 46% vs 34% ) , 8 weeks ( 49% vs 36% ) , and 12 weeks ( 48% vs 42% ) of treatment . fewer subjects in the ff / vi group ( 7/310 ; 2% ) experienced a copd exacerbation than in the tio group ( 11/313 ; 4% ) , and all were withdrawn from the study . holter monitoring was conducted in a subset of subjects ( 158 ff / vi , 153 tio ) , with no significant difference between ff / vi and tio groups in abnormalities of potential clinical significance , including mean heart rate , cardiac rhythm abnormalities ( ventricular ectopic events ) , or other prespecified abnormalities of potential clinical importance . following the run - in period , 310 and 313 subjects were randomized to treatment with ff / vi and tio , respectively , and constituted the intent - to - treat population . slightly under half of the subjects
baseline lung function results indicated that the study population had moderate - to - severe airflow limitation ( table 1 ) , and approximately one - third of the subjects demonstrated acute bronchodilator reversibility . baseline health - related quality of life ( qol ) and health status , indicated by sgrq - c and cat scores , respectively , suggested impairment and a high level of copd symptoms . results of the primary efficacy measure , change from baseline trough in 24-hour weighted mean fev1 after 12 weeks treatment , were similar between the treatment groups ( figure 2 ) , with a least squares ( ls ) mean change from baseline of 117 ml and 95 ml in the ff / vi and tio groups , respectively , with a difference of 0.022 l ( 95% confidence interval [ ci ] 0.012 to 0.055 ) ( p=0.201 ) . in the reversible subgroup , however , the mean change was greater in the ff / vi group compared with the tio group ( 181 ml and 110 ml , respectively ) , with a difference of 0.071 l ( 95% ci 0.0120.129 ) . table 2 shows that 79% and 82% of subjects receiving ff / vi or tio , respectively , achieved an increase in fev1 of 100 ml from baseline within 4 hours postdose on treatment day 1 , with a somewhat faster onset in the ff / vi group , with the median time to onset of 17.0 minutes compared with the median time of 20.5 minutes in the tio group . ff / vi and tio improved trough fev1 on day 84 to a similar degree . while sgrq - c baseline scores were similar between treatment groups ( table 1 ) and both groups improved with treatment ( figure 4a ) , ff / vi demonstrated a consistently greater numerical improvement in total score than tio treatment , and only ff / vi treatment reached the clinically significant mcid change score of 4 ( week 8) ; achievement of the total score mcid was not persistent at week 12 . with respect to individual sgrq - c components ( symptoms , impacts , activity ) , ff / vi consistently reduced each component score more than tio , and only the ff / vi group achieved the mcid for symptoms across all time points . a responder analysis for total sgrq - c score revealed that a higher percentage of ff / vi - treated than tio - treated subjects , respectively , achieved the mcid after 4 weeks ( 46% vs 34% ) , 8 weeks ( 49% vs 36% ) , and 12 weeks ( 48% vs 42% ) of treatment . fewer subjects in the ff / vi group ( 7/310 ; 2% ) experienced a copd exacerbation than in the tio group ( 11/313 ; 4% ) , and all were withdrawn from the study . holter monitoring was conducted in a subset of subjects ( 158 ff / vi , 153 tio ) , with no significant difference between ff / vi and tio groups in abnormalities of potential clinical significance , including mean heart rate , cardiac rhythm abnormalities ( ventricular ectopic events ) , or other prespecified abnormalities of potential clinical importance . our results in this head - to - head comparator trial between ff / vi and tio in subjects with copd who had or were at risk of cvd showed that both treatments resulted in lung function improvement > 12 weeks and were consistent with previous reports of ff / vi treatment effects from randomized controlled trials.1113,2224 the change from baseline in 24-hour weighted mean fev1 after 12-week treatment ( 117 ml for ff / vi and 95 ml for tio ) exceeded a proposed mcid for weighted mean fev125 and was within the putative mcid range of 100140 ml for trough
ff / vi;26 however , the treatment difference between the two therapies was not statistically significant . qol ( measured by mean sgrq - c total and component scores and by the percentage of sgrq - c responders ) and health status ( measured by mean cat scores ) improved with both treatments , but the improvement was greater with ff / vi treatment compared with tio . the total sgrq - c improvement with ff / vi was 5.0 at 8 weeks and 3.9 at 12 weeks , similar to the result of 4.3 at 12 weeks in another study.25 a previous comparison of another ics / laba with tio provided similar findings of a superior effect of ics / laba on qol.7 in our study , mean sgrq - c improvements reached or approached the mcid after treatment with ff / vi , but not tio . the safety profiles of ff / vi and tio in this study were similar , with a couple of exceptions . pneumonia was reported more frequently in ff / vi - treated subjects , all radiographically confirmed , consistent with previous reports of a higher incidence with ff / vi treatment13 or other ics treatment alone or in combination with a laba.32,33 more subjects failed to complete the study while receiving tio ( 12% vs 6% for ff / vi ) , including those who experienced copd exacerbations ( 4% vs 2% , respectively ) . likewise , in a 2-year study comparing tio with fp / salm , more tio - treated subjects withdrew , but the rates of copd exacerbation did not differ between groups.7 in our population of patients with comorbid cvd or increased risk of cvd , there were no apparent effects of ff / vi or tio treatment on the cv safety profile or differences between treatments , with the exception of two deaths from cv events in the tio - treated subjects . the cv risk profile of ff / vi versus tio has also been examined in a recent clinical study of ff / vi effects on arterial stiffness , a marker that is elevated in copd and is a predictor of cv events and mortality.42,43 arterial stiffness , as measured by aortic pulse wave velocity , was lowered by both ff / vi and tio in subjects with moderate - to - very severe copd and baseline aortic pulse wave velocity 11.0 m / s.44 these subjects also had a high incidence of baseline cvd , but the number of cv events over the 12-week treatment period was low and similar between treatments.44 results of the ongoing summit trial in 16,000 subjects with comorbid cvd risk and copd will further determine the impact of ff / vi on cvd and mortality.45 the major limitations of our study were its lack of placebo arm , relatively small sample size , and 12-week duration , making the study incapable of establishing effects on pneumonia incidence , cvd , or mortality . patients with copd demonstrate low adherence to study medication,46 which is significantly associated with increased risk of death and copd exacerbations.47 once - daily medications such as tio have been shown to improve adherence in medication use over medications with more frequent dosing regimens,4850 and it is expected that ff / vi will have similar benefits , especially in certain patient populations who prefer once - daily medications.51 adherence to study medication in the current study was high , with no difference between the ff / vi and tio groups . in conclusion , treatment with ff / vi or tio for 12 weeks improved lung function in copd subjects who have or are at risk for comorbid cvd , but there was no significant difference between the treatment groups . reduction in rescue medication use and dyspnea and improving sgrq - c - indicated qol favored ff / vi , whereas improvements in fvc and ic favored tio . other than more reports of pneumonia in ff / vi - treated subjects and cv aes in tio - treated subjects , the safety profiles of the treatments were similar . the risk of pneumonia should be weighed against the benefits of ff / vi treatment , such as the reduction in copd exacerbations , increased qol , and medication adherence , when clinicians consider the potential use of ff / vi for patients with copd . the results of an ongoing larger ff / vi trial in subjects with copd with cvd ( summit)45 are anticipated to provide additional evidence regarding efficacy and safety in this population . | [
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] |
zr - labeled
monoclonal antibodies ( mabs ) have recently
been useful in clinical trials for positron emission tomography ( pet )
imaging of various cancers due to the favorable pairing of the radioisotope s
half - life ( t1/2 = 3.3 days ) and the antibody s
pharmacokinetic properties , complemented by the high sensitivity and
quantitative properties of pet .
pertuzumab ( perjeta ) is an fda - approved
mab for the treatment of human epidermal growth factor receptor 2
( her2)-positive metastatic breast cancer , in combination with trastuzumab
( herceptin ) , another her2specific monoclonal antibody .
pertuzumab
binds to domain ii of the human epidermal growth factor receptor-2
( her2 ) epitope , distinct from the trastuzumab epitope located within
domain iv .
both antibodies elicit antibody - dependent cellular cytotoxicity
( adcc ) ; however , the predominant mechanisms responsible for the synergy
in downregulating her2 expression are that pertuzumab inhibits her2
dimerization with other epidermal growth factor receptors and trastuzumab
inhibits intracellular signaling .
zr - labeled trastuzumab has been evaluated as a pet
imaging agent for detecting her2 expression in trastuzumab - treated
or in nave patients , with promising results toward the stratification
of patients mostly likely to respond to trastuzumab therapy .
however , investigations with pertuzumab as a pet imaging agent have
not been explored , to our knowledge .
pertuzumab has only been investigated
in vivo as a spect imaging agent by radiolabeling with in and lu .
however , the sensitivity of pet
imaging may be advantageous for further developing pertuzumab as an
imaging agent .
a qualitative comparison of fluorescently labeled cy5-trastuzumab
and cy5-pertuzumab in the presence and absence of either unconjugated
trastuzumab or pertuzumab has been conducted in her2 + xenograft models ,
which suggests imaging with cy5-pertuzumab in the presence of unconjugated
trastuzumab and imaging with cy5-trastuzumab in the presence of unconjugated
pertuzumab do not alter the tumor accumulation of each cy5-labeled
antibody . however ,
a quantitative comparison
between the tumor accumulation of radiolabeled trastuzumab and pertuzumab
as imaging agents during trastuzumab therapy has yet to be investigated .
furthermore , in silico studies suggest the binding of trastuzumab
induces a change of her2 conformation , permitting greater exposure
of the pertuzumab binding site and enhancing pertuzumab affinity . toward this end
herein , we demonstrate the specificity of zr - pertuzumab in her2 + tumors in vivo and quantitatively
show enhanced tumor accumulation of zr - pertuzumab in
the presence of trastuzumab .
pertuzumab ( perjeta ) and
trastuzumab ( herceptin ) were purchased from roche ( south san francisco ,
ca ) .
desferrioxamine - p - benzyl - isothiocyanate ( dfo - bz - ncs )
was purchased from macrocyclics ( dallas , tx ) .
the automated production
of zr - oxalate was carried out in house , adapted from previously established methods and neutralized to ph 6.87.2 as previously developed for
optimal radiolabeling .
all other chemicals
were purchased from sigma - aldrich ( st . louis , mo ) unless stated otherwise .
her2expressing breast cancer cell lines bt-474 and skbr3 as
well as her2nonexpressing mda - mb-231 were purchased from the
american type culture collection ( atcc , manassas , va ) and cultured
in iscove s modified dulbecco s medium ( imdm ) containing
10% fetal bovine serum ( fbs ) and 50 g / ml gentamycin ( complete
media ) in a humidified incubator with 5% co2 at 37 c .
reagents for cell culture were purchased from life technologies ( grand
island , ny ) unless stated otherwise .
conjugation of dfo - bz - ncs
to pertuzumab and subsequent radiolabeling with zr - oxalate
were performed following previous methods .
briefly , pertuzumab
( 10 mg / ml ) was conjugated to 8-fold molar excess of dfo - bz - ncs dissolved
in dimethyl sulfoxide in 0.1 m sodium carbonate buffer ( ph 9 ) at 37
c for 1 h. the resulting dfo - pertuzumab conjugate was purified
and buffer exchanged into 1 m hepes buffer ( ph 7.1 ) using gel filtration
spin columns ( mwco 40 kda , thermo fisher scientific , rockford , il ) .
dfo - pertuzumab was radiolabeled with neutralized zr - oxalate
using a ratio of 296:1 kbq:g at 37 c for 3060
min for a final ph between 6.87.2.zr-pertuzumab with radiochemical yields 95%
as determined by instant thin - layer chromatography was used for in
vitro and in vivo studies without further purification .
dfo - bz - ncs - pertuzumab
conjugate and unmodified pertuzumab were buffer exchanged in 0.25
m nh4oac buffer twice using zeba spin desalting columns
( thermo scientific , rockford , il ) following supplier s instructions .
the final concentration of protein was quantified using a bicinchoninic
acid ( bca ) assay ( thermo scientific , rockford , il ) .
a 10 l
aliquot was loaded into an offline electrospray capillary pulled in
house using borosilicate tubing on a laser based puller ( both b100 - 58 - 10
tubing and p-2000 puller were from sutter instrument company , novato ,
ca ) to perform the native mass spectrometry measurement .
the collision energy at the trap and transfer
region was adjusted for desolvation of the ions .
the pulled capillaries
were coated with gold using a sputter coating machine in the electron
microscopy core at washington university in st .
the sample
solution was infused to a hybrid ion - mobility quadrupole time - of - flight
mass spectrometer ( q - im - tof , synapt g2 hdms , waters inc . , milford ,
ma ) .
the instrument was operated under gentle esi conditions ( capillary
voltage 1.51.8 kv , sampling cone 20 v , extraction cone 2 v ,
source temperature 30 c ) .
the collision energy at the trap and
transfer region was adjusted for dissociating the complex .
the instrument
was externally calibrated up to 8000 m / z with the clusters produced by esi of a nai solution .
the peak picking
and data processing was performed in masslynx ( v 4.1 ) and driftscope
software ( water inc . , milford , ma ) .
the number of dfo per pertuzumab
was calculated by ( m / z of conjugate
m / z of unconjugated)/(dfo
molecular weight ) z , where m / z represents the observed mass to charge ratio
and z represents the charge species .
the immunoreactivity
of zr - pertuzumab was determined using the lindmo et al .
other cell
uptake experiments were performed in her2 + bt-474 and skbr3 , and her2
mda - mb-231 cells , in which 250 l of 1 10 cells / ml were seeded in 24-well plates .
cells were allowed to adhere
overnight at 37 c in a 5% co2 atmosphere .
the media
was removed and replaced with 125 l of fresh complete media
and 125 l of 50 ng / ml of zr - pertuzumab , saving
aliquots in microcentrifuge tubes as a standard .
the cells were washed three
times with pbs , trypsinized , and transferred to microcentrifuge tubes .
radioactivity associated with cells was counted in a beckman 8000
gamma counter ( beckman coulter inc . ,
the percentage of
bound radioactivity was calculated as the ratio of bound to the total
radioactivity added per well multiplied by 100 .
equal number of cells
per well were confirmed by counting the cells using a cellometer auto
t-4 cell counter ( nexcelom , lawrence , ma ) .
competitive binding
assays were performed on skbr3 cells adhered to 24-well plates as
described above .
cells were incubated with 1.7 10 m zr - pertuzumab in the presence of different concentrations
( 1.7 101.7 10 m ) of unlabeled pertuzumab or trastuzumab competitor in triplicate
wells at 4 c for 1.5 h. the cells were washed with pbs and dissociated
from the wells as described above and assayed in a gamma counter .
the percentage of zr - pertuzumab bound in the presence
of competitor / no competitor ( b / b0 100 ) was plotted against the log concentration
of added competitor and fitted with a sigmoidal dose
skbr3 cells ( 1 10 cells in
400 l ) were seeded into 12-well plates and allowed to adhere
overnight in the cell culture incubator .
zr - pertuzumab
or zr - trastuzumab positive control antibody was diluted to 50 ng / ml using complete media
and 125 l of each radiolabeled antibody was added to their
designated wells .
the plates were incubated at 37 c in a 5%
co2 atmosphere for 0.5 , 1 , 3 , 17 , 24 , 28 , 42 , and 48 h.
as a control , some reactions were performed at 4 c . at each
time point , the cells were washed three times with 400 l of
cold pbs and the cells were dissociated from the well via incubation
with 400 l of trypsin for 3 min at 37 c . the cells were
washed again with 200 l of cold pbs , transferred to a microcentrifuge
tube via pipet , and sedimented at 7000 g using a benchtop
centrifuge .
the supernatant was collected and the cells were treated
with 100 l of 0.1 m sodium citrate ( ph 2 ) for 5 min to remove
any surface bound zr - mab
. the cells were sedimented at
7000 g , the supernatant was combined with the previously
collected supernatant , and assayed in the gamma counter .
the amount
of zr - mab bound on the cell surface and internalized
was calculated as previously described .
all animal experiments were conducted
according to the guidelines of the institutional animal care and use
committee ( iacuc ) and approved by the washington university animal
studies committee . in vivo pet imaging and biodistribution studies
were conducted in severely combined immunodeficient female nog mice
( taconic , hudson , ny ) .
mice were inoculated with 150 l of 13
10 cells / ml ( bt-474 , skbr3 , and mda - mb231 ) suspended
in saline with no artificial stimuli .
mice
were injected via tail - vein with 100 l of 3.7 mbq of zr - pertuzumab ( specific activity 222296 mbq / mg ) in saline .
studies in the presence of unlabeled antibodies were performed with
a tail vein injection of 100 l of 10 mg / ml of either pertuzumab
or trastuzumab in saline , between 560 min prior to the injection
of zr - pertuzumab in the bt-474 xenograft .
static pet
images were acquired for 20 min using an inveon micropet / ct scanner
( siemens , knoxville , tn ) at 7 days p.i .
the images were reconstructed
using maximum a posteriori probability ( map ) algorithm and coregistered
with ct images using image display software ( inveon research workplace
workstation , siemens , schenectady , ny ) .
regions of interest ( roi )
were drawn for tumor uptake and analyzed as standard uptake values
( suv ) using the formula suv = ( mbq / ml ) ( animal weight ( g))/injected
dose ( mbq ) .
mice were sacrificed and organs were harvested , weighed ,
and assayed in the gamma counter for biodistribution studies .
radioactivity
associated with each organ was expressed as percentage of injected
dose per gram of organ ( % id / g ) .
tumors and spleen were fixed with
4% paraformaldehyde at 4 c until radioactivity decayed for about
1 week .
tumors were paraffin - embedded , sliced , mounted on slides and
stained with hematoxylin and eosin ( h&e ) by the washington university
pulmonary morphology core facility .
immunohistochemical staining
was performed by first blocking the slides with goat serum following
instructions from a vectastain elite abc kit ( vector laboratories ,
burlingame , ca ) and incubated with 1:200 dilution of a polyclonal
goat anti - her2 antibody ( santa cruz biotechnology , santa cruz , ca )
in pbs for 1 h at room temperature .
secondary antibody incubation
was performed following the kit s instructions and colorimetric
development was achieved using a 3,3-diaminobenzidine ( dab )
substrate ( vector laboratories , burlingame , ca ) .
digital images were collected
on a nikon optiphot-2 microscope equipped with a zeiss axiocam digital
camera .
cox analysis indicated
that the variances among the test groups were unequal and a logarithm
transformation of the suv and biodistribution data was necessary for
variance stabilization .
a one - way anova
was performed on the log transformation data , followed by group comparisons
using the tukey method to control multiple comparisons .
statistical
analysis was performed using graphpad prism v6 ( graphpad software
inc . ,
la jolla , ca ) and sas v9.3 ( sas institute , cary , nc ) .
although
statistical significance was performed on the transformed data , the
results are presented in the original metric for ease of discussion .
the conjugation of dfo - bz - ncs to pertuzumab was performed by combining
8 mol equiv of chelator at ph 9 for 1 h at 37 c , followed by
immediate purification .
esi - ms determined up to 4 chelates per antibody
conjugate , calculated from the mass shift of the unconjugated pertuzumab
( figure 1 ) .
subsequent radiolabeling with zr yielded specific activities of 222370 mbq / mg with
95% radiochemical yield as determined by instant thin layer
chromatography .
( a ) unconjugated
pertuzumab depicted in + 25 , + 24 , and + 23 charge states and ( b ) dfo - bz - ncs - pertuzumab
conjugate in the same charge states as the unconjugated control .
the immunoreactivity of zr - pertuzumab
was determined using the lindmo assay in her2 + skbr3 cells .
the immunoreactive fraction was calculated to
be 72.2% , indicating retention of antigen - binding ( figure 2a ) .
the specificity of zr - pertuzumab
was investigated in vitro by comparing its binding in her2 + ( skbr3
and bt-474 ) and her2 ( mda - mb-231 ) cells .
cell binding of zr - pertuzumab to skbr3 , bt-474 , and mda - mb-231 cells was
94.7 2.6 , 95.2 19.1 , and 3.8 0.1% of total radioactivity
added , respectively ( figure 2b ) .
significantly
greater binding was observed in her2 + compared to her2 cells
( p < 0.0001 ) .
skbr3 and bt-474 cells have been
reported to have a similar number of her2 receptors , 647811 834
and 10 59513 917 fmol / mg of protein , respectively .
cell binding studies with zr - pertuzumab
in skbr3 and bt-474 were consistent with pegram et al .
studies , as no statistical difference was observed between
skbr3 and bt-474 cells ( p > 0.05 ) .
competitive binding studies showed decreased uptake of the radiolabeled
antibody with increasing concentrations of unlabeled pertuzumab , with
a half maximal effective concentration ( ec50 ) of 2.4
0.11 nm ( figure 2c ) .
the absence of competitive
binding with unlabeled trastuzumab is consistent with an her2 epitope
binding distinction between zr - pertuzumab to domain ii
and trastuzumab to domain iv of her2 .
furthermore ,
the binding of zr - pertuzumab is increased by 30% in the
presence of high concentrations ( 0.022 m ) of unlabeled
trastuzumab .
this observation is also in agreement with the in silico
studies by fuentes et al .
conformational changes occur
upon trastuzumab binding resulting in the increased affinity of pertuzumab
for its her2 binding epitope .
lastly ,
a comparative rate of cellular internalization study was conducted
at 37 c with zr - pertuzumab and zr - trastuzumab .
the internalization of both antibodies was similar ; with 50% of zr - pertuzumab internalizing at 23 h and zr - trastuzumab
at 21 h ( figure 2d ) .
the control reactions
at 4 c when receptor internalization is reduced showed minimal
internalization of both radiolabeled antibodies at 48 h and demonstrated
the efficiency of the acid wash treatment to separate surface - bound
from internalized antibody .
these studies showed zr - pertuzumab
was as effective as zr - trastuzumab in binding her2 in
vitro , despite different binding sites . in vitro cell
binding
studies show zr - pertuzumab retains
immunoreactivity and specifically binds to a different her2 binding
site than trastuzumab .
( a ) immunoreactivity of zr - pertuzumab in skbr3 cells
was determined with a y intercept of 1.39
0.04 and calculated an immunoreactive fraction of 72.2% .
( b ) cell
uptake studies at 37 c in bt-474 ( her2 + ) , skbr3 ( her2 + ) , and
mda - mb-231 ( her2 ) demonstrate significantly higher zr - pertuzumab uptake in the her2 + cell lines compared to the her2
control ( p < 0.001 ) .
no statistical difference
was observed between bt-474 and skbr3 ( p > 0.05 ) .
( c ) competitive binding studies of zr - pertuzumab with
unlabeled pertuzumab in skbr3 cells at 4 c yields an ec50 of 2.4 0.11 nm , and no competition was observed with
unlabeled trastuzumab .
( d ) cell internalization studies in skbr3 cells
at 37 c up to 48 h demonstrate 50% of zr - pertuzumab
internalizes at about 20 h , similar to zr - trastuzumab .
control reactions when receptor internalization is reduced at 4 c
show minimal internalization of radiolabeled antibodies at 48 h. subcutaneous tumor xenografts
in severely combined immunodeficient mice have been reported to have
excellent tumor take rates without the need for exogenous stimuli
such as matrigel or estrogen pellets .
we observed slow tumor growth for bt-474 and mda - mb-231 , which was
achieved in 46 weeks with tumor take rates of 93% ( 13/14 )
and 100% ( 7/7 ) , respectively .
the skbr3 tumor take rate , even at high
cell concentration ( 1 10 cells / ml ) was 22% ( 3/14 )
and this low sampling hampered our investigation of zr - pertuzumab
in this model .
, in bt-474 xenografts and zr - pertuzumab accumulation
in the bt-474 tumor was higher than in mda - mb-231 with max suv of
10.5 6.1 , and 2.2 0.8 , respectively ( p = 0.0006 , figure 3a , b ) .
these data were
analyzed as the logarithmic transformation to more precisely represent
the means due to the high variances observed .
max suv analysis showed
increased tumor uptake of zr - pertuzumab in bt-474 in
the presence of unlabeled trastuzumab ( 19.0 5.4 ) although not
statistically significant ( p > 0.05 ) and may be
attributed
to the high variance in each test group .
the accumulation of zr - pertuzumab was reduced in the presence of unlabeled pertuzumab
with suv of 2.2 2.5 ( p = 0.0004 ) , a value
equivalent to the tumor uptake in the mda - mb-231 model ( p > 0.05 ) .
post - pet biodistribution studies significantly correlate
with pet imaging quantification yielding pearson s coefficient
of r = 0.98 , with a linear fit of r = 0.98 ( p = 0.01 , supporting information figure s1 ) .
the tumor uptake was higher
for bt-474 with 47.5 32.9% id / g ( n = 7 ) compared
to the mda - mb-231 xenograft with 9.5 1.7% id / g ( n = 7 , p = 0.0009 ) . blocking with unlabeled pertuzumab
had a significant reduction in tumor uptake , 8.5 6.6% id / g
( n = 4 , p = 0.0006 ) .
the tumor accumulation
of zr - pertuzumab showed a significant increase of 173
74.5% id / g in the presence of unlabeled trastuzumab ( n = 3 , p = 0.01 ) .
the variance instability
for the biodistribution studies is shown in supporting
information figure s2 , showing the need to perform statistical
analyses on the logarithmic transformations of the data .
low accumulation was
observed in most normal organs particularly the blood , muscle , and
heart with 0.05 0.02 , 0.7 0.2 , and 1.0 0.1% id / g ,
respectively , for the bt-474 xenografts . in vivo studies
show
high and specific tumor uptake of zr - pertuzumab in her2 +
tumors , enhanced in the presence of trastuzumab .
( a ) maximum intensity projection of pet / ct images of zr - pertuzumab in bt-474 ( her2 + ) and mda - mb-231 ( her2 ) breast
cancer xenografts at 7 days p.i .
( b ) max suv quantification of zr - pertuzumab
tumor uptake in bt-474 was 10.5 6.1 , increased in the presence
of unlabeled trastuzumab ( 19.0 5.4 ) , decreased in the presence
of a blocking concentration of unlabeled pertuzumab ( 2.2 2.5 ) ,
and decreased in mda - mb-231 xenografts ( 2.2 0.8 ) .
( c ) biodistribution
studies correlate with pet imaging quantification with 47.5
32.9% id / g for zr - pertuzumab in bt-474 , 173 74.5%
id / g in the presence of unlabeled trastuzumab ( p =
0.01 ) , 8.5 6.6% id / g in the presence of unlabeled pertuzumab
( p = 0.0006 ) , and 9.5 1.7% id / g in the her2
mda - mb-231 .
biodistribution of zr - pertuzumab in her2 + bt-474 and
her2 mda - mb-231 xenografts .
a cohort of bt-474 xenografts were coinjected
with either unlabeled trastuzumab ( checkered bars ) or unlabeled pertuzumab
( horizontal line bars ) .
zr - pertuzumab alone with no other
cotreatments added were studied in bt-474 ( black bars ) or mda - mb-231
( unfilled bars ) .
interestingly , the spleen
showed a relatively high tracer accumulation ,
greater than 100% id / g , in these severely combined immunodeficient
mice .
this unexpected outcome led us to investigate whether this phenomenon
was a consequence of these tumors ; thus , we performed biodistribution
studies of zr - pertuzumab in nontumor bearing nogs and
in the conventional athymic nu / nu mice as a control .
similar spleen uptake of 119.7 17.9% id / g ( n = 2 ) was observed in nontumor bearing nog mice at 7 days p.i . however ,
only 2.7 1.7% id / g ( n = 2 ) was observed in
nontumor bearing athymic nu / nu mice ( supporting information figure s3 ) .
thus , the
high spleen uptake is unique to the severely combined immunodeficient
mice .
we hypothesize that the absence of immunogenic response ( e.g. ,
the lack of t , b , and natural killer cell functions ) as well as the
abnormal macrophage activity in the nog strain are involved in the
catabolism of zr - pertuzumab to the spleen . in contrast ,
conventional strains of mice that still have some intact immunogenic
response ( e.g. , with functional natural killer cells ) , catabolize
antibodies primarily in the liver .
further investigations
are needed to determine the pharmacokinetic properties of humanized
antibodies in different types of preclinical models .
following biodistribution
studies , tumor slices from the bt-474 and mda - mb-231 groups were stained
for her2 expression ( brown ) using a commercially available anti - her2
antibody and counterstained with tartrazine ( yellow ) ( supporting information figure s4 ) .
her2 expression
in bt-474 tumors was pronounced , visibly outlining the her2 extracellular
membrane , whereas the mda - mb-231 tissue stained negative for her2
expression .
furthermore , we observed negative her2 staining in the
spleen , confirming the absence of human her2 in this organ ( data not
shown ) . h&e stains confirmed tumor viability .
these results showed
that the specific targeting of zr - pertuzumab to her2
in vivo were in agreement with the her2 expression levels ex vivo .
our findings illustrate zr - pertuzumab
specifically
accumulates in her2 + tumors , enhanced by the presence of trastuzumab .
this study is consistent with in silico studies conducted by fuentes
et al . , where a conformational change in her2 occurs upon trastuzumab
binding , likely inducing greater exposure of this binding site leading
to the enhanced affinity of pertuzumab .
our in vitro and in vivo studies show increased cellular and tumor
uptake of zr - pertuzumab in the presence of excess unlabeled
trastuzumab , respectively ( figure 2c , figure 3 ) .
these different studies did not show a comparable
degree of enhanced uptake of zr - pertuzumab , with only
30% increased cellular uptake in vitro at 4 c within 1.5 h of
incubation and almost 400% increased tumor uptake in vivo within 7
days of bloodstream circulation .
this apparent discrepancy may be
due to differences in the kinetic properties of zr - pertuzumab
in the more artificial environment in vitro compared to the complex
environment in vivo over several days .
the 7 day pet imaging after
administration of zr - pertuzumab was performed to allow
clearance of the radiolabeled antibody as exemplified by previously
studied zr - labeled monoclonal antibodies , one of the
advantages of imaging with zr over shorter - lived radioisotopes .
an earlier imaging time point at 5 days p.i . was performed in a
small cohort of mice , and no differences in tumor uptake were observed
between 5 and 7 days p.i .
( data not shown ) , which confirms the in
vivo stability of this radiopharmaceutical .
additionally , the
increased bloodstream residence of zr - pertuzumab may
also contribute to its increased tumor uptake in
the presence of trastuzumab . the absence of t , b , and nk cells part
of the immune response and the abnormal macrophage activity in this
nog host may contribute to the inefficiency of the clearance mechanism
for antibodies , as evidenced by the high spleen
uptake of zr - pertuzumab .
in contrast , the less immunocompromised
strains such as athymic mice which require estrogen pellet implantation
for bt-474 tumor engraftment have some functional immunogenic response
to mediate the hepatobiliary clearance of antibodies more efficiently . as such
, higher liver and lower spleen uptake were observed for
nontumor bearing athymic mice compared to the nog strain ( supporting information figure s3 ) .
differences
in clearance mechanisms for antibodies in different preclinical models
may be an interesting future study ; however , this should not impede
the translation of zr - pertuzumab to clinical trials .
in a previous study , scheuer et al . investigated pertuzumab as
an optical imaging agent labeled with a cy5 fluorescent dye , to assess
the synergistic therapeutic effect of the combination of pertuzumab
and trastuzumab in kpl-4 human breast cancer xenografts .
this study found similar tumor uptake between
cy5-pertuzumab alone and cy5-pertuzumab in combination with unlabeled
trastuzumab .
no enhancement of cy5-pertuzumab tumor uptake in the
presence of trastuzumab was reported , possibly due to differences
in experimental conditions such as the increased injected mass of
the cy5-pertuzumab , in which its unlabeled fraction may block some
her2 binding sites or the reduced sensitivity of optical imaging compared
to pet .
a limitation of our study is the high variance in zr - pertuzumab tumor uptake in the bt-474 xenografts .
this
observation
may be a consequence of tumor growth variability among the nogs . despite
the high variance in the means , zr - pertuzumab demonstrates
high affinity and selectivity for her2 + tumors in vivo .
although radiolabeled
trastuzumab has had much success in imaging her2 expression in breast
cancer patients who were trastuzumab nave or were undergoing
trastuzumab therapy , radiolabeled pertuzumab may be
a more favorable alternative as it binds to a distinct her2 epitope .
imaging with pertuzumab while treating with trastuzumab or the trastuzumab
antibody drug conjugate , t - dm1 , would allow a noncompetitive and cooperative
binding interaction with her2 .
currently , in - labeled
pertuzumab is being investigated in breast cancer patients using single - photon
emission computed tomography ; however ,
the sensitivity of pet and the resolution of zr will
provide excellent images with reduced radioactivity exposure to the
patient . thus , pet imaging using zr - pertuzumab may be
advantageous to monitor patient response to her2-targeted therapy
involving trastuzumab to more sensitively detect her2 expression .
zr - pertuzumab as a pet imaging agent detects her2
expression selectively in breast cancer xenograft models with increased
tumor uptake in the presence of trastuzumab .
zr - pertuzumab
is a promising radiopharmaceutical for noninvasive imaging of her2 +
breast cancer undergoing trastuzumab therapy to enhance the sensitivity
of her2 detection in vivo . | pertuzumab is a monoclonal antibody that binds to her2 and is used
in combination with another her2specific monoclonal antibody ,
trastuzumab , for the treatment of her2 + metastatic breast cancer .
pertuzumab binds to an her2 binding site distinct from that of trastuzumab ,
and its affinity is enhanced when trastuzumab is present .
we aim to
exploit this enhanced affinity of pertuzumab for its her2 binding
epitope and adapt this antibody as a pet imaging agent by radiolabeling
with 89zr to increase the sensitivity of her2 detection
in vivo . here
, we investigate the biodistribution of 89zr - pertuzumab in her2expressing bt-474 and her2nonexpressing
mda - mb-231 xenografts to quantitatively assess her2 expression in
vivo . in vitro cell binding studies
were performed resulting in retained
immunoreactivity and specificity for her2expressing cells .
in vivo evaluation of 89zr - pertuzumab
was conducted in
severely combined immunodeficient mice , subcutaneously inoculated
with bt-474 and mda - mb-231 cells .
89zr - pertuzumab was systemically
administered and imaged at 7 days postinjection ( p.i . )
followed by
terminal biodistribution studies .
higher tumor uptake was observed
in bt-474 compared to mda - mb-231 xenografts with 47.5 32.9
and 9.5 1.7% id / g , respectively at 7 days p.i ( p = 0.0009 ) and blocking studies with excess unlabeled pertuzumab
showed a 5-fold decrease in bt-474 tumor uptake ( p = 0.0006 ) , confirming the in vivo specificity of this radiotracer .
importantly , we observed that the tumor accumulation of 89zr - pertuzumab was increased in the presence of unlabeled trastuzumab ,
at 173 74.5% id / g ( p = 0.01 ) .
biodistribution
studies correlate with pet imaging quantification using max suv ( r = 0.98 , p = 0.01 ) .
collectively , these
results illustrate that 89zr - pertuzumab as a pet imaging
agent may be beneficial for the quantitative and noninvasive assessment
of her2 expression in vivo especially for patients undergoing trastuzumab
therapy . | Introduction
Materials and Methods
Results
Discussion
Conclusions | pertuzumab ( perjeta ) is an fda - approved
mab for the treatment of human epidermal growth factor receptor 2
( her2)-positive metastatic breast cancer , in combination with trastuzumab
( herceptin ) , another her2specific monoclonal antibody . pertuzumab
binds to domain ii of the human epidermal growth factor receptor-2
( her2 ) epitope , distinct from the trastuzumab epitope located within
domain iv . zr - labeled trastuzumab has been evaluated as a pet
imaging agent for detecting her2 expression in trastuzumab - treated
or in nave patients , with promising results toward the stratification
of patients mostly likely to respond to trastuzumab therapy . however , investigations with pertuzumab as a pet imaging agent have
not been explored , to our knowledge . pertuzumab has only been investigated
in vivo as a spect imaging agent by radiolabeling with in and lu . however , the sensitivity of pet
imaging may be advantageous for further developing pertuzumab as an
imaging agent . a qualitative comparison of fluorescently labeled cy5-trastuzumab
and cy5-pertuzumab in the presence and absence of either unconjugated
trastuzumab or pertuzumab has been conducted in her2 + xenograft models ,
which suggests imaging with cy5-pertuzumab in the presence of unconjugated
trastuzumab and imaging with cy5-trastuzumab in the presence of unconjugated
pertuzumab do not alter the tumor accumulation of each cy5-labeled
antibody . however ,
a quantitative comparison
between the tumor accumulation of radiolabeled trastuzumab and pertuzumab
as imaging agents during trastuzumab therapy has yet to be investigated . toward this end
herein , we demonstrate the specificity of zr - pertuzumab in her2 + tumors in vivo and quantitatively
show enhanced tumor accumulation of zr - pertuzumab in
the presence of trastuzumab . her2expressing breast cancer cell lines bt-474 and skbr3 as
well as her2nonexpressing mda - mb-231 were purchased from the
american type culture collection ( atcc , manassas , va ) and cultured
in iscove s modified dulbecco s medium ( imdm ) containing
10% fetal bovine serum ( fbs ) and 50 g / ml gentamycin ( complete
media ) in a humidified incubator with 5% co2 at 37 c . dfo - pertuzumab was radiolabeled with neutralized zr - oxalate
using a ratio of 296:1 kbq:g at 37 c for 3060
min for a final ph between 6.87.2.zr-pertuzumab with radiochemical yields 95%
as determined by instant thin - layer chromatography was used for in
vitro and in vivo studies without further purification . other cell
uptake experiments were performed in her2 + bt-474 and skbr3 , and her2
mda - mb-231 cells , in which 250 l of 1 10 cells / ml were seeded in 24-well plates . cells were incubated with 1.7 10 m zr - pertuzumab in the presence of different concentrations
( 1.7 101.7 10 m ) of unlabeled pertuzumab or trastuzumab competitor in triplicate
wells at 4 c for 1.5 h. the cells were washed with pbs and dissociated
from the wells as described above and assayed in a gamma counter . the percentage of zr - pertuzumab bound in the presence
of competitor / no competitor ( b / b0 100 ) was plotted against the log concentration
of added competitor and fitted with a sigmoidal dose
skbr3 cells ( 1 10 cells in
400 l ) were seeded into 12-well plates and allowed to adhere
overnight in the cell culture incubator . in vivo pet imaging and biodistribution studies
were conducted in severely combined immunodeficient female nog mice
( taconic , hudson , ny ) . mice were inoculated with 150 l of 13
10 cells / ml ( bt-474 , skbr3 , and mda - mb231 ) suspended
in saline with no artificial stimuli . studies in the presence of unlabeled antibodies were performed with
a tail vein injection of 100 l of 10 mg / ml of either pertuzumab
or trastuzumab in saline , between 560 min prior to the injection
of zr - pertuzumab in the bt-474 xenograft . static pet
images were acquired for 20 min using an inveon micropet / ct scanner
( siemens , knoxville , tn ) at 7 days p.i . ( a ) unconjugated
pertuzumab depicted in + 25 , + 24 , and + 23 charge states and ( b ) dfo - bz - ncs - pertuzumab
conjugate in the same charge states as the unconjugated control . the specificity of zr - pertuzumab
was investigated in vitro by comparing its binding in her2 + ( skbr3
and bt-474 ) and her2 ( mda - mb-231 ) cells . cell binding of zr - pertuzumab to skbr3 , bt-474 , and mda - mb-231 cells was
94.7 2.6 , 95.2 19.1 , and 3.8 0.1% of total radioactivity
added , respectively ( figure 2b ) . significantly
greater binding was observed in her2 + compared to her2 cells
( p < 0.0001 ) . cell binding studies with zr - pertuzumab
in skbr3 and bt-474 were consistent with pegram et al . the absence of competitive
binding with unlabeled trastuzumab is consistent with an her2 epitope
binding distinction between zr - pertuzumab to domain ii
and trastuzumab to domain iv of her2 . furthermore ,
the binding of zr - pertuzumab is increased by 30% in the
presence of high concentrations ( 0.022 m ) of unlabeled
trastuzumab . conformational changes occur
upon trastuzumab binding resulting in the increased affinity of pertuzumab
for its her2 binding epitope . in vitro cell
binding
studies show zr - pertuzumab retains
immunoreactivity and specifically binds to a different her2 binding
site than trastuzumab . ( b ) cell
uptake studies at 37 c in bt-474 ( her2 + ) , skbr3 ( her2 + ) , and
mda - mb-231 ( her2 ) demonstrate significantly higher zr - pertuzumab uptake in the her2 + cell lines compared to the her2
control ( p < 0.001 ) . no statistical difference
was observed between bt-474 and skbr3 ( p > 0.05 ) . ( c ) competitive binding studies of zr - pertuzumab with
unlabeled pertuzumab in skbr3 cells at 4 c yields an ec50 of 2.4 0.11 nm , and no competition was observed with
unlabeled trastuzumab . control reactions when receptor internalization is reduced at 4 c
show minimal internalization of radiolabeled antibodies at 48 h. subcutaneous tumor xenografts
in severely combined immunodeficient mice have been reported to have
excellent tumor take rates without the need for exogenous stimuli
such as matrigel or estrogen pellets . we observed slow tumor growth for bt-474 and mda - mb-231 , which was
achieved in 46 weeks with tumor take rates of 93% ( 13/14 )
and 100% ( 7/7 ) , respectively . , in bt-474 xenografts and zr - pertuzumab accumulation
in the bt-474 tumor was higher than in mda - mb-231 with max suv of
10.5 6.1 , and 2.2 0.8 , respectively ( p = 0.0006 , figure 3a , b ) . max suv analysis showed
increased tumor uptake of zr - pertuzumab in bt-474 in
the presence of unlabeled trastuzumab ( 19.0 5.4 ) although not
statistically significant ( p > 0.05 ) and may be
attributed
to the high variance in each test group . the accumulation of zr - pertuzumab was reduced in the presence of unlabeled pertuzumab
with suv of 2.2 2.5 ( p = 0.0004 ) , a value
equivalent to the tumor uptake in the mda - mb-231 model ( p > 0.05 ) . post - pet biodistribution studies significantly correlate
with pet imaging quantification yielding pearson s coefficient
of r = 0.98 , with a linear fit of r = 0.98 ( p = 0.01 , supporting information figure s1 ) . the tumor uptake was higher
for bt-474 with 47.5 32.9% id / g ( n = 7 ) compared
to the mda - mb-231 xenograft with 9.5 1.7% id / g ( n = 7 , p = 0.0009 ) . blocking with unlabeled pertuzumab
had a significant reduction in tumor uptake , 8.5 6.6% id / g
( n = 4 , p = 0.0006 ) . the tumor accumulation
of zr - pertuzumab showed a significant increase of 173
74.5% id / g in the presence of unlabeled trastuzumab ( n = 3 , p = 0.01 ) . low accumulation was
observed in most normal organs particularly the blood , muscle , and
heart with 0.05 0.02 , 0.7 0.2 , and 1.0 0.1% id / g ,
respectively , for the bt-474 xenografts . in vivo studies
show
high and specific tumor uptake of zr - pertuzumab in her2 +
tumors , enhanced in the presence of trastuzumab . ( a ) maximum intensity projection of pet / ct images of zr - pertuzumab in bt-474 ( her2 + ) and mda - mb-231 ( her2 ) breast
cancer xenografts at 7 days p.i . ( b ) max suv quantification of zr - pertuzumab
tumor uptake in bt-474 was 10.5 6.1 , increased in the presence
of unlabeled trastuzumab ( 19.0 5.4 ) , decreased in the presence
of a blocking concentration of unlabeled pertuzumab ( 2.2 2.5 ) ,
and decreased in mda - mb-231 xenografts ( 2.2 0.8 ) . ( c ) biodistribution
studies correlate with pet imaging quantification with 47.5
32.9% id / g for zr - pertuzumab in bt-474 , 173 74.5%
id / g in the presence of unlabeled trastuzumab ( p =
0.01 ) , 8.5 6.6% id / g in the presence of unlabeled pertuzumab
( p = 0.0006 ) , and 9.5 1.7% id / g in the her2
mda - mb-231 . biodistribution of zr - pertuzumab in her2 + bt-474 and
her2 mda - mb-231 xenografts . zr - pertuzumab alone with no other
cotreatments added were studied in bt-474 ( black bars ) or mda - mb-231
( unfilled bars ) . interestingly , the spleen
showed a relatively high tracer accumulation ,
greater than 100% id / g , in these severely combined immunodeficient
mice . this unexpected outcome led us to investigate whether this phenomenon
was a consequence of these tumors ; thus , we performed biodistribution
studies of zr - pertuzumab in nontumor bearing nogs and
in the conventional athymic nu / nu mice as a control . similar spleen uptake of 119.7 17.9% id / g ( n = 2 ) was observed in nontumor bearing nog mice at 7 days p.i . however ,
only 2.7 1.7% id / g ( n = 2 ) was observed in
nontumor bearing athymic nu / nu mice ( supporting information figure s3 ) . thus , the
high spleen uptake is unique to the severely combined immunodeficient
mice . ,
the lack of t , b , and natural killer cell functions ) as well as the
abnormal macrophage activity in the nog strain are involved in the
catabolism of zr - pertuzumab to the spleen . following biodistribution
studies , tumor slices from the bt-474 and mda - mb-231 groups were stained
for her2 expression ( brown ) using a commercially available anti - her2
antibody and counterstained with tartrazine ( yellow ) ( supporting information figure s4 ) . her2 expression
in bt-474 tumors was pronounced , visibly outlining the her2 extracellular
membrane , whereas the mda - mb-231 tissue stained negative for her2
expression . furthermore , we observed negative her2 staining in the
spleen , confirming the absence of human her2 in this organ ( data not
shown ) . these results showed
that the specific targeting of zr - pertuzumab to her2
in vivo were in agreement with the her2 expression levels ex vivo . our findings illustrate zr - pertuzumab
specifically
accumulates in her2 + tumors , enhanced by the presence of trastuzumab . , where a conformational change in her2 occurs upon trastuzumab
binding , likely inducing greater exposure of this binding site leading
to the enhanced affinity of pertuzumab . our in vitro and in vivo studies show increased cellular and tumor
uptake of zr - pertuzumab in the presence of excess unlabeled
trastuzumab , respectively ( figure 2c , figure 3 ) . these different studies did not show a comparable
degree of enhanced uptake of zr - pertuzumab , with only
30% increased cellular uptake in vitro at 4 c within 1.5 h of
incubation and almost 400% increased tumor uptake in vivo within 7
days of bloodstream circulation . this apparent discrepancy may be
due to differences in the kinetic properties of zr - pertuzumab
in the more artificial environment in vitro compared to the complex
environment in vivo over several days . the 7 day pet imaging after
administration of zr - pertuzumab was performed to allow
clearance of the radiolabeled antibody as exemplified by previously
studied zr - labeled monoclonal antibodies , one of the
advantages of imaging with zr over shorter - lived radioisotopes . was performed in a
small cohort of mice , and no differences in tumor uptake were observed
between 5 and 7 days p.i . ( data not shown ) , which confirms the in
vivo stability of this radiopharmaceutical . additionally , the
increased bloodstream residence of zr - pertuzumab may
also contribute to its increased tumor uptake in
the presence of trastuzumab . investigated pertuzumab as
an optical imaging agent labeled with a cy5 fluorescent dye , to assess
the synergistic therapeutic effect of the combination of pertuzumab
and trastuzumab in kpl-4 human breast cancer xenografts . this study found similar tumor uptake between
cy5-pertuzumab alone and cy5-pertuzumab in combination with unlabeled
trastuzumab . no enhancement of cy5-pertuzumab tumor uptake in the
presence of trastuzumab was reported , possibly due to differences
in experimental conditions such as the increased injected mass of
the cy5-pertuzumab , in which its unlabeled fraction may block some
her2 binding sites or the reduced sensitivity of optical imaging compared
to pet . despite
the high variance in the means , zr - pertuzumab demonstrates
high affinity and selectivity for her2 + tumors in vivo . although radiolabeled
trastuzumab has had much success in imaging her2 expression in breast
cancer patients who were trastuzumab nave or were undergoing
trastuzumab therapy , radiolabeled pertuzumab may be
a more favorable alternative as it binds to a distinct her2 epitope . currently , in - labeled
pertuzumab is being investigated in breast cancer patients using single - photon
emission computed tomography ; however ,
the sensitivity of pet and the resolution of zr will
provide excellent images with reduced radioactivity exposure to the
patient . thus , pet imaging using zr - pertuzumab may be
advantageous to monitor patient response to her2-targeted therapy
involving trastuzumab to more sensitively detect her2 expression . zr - pertuzumab as a pet imaging agent detects her2
expression selectively in breast cancer xenograft models with increased
tumor uptake in the presence of trastuzumab . zr - pertuzumab
is a promising radiopharmaceutical for noninvasive imaging of her2 +
breast cancer undergoing trastuzumab therapy to enhance the sensitivity
of her2 detection in vivo . | [
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they cause significant morbidity through a compression of regional structures and an inappropriate expression of pituitary hormones that disturb the multiple hypothalamic - pituitary - target organ axis systems .
pituitary adenomas are clinically classified as functional ( 70% ; such as acromegaly and prolactinoma ) and nonfunctional ( 30% ) . unlike functional pituitary adenomas , nonfunctional ( nf ) pituitary adenomas are typically quite large , do not cause any clinical hormone hypersecretion , and result in hypopituitarism or blindness from regional compression .
therefore , an early - stage diagnosis of nf pituitary adenomas is difficult because there is no hormone hypersecretion and there is no effective chemical therapy for nf pituitary adenomas .
predictive diagnosis / prevention and early - stage diagnosis / clinical therapy are the crucial aspects needed to conquer a tumor because most of nf pituitary adenomas remain as late - stage diagnosed and neurosurgically / radiation treated .
modern proteomics methods provide insight into the proteome , which is a dynamically changing protein - network system that progresses with disease .
study of the proteomic variations will result in new concepts and models for the more - accurate prediction , prevention , and personalized patient therapy for nf pituitary adenomas .
this review article addresses the contribution of tissue - proteomic variations to the interventional prevention and personalized patient treatment , and the contribution of body - fluid proteomic variations to the predictive diagnosis and the assessment of preventive and therapeutic responses .
pituitary adenomas are monoclonal in the cell of origin [ 2 , 3 ] , and might arise from any one of five pituitary anterior cell types ( fig . 1 ) [ 4 , 5]including corticotrophs [ secrete adrenocorticotropic hormone ( acth ) ] , somatotrophs [ secrete growth hormone ( gh ) ] , lactotrophs [ secrete prolactin ( prl ) ] , thyrotrophs [ secrete thyrotropin - stimulating hormone , or thyrotropin ( tsh ) ] , and gonadotrophs [ secrete follicle - stimulating hormone , or follitropin ( fsh ) and luteinizing hormone , or lutropin ( lh)]to produce an imbalance in hormone levels that will disturb the multiple regulatory hypothalamic - anterior pituitary - target organ axes . each axis performs a range of important physiologic functions in the human body ( fig . 1 ) .
mixed - tumors ( co - secrete gh with prl , tsh , or acth ) might also arise from single cells .
acth oversecretion results in cushing s disease , with features of hypercortisolism ; gh hypersecretion leads to acral overgrowth and metabolic dysfunction associated with acromegaly ; and prl oversecretion leads to gonadal failure , secondary infertility , and galactorrhea .
more rarely , tsh hypersecretion leads to hyperthyroxinemia and goiter , and hypersecreted gh ( or gh subunits ) leads to gonadal dysfunction .
in contrast , tumors that arise from gonadotroph cells do not efficiently secrete their gene products , and they are usually clinically silent .
they are commonly referred to as nf pituitary adenomas , which can not be diagnosed early because no hormone change is observed . however , nf pituitary adenomas are highly heterogeneous in the cell of origin ( table 1 ) . except for a gonadotroph - originated intact lh / fsh adenoma ( up to 79% of nf adnomas ) ,
immunocytochemical staining of hormones also reveals that some nf pituitary adenomas might arise from a silent corticotroph ( acth - positive ; 8% ) , silent somatotroph ( gh - positive ; 3% ) , no hormone - expressed oncocytoma ( 6% ) , and null cell ( 17% ) .
fig .
1scheme of the hypothalamic - anterior pituitary - target organ axis systems and pituitary adenoma pathogenesis .
( + ) = stimulatory regulation ; ( ) = inhibitory regulation . reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005 ; and modified from melmed , with permission from copyright clearance center inc .
( re : journal of clinical investigation ) , copyright 2003table 1classification of nonfunctional pituitary adenomas by cell of origincell typehormone expression% nonfunctional tumorsgonadotrophsintact lh / fsh or subunits4079null cellnone17oncocytomanone6silent corticotrophacth8silent somatotrophgh3reproduced from oyesiku , with permission from aacr , copyright 2005 .
scheme of the hypothalamic - anterior pituitary - target organ axis systems and pituitary adenoma pathogenesis .
( + ) = stimulatory regulation ; ( ) = inhibitory regulation . reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005 ; and modified from melmed , with permission from copyright clearance center inc .
( re : journal of clinical investigation ) , copyright 2003 classification of nonfunctional pituitary adenomas by cell of origin reproduced from oyesiku , with permission from aacr , copyright 2005 .
those heterogeneous cells of origin of nf pituitary adenomas produce differences in a proteome relative to the control and among different nf pituitary adenoma patients , and that difference in the proteome dynamically changes with tumor progression . the proteomic difference among different types of nf pituitary adenomas , and the dynamic alteration with tumor progression ,
both contribute to the prediction , interventional prevention , and personalized patient treatment of nf pituitary adenoma patients .
the pituitary is the most - protected organ in the body , and is the master regulatory gland ; several different hypothalamic - pituitary - target organ axes exist .
based on the new concept of prediction , prevention , and personalized patient treatment of an nf pituitary adenoma , pituitary tissues , cerebrospinal fluid ( csf ) , and blood plasma must be analyzed .
any tissue - proteome variation directly reflects the pathophysiological changes of an nf pituitary adenoma .
those tissue - proteome variations , and the corresponding changes in the biological system , can be developed into an accurate molecular classification , interventional prevention , and personalized patient treatment .
in addition , in an nf pituitary adenoma patient , some secreted proteins and peptides will enter into the csf and blood circulation .
comparative proteomics can detect those different components ( proteins or peptides ) in the patient s csf and blood plasma .
those csf and blood plasma proteomic and peptidomic variations will lead to the development of an accurate predictive diagnosis and the measurement of interventional prevention and therapy response based on accurate molecular data .
pituitary tumor tissues are generally acquired with neurosurgery from patients , whereas control tissues are generally obtained via forensic anatomy from post - mortem cardavers .
therefore , there is some uncontrollable bias among pituitary adenoma tissues and so - called control tissue proteome such as sex , age , race , and time of tissue acquisition ; and some biochemical changes might occur in post - mortem tissue compared to surgical tissue . also , an nf pituitary adenoma is monoclonal in origin , and is generally benign .
an nf pituitary adenoma tissue contains a relatively pure cell population , whereas control post - mortem pituitary tissues contain different types of cells , including at least corticotrophs , somatotrophs , lactotrophs , thyrotrophs , and gonadotrophs , etc .
when the tumor tissues and post - mortem tissues are used to analyze the proteomic variation , another bias is possible .
the use of laser - capture microdissection ( lcm ) to preferentially enrich a specific tumor cell type from tumor tissues and the corresponding normal cells from post - mortem tissues overcomes this bias .
compared to a tissue proteome , those limitations do not exist for the csf and blood specimens .
also , csf and blood specimens are much more accessible from patients and controls than the pituitary tissues .
modern proteomics uses protein - separation techniques such as gel and non - gel methods , protein - identification techniques such as mass spectrometry ( ms ) , multiple protein - preparation techniques , and systems biology techniques to quantitatively measure proteomic variation in tissue , csf , and blood plasma samples from patients and controls .
gel methods include two - dimensional gel electrophoresis ( 2dge ) and 2d differential in - gel electrophoresis ( 2d dige ) .
2dge - based comparative proteomics is a classical , reliable method to quantify accurately any proteomic variation .
briefly , proteins are arrayed with 2dge according to their pi and mr , and are stained .
each 2d gel spot - volume reflects the abundance of the protein contained in that spot .
each individual gel spot - volume is generally normalized with the total spot - volumes of each gel image [ 6 , 7 ] .
2dge images from tumors and controls are compared with 2d gel - analysis software to obtain statistically significant differential 2d gel spot - volume between tumors and controls .
the amino acid sequence of a protein in each 2d gel spot is obtained with ms , including peptide mass fingerprint ( pmf ) ( which determines the mw of each tryptic peptide from that protein ) and/or tandem mass spectrometry ( ms / ms ) ( which determines the amino acid sequence of each tryptic peptide ) , and corresponding protein database analysis .
generally , the tumor and control samples are labeled with different fluorescent dyes such as cydye 3 and cydye 5 .
the cydye - labeled samples are mixed equally ( 1:1 ) , and the mixed sample is separated with 2dge according to pi and mr .
the proteins are visualized , and the image is scanned with a cydye 3 channel and cydye 5 channel to form two fluorescent images , which are processed with decyder dige analysis software or delta 2d software .
any color difference in a gel spot defines and quantifies the protein content between tumors and controls .
the amino acid sequence of tryptic peptides from each protein in each gel spot is determined with ms / ms .
2d dige is more useful to analyze precious lcm - enriched samples because 2d dige requires less protein amount and fewer gel - analysis procedures relative to the traditional 2dge - based comparative proteomics .
non - gel methods mainly include the analysis of trypsin - digests from a complex mixture of proteins with multidimensional chromatography coupled on - line with ms [ 12 , 13 ] .
that strategy generates tryptic peptides that are separated in the first dimension with any one of several different chromatographies , and with reversed - phase chromatography in the second dimension .
the separated peptides are analyzed with ms / ms , and the amino acid sequences are analyzed with a database analysis for protein identification .
for quantification of proteomic variation , that technique must be coupled with stable - isotope labeling prior to multidimensional - chromatography analysis .
the light and heavy isotope - labeled peptides were quantified with ms / ms to quantify and identify the protein amount between tumor and control proteomes .
a wide variety of stable isotope - labeling techniques have been developed ; for example , cysteine - specific tagging such as icat , lysine - specific tagging [ 17 , 18 ] , and amine - specific tagging such as itraq [ 1921 ] .
moreover , some software has been developed to quantitatively analyze the stable isotope - labeled peptides ; for example , xpress software for icat - labeled peptides .
non - gel methods effectively detect low - abundance proteins , extremely acidic / basic proteins , and low / high - mr proteins .
although the gel methods are limited by the extremely acidic ( pi < 3.5 or 4)/basic ( pi > 7.5 or 8) proteins , extremely high - mass ( > 150 kda ) or low - mass ( < 10 kda ) proteins , and hydrophobic proteins , the non - gel techniques are complementary with the gel - based methods , and improve the proteomic coverage of an nf pituitary adenoma proteome . systems biology techniques such as pathway analysis help to elucidate each protein variation within a pathway network system .
a wide - range of pathway analysis programs has been developed such as ingenuity pathway analysis ( ipa ) ( www.ingenuity.com ) and metacore ( www.genego.com/metacore.php ) .
pathway analysis has been used to mine significant pathway networks from pituitary adenoma tissue comparative proteomic data and proteomic modification data .
the recovery of the tumor - altered pathway system to the normal pathway system would contribute significantly to interventional prevention and effective chemical therapy .
those proteomic variations include variations in protein expression and post - translational modifications ( ptms ) .
a tissue proteome is a systematic and tightly regulated protein - network system that dynamically changes during pathophysiological processes .
multiple endogenous and exogenous factors cause an alteration of certain elements within a proteome relative to controls .
those altered elements include protein differential expression ( deps ) , splicing isoforms ( such as hormone isoforms ) , and protein modifications ( such as tyrosine nitration and phosphorylation ) , etc .
a total of 56 deps has been identified among nf adenomas and controls with 2dge comparative proteomics strategies ( table 2 ) .
a large - scale comparative proteomics study was performed on a set of human pituitary samples [ 1 , 5 , 23 ] : controls ( n = 8) versus several different cell - types of nf pituitary adenomas ( nf- , n = 3 ; lh+ , n = 3 ; fsh+ , n = 3 ; fsh+/lh+ , n = 3 ) and hyperprolactinomas ( n = 4 ) .
a total of 93 differential protein - spots ( 65 decreased spot volumes , 28 increased ) were found in nf pituitary adenomas and prolactinomas .
seventy - two spots ( 50 decreased , 22 increased ) that represented 56 dep s ( 34 down - regulated , 22 up - regulated ) were characterized with ms and database analysis .
results indicated that : ( i ) neuroendocrine - related proteins , including somatotropin , secretagogin , and mu - crystallin homolog , were down - regulated in nf pituitary adenomas and the hyperprolactinoma ; ( ii ) somatotropin existed in at least 17 isoforms that were down - regulated in nf adenomas and the hyperprolactinomas ; ( iii ) prolactin existed in six isoforms that were down - regulated in nf adenomas , and no - change in the hyperprolactinomas ; ( iv ) immunologic regulation proteins and tumor - related antigen ( for example , immunoglobulin , tumor - rejection antigen 1 ) were down - regulated in nf adenomas ; ( v ) cell proliferation , differentiation , and apoptosis - related proteins were down - regulated in the nf - adenomas and the hyperprolactinomas ; ( vi ) some cell - defense and stress - resistance proteins ( for example , phospholipid hydroperoxide glutathione peroxidase , cd59 glycoprotein , and heat - shock 27 kda protein ) were down - regulated in the nf pituitary adenomas ; ( vii ) some metabolic enzyme - related proteins ( for example , cytoplasmic isocitrate dehydrogenase [ nadp ] , tryptophan 5-hydroxylase 2 , matrix metalloproteinase-9 , aldose reductase , lactoylglutathione lyase , acyl - coa - binding protein , etc . ) were up - regulated in the nf pituitary adenomas ; and ( viii ) for cell - signal proteins , some were down - regulated and some were up - regulated in nf adenomas ; those cell signals are involved in the complex biological roles in the cell growth , proliferation , differentiation , apoptosis , and death cycles .
table 2differentially expressed proteins identified in human nonfunctional pituitary adenoma tissues with 2dge - based comparative proteomicsidmoleculesfold - changedescriptionlocationfunctionp49753acot25.8acyl - coa thioesterase 2cenzymep15121akr1b114.6aldo - keto reductase family 1b1cenzymep02647apoa13.2apolipoprotein a - iectransporterp06576atp5b5.0atp synthase , h + transporting , mitochondrial f1 complex , beta polypeptidectransporterp47756capzb6.5capping protein ( actin filament ) muscle z - line , betacothercapzbcapzb6.5capping protein ( actin filament ) muscle z - line , betacotherp13987cd599.5cd59 molecule , complement regulatory proteinpmotherp12110col6a214.7collagen , type vi , alpha 2ecotherp14854cox6b19.3cytochrome c oxidase subunit vib polypeptide 1 ( ubiquitous)cenzymeq14894crym35.3crystallin , mucenzymecsn1s1csn1s1 ( includes eg:1446)17.8casein alpha s1ecotherp07108dbi4.3acyl - coenzyme a binding proteincotherq9uhl4dpp79.3dipeptidyl - peptidase 7cpeptidaseq14259erh5.0enhancer of rudimentary homolognotherp30040erp294.6endoplasmic reticulum protein 29ctransporterp00742f1083.0coagulation factor xecpeptidasep06241fyn3.9fyn oncogene related to src , fgr , yespmkinasep31150gdi19.4gdp dissociation inhibitor 1cotherp01241gh1180.6growth hormone 1eccytokinep01241gh113.3growth hormone 1eccytokinep01241gh113.3growth hormone 1eccytokinep01241gh120.2growth hormone 1eccytokinep01241gh114.1growth hormone 1eccytokinep01241gh1128.3growth hormone 1eccytokinep01241gh114.8growth hormone 1eccytokinep01241gh148.1growth hormone 1eccytokinep01241gh117.3growth hormone 1eccytokinep01241gh116.9growth hormone 1eccytokinep01241gh188.8growth hormone 1eccytokinep01241gh127.8growth hormone 1eccytokinep01241gh151.2growth hormone 1eccytokinep01241gh132.1growth hormone 1eccytokinep01241gh1478.6growth hormone 1eccytokinep01241gh113.4growth hormone 1eccytokinep01241gh1161.0growth hormone 1eccytokinep01241gh137.0growth hormone 1eccytokinep01242gh214.1growth hormone 2ecotherq04760glo110.2glyoxalase icenzymeq04760glo18.3glyoxalase icenzymep09471gnao19.5 g protein , alpha activating activity polypeptide opmenzymep29777gnao19.5 g protein , alpha activating activity polypeptide opmenzymep36969gpx426.1glutathione peroxidase 4cenzymep28161gstm24.2glutathione s - transferase m2 ( muscle)cenzymehbbhbb ( includes eg:3043)3.1hemoglobin , betactransporterhbbhbb ( includes eg:3043)99.4hemoglobin , betactransporterp02081hbd3.1hemoglobin , deltactransporterp14625hsp90b111.0hsp90 beta ( grp94 ) , member 1cotherp04792hspb15.1heat shock 27 kda protein 1cotherq9ujy1hspb83.7heat shock 22 kda protein 8ckinaseo75874idh18.7isocitrate dehydrogenase 1 ( nadp+ ) , solublecenzymep24592igfbp620.9insulin - like growth factor binding protein 6ecotheriglc1iglc132.1immunoglobulin lambda constant 1cotheriglc3iglc332.1immunoglobulin lambda constant 3naotherp08779krt1628.8keratin 16 ( focal non - epidermolytic palmoplantar keratoderma)cotherq86u44mettl38.3methyltransferase like 3nenzymeq99542mmp193.1matrix metallopeptidase 19ecpeptidasep12524mycl117.8v - myc myelocytomatosis viral oncogene homolog 1 , lung carcinoma derived ( avian)ntranscription regulatoro00217ndufs85.2nadh - coenzyme q reductasecenzymep20774ogn38.0osteoglycinecgrowth factorq08752ppid5.1peptidylprolyl isomerase d ( cyclophilin d)cenzymeq00007ppp2r2a8.2protein phosphatase 2 ( formerly 2a ) , regulatory subunit b , alpha isoformcphosphatasep01236prl99.9prolactineccytokinep01236prl9.7prolactineccytokinep01236prl26.2prolactineccytokinep01236prl20.1prolactineccytokinep01236prl36.7prolactineccytokinep01236prl33.6prolactineccytokineo76038scgn6.6secretagogincotherq9ui15tagln35.6transgelin 3naotherp21980tgm217.1transglutaminase 2cenzymeq8iwu9tph210.6tryptophan hydroxylase 2naenzymeo14530txndc911.4thioredoxin domain containing 9naotherp08670vim5.5vimentincotherp27348ywhaq ( includes eg:10971)43.5tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein , theta polypeptide ( 14 - 3 - 3 protein tau)cotherq14584znf2667.3zinc finger protein 266notheril15-s21aap40933 - 22.7splice isoform il15-s21aa of interleukin-15p0162032.1ig kappa chain v - iii region sieiglc2p0184232.1ig lambda chain c regionsp0162132.1ig kappa chain v - iii region ng9p0443332.1ig kappa chain v - iii region vghbb2p189883.1hemoglobin beta-2 chain ( panle)gi 1066765176.7hemoglobin beta unit varianthbb2p1898899.4hemoglobin beta-2 chain ( panle)p0193599.4hemoglobin alpha-3 chain ( pantr)p0196820.2hemoglobin alpha-2 chain ( bosmu)hbb2p1898820.2hemoglobin beta-2 chain ( panle)c , cytoplasm ; ec , extracellular space ; pm , plasma membrane ; n , nucleus ; na , unknown . modified from zhan and desiderio [ 1 , 22 ] , with permission from aacr , copyright 2005 ; and with permission from biomed central open access journal , copyright remains with the authors.fig .
2functional categories of the 56 deps . a down - regulated proteins in pituitary adenoma ( 34 ) : i. neuro - endocrine and hormones ; ii . cytokine and cellular signal - related proteins ; iii .
mrna splicing , transport , or translation - related enzymes ; v. dna - binding proteins ; vi .
b up - regulated proteins in pituitary adenomas ( 22 ) : i. metabolic enzyme - related proteins ; ii .
cell cycle , cell growth and proliferation proteins ; v. cellular defense response ; vi .
others . reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005 differentially expressed proteins identified in human nonfunctional pituitary adenoma tissues with 2dge - based comparative proteomics c , cytoplasm ; ec , extracellular space ; pm , plasma membrane ; n , nucleus ; na , unknown . modified from zhan and desiderio [ 1 , 22 ] , with permission from aacr , copyright 2005 ; and with permission from biomed central open access journal
functional categories of the 56 deps . a down - regulated proteins in pituitary adenoma ( 34 ) : i. neuro - endocrine and hormones ; ii . cytokine and cellular signal - related proteins ; iii .
mrna splicing , transport , or translation - related enzymes ; v. dna - binding proteins ; vi .
ix . cell proliferation , differentiation , and apoptosis - related proteins ; x. others .
b up - regulated proteins in pituitary adenomas ( 22 ) : i. metabolic enzyme - related proteins ; ii . energy metabolism ; iii . cellular signal proteins ; iv .
cell cycle , cell growth and proliferation proteins ; v. cellular defense response ; vi .
others . reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005 the heterogeneity of hormone expression is an important characteristic in an nf pituitary adenoma .
a comparative proteomics study of nf adenomas demonstrated that two hormones ( gh and prl ) had multiple isoforms [ 1 , 23 ] .
twenty - four 2d gel spots that contained gh were found in control pituitary tissues ( fig .
those hghs in the 24 2d gel spots were classified into four types of hgh - splicing isoforms , 14 .
the expression proportion of those four isoforms were isoform 1 ( 87.5 % ) > isoform 2 ( 8.1 % ) > isoform 3 ( 3.3 % ) > isoform 4 ( 1.1 % ) ( fig .
somatotropin was significantly down - regulated at the protein and the mrna levels in the nf pituitary adenoma and in prolactinomas ; that finding is consistent with their monoclonal origin [ 2 , 3]an nf adenoma originated from gonadotrophs , and a prolactinoma from lactotrophs ; the gh receptor gene was unchanged .
those data showed that gh hyposecretion in an nf - adenoma results from the hypo - expression of the gh gene .
however , the more important finding is that the multiple gh isoforms ( 17 spots contain somatotropin ) were detected [ 1 , 23 ] ; those data can not be interpreted from transcriptomics data .
the down - regulated ratio of the different gh isoforms was different in each cell - type of nf pituitary adenoma relative to the controls .
those data suggested that the proportion of the different gh isoforms changed in each cell - type nf adenoma compared to controls .
other researchers showed that the proportion of a circulating gh isoform significantly changed in pituitary adenomas and other pituitary diseases [ 25 , 26 ] .
the proportional change of the different gh isoforms might have an important value in the clinical evaluation of nf adenomas .
recently , studies found that gh isoforms were derived from a variety of splicing variants and ptms , including phosphorylation .
the phosphorylation of endogenous gh in the human pituitary provided new insights into the mechanisms of gh that participate in the neuroendocine signal pathways .
ief was performed with an ipg dry strip ( 18 cm , ph 310 nonlinear ) .
reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005fig .
4expression proportion of the four splicing isoforms of gh in human pituitary glands . reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005 ms - characterized gh isoforms in a 2-de map of human pituitary .
ief was performed with an ipg dry strip ( 18 cm , ph 310 nonlinear ) .
reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005 expression proportion of the four splicing isoforms of gh in human pituitary glands .
reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005 prolactin is another important pituitary hormone .
similar to gh , each prolactin isoform was down - regulated in each cell - type nf adenoma , with a different down - regulated ratio relative to controls .
there was no significant expression change of the prolactin gene at the protein and mrna levels in the prolactinoma relative to controls ; that finding is consistent with a prolactinoma s monoclonal origin .
however , the proportion of those six prolactin isoforms changed in nf adenomas and prolactinomas compared to controls .
other researchers showed that glycosylation is an important prolactin modification that produces different isoforms ; glycosylation of human prolactin might down - regulate its hormone bioactivity and promote its metabolic clearance .
other studies showed that the main variant of prolactin was the non - glycosylated form of prl in human prolactinomas ; that finding is consistent with the result that some prolactin isoforms were down - regulated in prolactinomas compared to controls .
thus , it has been speculated that the prolactin isoform in those down - regulated spots was possibly a glycosylated prolactin .
therefore , further detailed study of the different isoforms and ptms of gh and prolactin could lead to new insights into the clinical importance of pituitary hormones in the formation of an nf adenoma .
the ratio of the change of each isoform in a human pituitary tissue has value for clinical research . except for protein - expression variation and splicing isoforms ,
protein ptms are an important source of proteome variation that is associated with different pathophysiological processes .
also , the degree and the sites of the ptms could dynamically change with pathophysiological processes .
dep analyses demonstrated that many reactive - oxygen species ( ros)-related proteins were changed in human pituitary adenomas , including heat shock protein , cytochrome p450 , gst , and phospholipid hydroperoxide glutathione peroxidase ( table 2 ) .
tyrosine nitration was studied in an nf adenoma and a post - mortem control [ 32 , 33 ] .
2dge - based nitrotyrosine western blot and ms / ms identified eight nitroproteins from a pituitary control ( table 3 ) [ 32 , 33 ] .
those control nitroproteins are involved in the transforming growth factor beta 1 ( tfgb1 ) and actin cellular skeleton signaling networks ( fig .
5a ) , which function in gene expression , cellular development , and connective - tissue development .
the nitroproteins in that network include snap91 , fcar , actin , prkg2 , stc1 , paqr3 , and psma2 .
nitrotyrosine affinity column ( ntac)-based ms / ms identified nine nitroproteins , and three non - nitrated proteins that interact with nitroproteins , from a nf adenoma ( table 3 ) . those nf adenoma nitroproteins and their complexes are involved in the tumor necrosis factor ( tnf ) and interleukin 1 ( il1 ) signaling networks ( fig .
5b ) , which function in cancer , cell cycle , and reproductive - system disease .
the nitroproteins in that network include arhgap5 , prkar1b , psma2 , il1f6 , and rhpn2 .
the non - nitrated proteins that interact with nitroproteins include irak2 , grip2 , and ubiquitin .
three nitroprotein - protein complexes were identified : the nitrated proteasome - ubiquitin complex , the nitrated beta - subunit of pka complex , and the nitrated il1f6-il1 receptor - il1 receptor - associated kinase - like 2 ( il1f6-il1r - irak2 ) complex .
5 a and b ) include a same beta - estradiol signal pathway , which indicates that hormone metabolism involves the control pituitary and pituitary adenoma .
this result is consistent with the fact that no participates in pituitary hormone metabolism in normal physiology , and that a tumor interferes with hormone metabolism .
based on the documented functions of those identified proteins , each nitroprotein and nitroprotein - protein complex was rationalized into the corresponding functional system ( fig .
the nitrated cent1 and the nitrated pkar1 are involved in the camp - dependent protein - kinase signal pathway .
the nitrated rhogap5 and the nitrated rhophilin 2 are involved in the gtpase signal pathway .
irak-2 in the il1-r complex and the nitrated il1-f6 are involved in the cytokine system .
the nitrated s1p lyase 1 participates in sphingolipid metabolism to regulate cell proliferation , survival , and cell death as well as the immune system [ 35 , 36 ] .
the nitrated proteasome - ubiquitin complex is an important enzymatic complex that is involved in the intracellular nonlysosomal proteolytic pathway [ 37 , 38 ] .
these data demonstrate that those nitroproteins and nitroprotein - protein complexes might participate in several different and very important functional pathways in human pituitary tumor cells .
table 3nitroproteins and non - nitrated proteins identified in a pituitary adenoma , and control tissue [ 32 , 33]pituitary adenomapituitary controlprotein nameny siteprotein nameny sitenitrated protein : nitrated protein : rho - gtpase - activating 5 [ q13017 ] ( arhgap5)y synaptosomal - associated protein ( snap91)y leukocyte immunoglobulin - like receptor a4 [ p59901]y ig alpha fc receptor [ p24071 ] ( fcar)y zinc finger protein 432 [ o94892]y actin [ p03996 ] ( acta2 , actg2 , actc1)y pka beta regulatory subunit [ p31321 ] ( prkar1b)y pkg 2 [ q13237 ] ( prkg2)y sphingosine-1-phosphate lyase 1 [ o95470]y , y mitochondrial co - chaperone protein hscb [ q8iwl3]y centaurin beta 1 [ q15027]y stanniocalcin 1[p52823 ] ( stc1)y proteasome subunit alpha type 2 [ p25787 ] ( psma2)y proteasome subunit alpha type 2 ( psma2)y interleukin 1 family member 6 [ q9uha7 ] ( il1f6)y progestin and adipoq receptor family member iii [ q6tch7 ] ( paqr3)y rhophilin 2 [ q8iuc4 ] ( rhpn2)ynitroprotein - interacting protein interleukin-1 receptor - associated kinase - like 2 ( irak-2 ) [ o43187 ] ( irak2 ) glutamate receptor interacting protein 2 [ q9c0e4 ] ( grip2 ) ubiquitin [ p62988 ] ( ubb or ubc)modified from zhan and desiderio [ 3133 ] , with permission from elsevier , copyright 2004 , 2006 , and 2007.fig . 5significant signaling pathway networks that are involved in human pituitary adenoma nitroproteomic data . network a derives from control nitroproteomic data , and functions in gene expression , cellular development , and connective - tissue development and function .
network b derives from adenoma nitroproteomic data , and functions in cancer , cell cycle , and reproductive - system disease .
a gray node denotes an identified nitroprotein , or a protein that interacts with nitroproteins .
an orange solid - edge denotes a direct relationship between two nodes ( molecules : proteins ; genes ) .
an orange nonsolid - edge denotes an indirect relationship between two nodes ( molecules : proteins ; genes ) .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authorsfig .
6experimental data - based model of nitroproteins and their functions in human nonfunctional pituitary adenomas .
no2 , nitroprotein . reproduced from zhan and desiderio , with permission from elsevier , copyright 2006 nitroproteins and non - nitrated proteins identified in a pituitary adenoma , and control tissue [ 32 , 33 ] modified from zhan and desiderio [ 3133 ] , with permission from elsevier ,
significant signaling pathway networks that are involved in human pituitary adenoma nitroproteomic data . network a derives from control nitroproteomic data , and functions in gene expression , cellular development , and connective - tissue development and function .
network b derives from adenoma nitroproteomic data , and functions in cancer , cell cycle , and reproductive - system disease .
a gray node denotes an identified nitroprotein , or a protein that interacts with nitroproteins .
an orange solid - edge denotes a direct relationship between two nodes ( molecules : proteins ; genes ) .
an orange nonsolid - edge denotes an indirect relationship between two nodes ( molecules : proteins ; genes ) .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authors experimental data - based model of nitroproteins and their functions in human nonfunctional pituitary adenomas .
no2 , nitroprotein . reproduced from zhan and desiderio , with permission from elsevier , copyright 2006 four pathway - network systems , including mitochondrial dysfunction , oxidative stress , cell - cycle dysregulation , and mapk - signal abnormality for the nf adenoma , were generated from those dep and nitroprotein data .
( i ) mitochondrial dysfunctions underlie a broad spectrum of human diseases [ 39 , 40 ] that includes cancer [ 41 , 42 ] , neurodegenerative diseases , cardiovascular diseases , diabetes mellitus , and inflammatory diseases [ 46 , 47 ] .
notable differences in the structure and function of mitochondria appear between cancer and normal cells [ 41 , 42 ] .
mitochondrial dysfunctions have been proposed as a cause of cancer , are a biomarker for the early detection of cancer , and are a therapeutic target for cancer .
mitochondrial dysfunction could be confirmed with a mitochondrial morphological change and the increased number of mitochondria in a human pituitary tumor [ 4952 ] .
mitochondria are emerging as biomarker targets for the early detection of , and for novel therapeutic targets in , cancer [ 48 , 5356 ] .
( ii ) oxidative stress occurs when the balance is disturbed between an upload of free - radical / reactive oxygen / nitrogen species ( ros / rns ) from in vivo formation and from in vitro environmental carcinogens , and the capability of endogenous antioxidant defense mechanisms to remove those reactive species [ 57 , 58 ] .
many studies have indicated that ros and rns are involved in pituitary adenoma pathology [ 5961 ] .
a nitroproteomics study discovered nine tyrosine - nitrated proteins in human pituitary adenoma tissues , and each tyrosine nitration site is located within an important protein domain to alter protein functions .
however , with the formation of ros and rns , the in vivo antioxidative mechanism is also initiated against ros / rns .
pivotal to the antioxidant response is the transcription factor nrf2 ( nuclear factor - e2-related factor-2 ) .
dep and nitroproteomic data clearly reveal that the nrf2-mediated oxidative - stress response pathway system ( fig .
any decrease in the capability of this antioxidant protective system could increase the susceptibility to oxidative stress , tumor inflammation , carcinogen toxicity , and tumorigenesis .
therefore , the oxidative stress - antioxidative stress - response system could be the novel target to develop effective therapeutic agents that could be used for human pituitary adenomas [ 63 , 64 ] .
the basic biological characteristics of tumor cells are the unrestricted proliferation and growth that are controlled by the dysregulated cell - cycle .
the cell cycle is regulated by a cyclically operating biochemical system that includes cyclins , cyclin - dependent kinases ( cdk ) , and their inhibitors ( cdki ) .
studies have demonstrated that the ectopic expression of cyclin d and the overexpresion of cyclins a , b , and e occur in an nf pituitary adenoma to regulate different phases of the cell cycle , and to accelerate the progression of the cell - cycle .
the details on cell - cycle dysregulation in a human pituitary adenoma have been reviewed [ 6669 ] .
the pathway analysis of nf pituitary adenoma nitroproteomic data and dep data clearly revealed the cell - cycle g2/m dna damage checkpoint - regulation pathway ( fig .
the important cell - cycle regulator 14 - 3 - 3 protein was down - regulated ( 44-fold ) in nf adenomas compared to controls ( table 2 ) .
moreover , nitroproteomic data demonstrate that a nitrated proteasome could interfere with the functions of the ubiquitin - proteasome system in the regulation of the cell cycle .
the proteasome inhibitors can induce apoptosis in gh - and prolactin - secreting rat pituitary tumor cells through a blocking of the cell cycle at the g2/m transition .
mapk - signaling pathways link the extracelluar signal stimuli to functional cellular processes such as growth , proliferation , migration , and apoptosis .
the basic mapk pathway is stimulus ( mitogens , growth factors , cytokines , stress , etc . )
g - protein ( ras , rac , cdc42 , rho ) mapkkk ( raf , tpl2 , mekk , mlk , tak , ask , tao ) mapkk ( mek ) mapk ( erk , jnk , p38 ) to generate responses ( proliferation , differentiation , apoptosis , and migration ) .
erks ( extracellular signal - regulated kinases ) , jnks ( c - jun n - terminal kinases ) , and p38-mapks are the three main subfamilies of mapks .
the details of mapk - signaling pathways in cancer have been reviewed [ 7173 ] .
the mapk pathways are emerging as potential therapeutic targets for cancer [ 74 , 75 ] , and the development of inhibitors of mapk pathways has a growing importance in cancer therapy .
the pathway analyses of nf adenoma proteomic data clearly demonstrate that mapk - signaling pathways are involved in pituitary tumorigenesis .
dep proteomic data show that ras , erk , jnk , p38-mapk , akt , tnf , tgfb1 , mapk , and nfkb are the key nodes in their pathway networks , and that erk / mapk signaling ( fig .
the nitroproteomic data of nf adenomas show that tnf and il1b are the key nodes in their pathway networks , and that p38-mapk signaling ( fig .
11 ) is the significant canonical pathway that participates in an oxidative - stress response in an adenoma .
moreover , the pka type i beta regulatory subunit is nitrated in human pituitary adenomas ( fig . 6 ; table 3 ) , and tyrosine nitration occurs within the dimerization region ; those nitrations could interfere with dimerization and affect pka activity to suppress raf activity .
recent studies demonstrate that an overexpression of b - raf mrna and protein is a feature of nf adenomas ; that overactivity highlights an overactivity of the ras - b - raf - map kinase pathway to promote pituitary tumorigenesis , and that the low levels of raf kinase inhibitory protein ( rkip ) in a gh - pituitary adenoma correlate with a poor clinical response to somatostatin analog therapy because rkip can bind to and inhibit raf1 kinase to attenuate mapk signaling .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authorsfig .
reproduced from zhan and desiderio , with permission from biomed central open access journal , copyright remains with the authorsfig .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authorsfig .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authorsfig .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authors mitochondrial dysfunctional pathway network .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authors nrf2-mediated oxidative - stress response pathway .
reproduced from zhan and desiderio , with permission from biomed central open access journal , copyright remains with the authors pathway component for cell cycle g2/m dna damage checkpoint regulation .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authors erk / mapk signaling pathway .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authors the p38 mapk signaling pathway .
reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authors those nf pituitary adenoma - related tissue proteome data demonstrate clearly that crucial elements in the proteome - network system occur as variations in the nf adenoma pathology , offer systematic concept to treat nf pituitary adenoma patients , and could improve patient healthcare . from a prospective viewpoint ,
those altered crucial elements in the nf pituitary adenoma tissue proteomic network system could have three main uses : ( i ) the accurate molecular classification of nf pituitary adenoma , which is necessary for an nf pituitary adenoma , because an nf pituitary adenoma is highly heterogeneous ( table 1 ) .
( ii ) interventional prevention of nf pituitary adenomas , which will be an important strategy to eliminate nf adenoma in the pre - clinical stage for population healthcare .
tissue quantitative and comparative proteomics can identify the crucial protein elements unique to an nf pituitary adenoma in the proteome - network systems . from those crucial protein elements
the high - risk population that will be identified with body fluid ( serum ; csf ) biomarkers will benefit from those drugs to recover their proteome to the normal status , and to eliminate nf pituitary adenomas at the pre - clinical stage , which will significantly benefit the pituitary adenoma populations and reduce social economical load .
the proteome differs among different type of nf pituitary adenomas , and dynamically alters with disease pathophysiological processes . with the development of proteomic techniques , computation biology , and system biology ,
the complete proteomic databases unique to each cell - type of nf pituitary adenomas , and different pathophysiological stages , will be accurately established .
the individualized unique proteomic elements will be determined for each patient . toward those personalized proteomic elements
, one could apply protein - engineering techniques to correct for those altered proteomic elements to the normal status in the proteome - network system in order to realize personalized patient treatment and to halt disease progression , which will provide a highly efficacious therapy , and will reduce the patient costs compared to traditional therapy strategy .
csf and blood serum are two important body fluids that are used for pituitary adenoma studies .
csf around the brain and spinal cord contains peptides ( several hundred ) and proteins ( about 1000 ) that are important for brain physiology , and is an excellent source of peptides and proteins that are secreted during brain - tumor development , and also potentially relevant to brain pathology .
serum is another important biological fluid that is an excellent source of peptides and proteins that are metabolized during pituitary - tumor pathological processes .
serum proteomes and peptidomes contain resident hemostatic proteins , immunoglobulins , cytokines , hormones , secreted proteins , exogenous proteins , peptides ; infection indictors ; and also tissue - leakage proteins such as those released from damaged or dying cells
. therefore , blood serum contains accurate molecular information on the physiological state of all tissues / organs in the body , and changes with disease status . monitoring the changes in the peptide and protein composition of an nf pituitary adenoma patient s csf and serum can be used as a sensitive and accurate molecular indicator of tumor pathology .
also , csf and blood specimens are easily accessible from patients and controls , with minimal invasion .
moreover , csf and blood specimens that are from different pathological stages can be obtained at different time - points .
therefore , csf and blood serum / plasma are excellent targets to develop early - stage diagnostic markers , and to develop the assessment indices to measure the interventional prevention and therapy response for nf pituitary adenomas .
we state here an important paradigm shift : in the future , multiple - parameter pattern - diagnostic markers and assessment indices of prevention and therapy response will replace the single - parameter biomarker for the prediction , prevention , and personalized treatment of nf pituitary adenomas .
the reason for that paradigm shift is that a traditional single - molecule biomarker is based on an unrealistic assumption that an increase in the amount of a single compound can unambiguously specify a disease .
however , the reality is that nf pituitary adenomas are characterized by a heterogeneity among patients ; pituitary adenomas are initiated by numerous factors , and cause a range of different molecular changes . for example , a significant elevation of blood gh and prolactin has been useful to predict and diagnose acromegaly and prolatinoma , respectively .
however , a large number of acromegaly and prolactinoma patients are still detected at an advanced stage . for the nf pituitary adenomas ,
no blood hormone levels are elevated , almost all nf pituitary adenomas are diagnosed at a late stage , and no molecular indices measure the therapy response and prognosis .
multiple - parameter biomarkers from serum or csf will resolve these problems in the prediction , prevention , and personalized treatment assessment of an nf pituitary adenoma .
several promising multiparameter strategies from serum or csf proteomes and peptidomes might meet those requirements , and include protein / peptide pattern diagnostics , protein microarrays , and an antibody microarray .
this diagnostic is an ms - based technique ; namely , use the pattern variation of ms peaks to diagnose disease .
the concept behind pattern diagnostics is that the csf / blood serum proteome / peptidome reflects the pituitary tumor tissue and organ pathology that cause patterns of body - fluid protein / peptide changes that have diagnostic potential without even knowing the identities of the individual proteins / peptides .
ms can be used to detect that pattern of peaks that correspond to the intact proteins or peptides , and that peak pattern can be used to discriminate among different stages of tumor from controls . the prediction diagnosis and prognostic monitoring becomes the comparison of the pattern or
a serum - protein pattern has been used to predict and assess laryngeal squamous cell carcinoma and ovarian cancer .
surface - enhanced laser desorption / ionization time - of - flight mass spectrometry ( seldi - tof - ms ) has been used in this protein - pattern recognition [ 80 , 81 ] .
serum or csf samples from tumor and controls were individually applied to a seldi chip .
the retained proteins on the chip were subsequently ionized ( maldi ) and detected with tof - ms .
corresponding bioinformatics software was used to compare the ms spectra and to determine the differential pattern of peaks to discriminate tumors from controls .
protein pattern within a training set of seldi - tof ms spectra that contained 50 female ovarian cancer sera and 50 female control sera .
the identified signature protein pattern was used to identify ovarian cancer in individuals within a new independent validation set that contained 50 female ovarian cancer sera and 66 female control sera , with a positive predictive value of 94% , sensitivity of 100% , and specificity of 95% . however , even though seldi - tof ms was the most commonly used instrument for this protein - pattern recognition , some problems remain to be resolved for the seldi method : ( i ) what is the total amount ( moles ) of ionizable groups ( positive , negative ) due to lys , arg , asp , glu , etc . in the biological samples ?
( ii ) what is the total number ( moles ) of ionizable groups on the seldi sample probe surface ? ( iii ) what is the ratio of ( i)/(ii ) in the seldi experiments ? ( iv ) does the sample exceed , and/or chemically modify , the chromatography capability of the seldi probe surface ? ( v ) seldi does not allow a direct identification of the discriminatory proteins and suffers from low reproducibility , sensitivity , and accuracy [ 8286 ] . to overcome some of those problems , dekker et al
. used maldi - tof - ms to replace seldi - tof - ms ; the body - fluid samples were first digested with trypsin , and the tryptic peptide mixtures were analyzed with maldi - tof - ms to obtain the tryptic - peptide profile for each sample to diagnose leptomeningeal metastases in breast cancer patients with an accuracy of 77% , a sensitivity of 79% , and a specificity of 76% [ 82 , 87 ] .
developed a signature serum - peptide pattern to predict metastatic thyroid carcinomas [ 88 , 89 ] .
the plasma peptides were isolated with magnetic beads , and were analyzed with a maldi - ms scan to obtain ms spectra .
the pattern of peaks that corresponded to the serum peptides were identified in a training setting ; subsequently , the serum - peptide pattern was used to discriminate the tumors from controls in an independent validating setting .
this serum - peptidome pattern was used to distinguish metastatic thyroid carcinomas from cancer - free controls with 95% sensitivity and 95% specificity ( binomial confidence intervals , 75.199.9% ) .
csf protein pattern and peptide patterns are also under development to diagnose cns system disease with a similar strategy [ 78 , 82 ] .
the serum / csf protein / peptide pattern - recognition approach is emerging as a promising method for cancer prediction and diagnosis , and also offers the promise to measure the interventional prevention and therapeutic response .
however , one should clearly realize that the standardized common serum / csf - sample handing procedure and the large - scale samples analyzed are essential , and that this pattern - recognition method would have more potential in combination with other genetic factor analysis , and with amino acid sequence data .
this technique is derived from dna - microarray technologies without the use of ms to detect any proteomic variation .
it is another promising approach for prediction diagnosis and for the measurement of interventional prevention and therapeutic response .
briefly , a protein microarray immobilizes thousands of protein - detecting features on a microchip .
this microchip is incubated with a biological sample to detect any proteomic pattern variation in the disease relative to controls .
the common protein array refers to an antibody array that hosts different antibodies such as monoclonal , polyclonal , and antibody fragments to detect their respective antigens from human blood samples .
another type of protein microarray is to directly immobilize multiple functional proteins onto a microchip to detect protein - protein , protein - ligand , enzyme - substance , protein - dna interactions , and ptms , which is also called a functional protein array [ 9294 ] .
an example is the whole - proteome array from yeast that contains 5,000 purified proteins immobilized onto a glass microscope slide . with the prospective to identify the serum / csf protein / peptide patterns of nf pituitary adenoma ,
those signature protein / peptides or their antibodies could be immobilized onto a microchip for the predictive diagnosis and the measurement of preventive and therapeutic response for nf pituitary adenoma patients .
the traditional assessment of the level of one serum - hormone is difficult to detect clinical nf pituitary adenomas ; furthermore , no effective chemical therapy exists to treat an nf pituitary adenoma .
an nf pituitary adenoma contains highly heterogeneous cell types that are the distinguishing pathophysiological basis that causes the proteomic variation . in combination with multiple endogenous and exogenous factors ,
a more accurate multiple - parameter global systematic strategy will replace the limited single - factor strategy in the prediction , prevention , and personalized treatment of nf pituitary adenomas .
the global - proteomic strategy offers the promise for the multiple - parameter assessment for the predictive diagnosis , measurement of preventive and therapeutic response , and personalized patient treatment .
the diagram of the use of proteomic variations to predict , prevent , and personalize treatment of nf adenomas is shown ( fig .
12 ) . a tissue proteomic - variation study would lead to the accurate molecular classification of an nf adenoma , and the modality of proteomic variation for interventional prevention and halting the tumor progression .
the serum / csf proteomic / peptidomic variation would lead to serum / csf protein / peptide pattern diagnostics , and the protein / antibody microarray for the predictive diagnosis and measurement of interventional prevention and personalized therapeutic response .
briefly , a tumor - tissue proteomic - variation study would lead to an accurate molecular classification that will contribute to identification of personalized patients .
modalities of the altered tissue - proteome of a personalized patient will realize the personalized treatment of nf pituitary adenomas .
this therapeutic response will be assessed with body - fluid ( serum and/or csf ) proteomic / peptidomic variations to determine whether tumor progression is inhibited or halted .
the high - risk population that might develop an nf pituitary adenoma will be determined with a predictive diagnosis strategy body - fluid ( serum and/or csf ) proteomic / peptidomic variations .
modalities of the altered tissue - proteome of the high - risk population will realize the interventional prevention .
this preventive response will be assessed with body - fluid ( serum and/or csf ) proteomic / peptidomic variations to determine the preventive effects and whether tumor occurrence is halted . in summary ,
the detection and modalities of proteomic variation will serve an effective role in the prediction , prevention , and personalized treatment of nf pituitary adenoma patients .
12diagram of the use of proteomic variations to predict , prevent , and personalize treatment for nf pituitary adenomas diagram of the use of proteomic variations to predict , prevent , and personalize treatment for nf pituitary adenomas proteomic variations offer numerous opportunities to predict , prevent , and provide a personalized treatment ( pppm ) for nf pituitary adenomas .
currently , whole tissue - proteome variations ( protein expression ; hormone isoforms ; modifications ) are studied in nf pituitary adenomas , and the significant signal pathway networks associated with nf pituitary adenomas are mined from those whole tissue - proteome variation data .
four important pathway - network systems , including mitochondrial dysfunction , oxidative stress , cell - cycle dysregulation , and mapk - signal abnormality , for the nf adenoma are identified .
the follow - up studies of those tissue proteome data would contribute to the accurate molecular classification for the diagnosis of personalized patients and their personalized treatment .
however , some problems remain to be resolved towards that pppm goal : ( i ) the complete tissue - proteomic databases unique to single - cell type pituitary adenomas , and different pathophysiological stages , must be accurately established towards the development of individualized unique proteomic elements for each patient .
recently , laser - captured single - cell type pituitary prolactinoma cells have been used for proteomics analysis .
( ii ) the complete body - fluid ( serum ; csf ) proteomic / peptidomic databases unique to single - cell type pituitary adenomas , and different pathophysiological stages , must be accurately established . for body - fluid proteomic
/ peptidomic databases , a large - scale of normal population is needed towards the development of the predictive diagnostic pattern .
( iii ) the novel techniques such as protein - engineering techniques must be developed to correct for those altered personalized proteomic elements to the normal status in the proteome - network system towards an interventional prevention in the high - risk population and for the personalized treatment of nf pituitary adenomas .
( iv ) the comprehensive computation model system needs to be developed to integrate all proteomic data , personal data , clinical data , and other genetic data towards the facile and accurate predictive diagnosis of the high - risk population and an accurate assessment of preventive and therapeutic responses . | pituitary adenomas account for 10% of intracranial tumors , and they cause the compression of nearby structures and the inappropriate expression of pituitary hormones . unlike functional pituitary adenomas ,
nonfunctional ( nf ) pituitary adenomas account for 30% of pituitary tumors , and are large enough to cause blindness ; because they do not cause any clinical hormone hypersecretion , they are difficult to detect at an early stage ; and hypopituitarism results . no effective molecular biomarkers or chemical therapy have been approved for the clinical setting .
because an nf pituitary adenoma is highly heterogeneous , differences in the proteins ( the proteome ) can distinguish among those heterogeneity structures .
the components of a proteome dynamically change as an nf adenoma progresses .
changes in protein expression and protein modifications , individually or in combination , might be biomarkers to predict the disease , monitor the tumor progression , and develop an accurate molecular classification for personalized patient treatment .
the modalities of proteomic variation might also be useful in the interventional prevention and personalized treatment of patients to halt the occurrence and progression of nf pituitary adenomas . | Introduction
Pathophysiological basis for the variation of the proteome in an NF pituitary adenoma
Targeted organs of proteomic variation that contribute to the prediction, prevention, and personalized treatment of an NF pituitary adenoma
Techniques to measure proteomic variation
Tissue proteomic variations that contribute to accurate molecular classification, interventional prevention, and personalized patient treatment
Body-fluid proteomic and peptidomic variations that potentially will contribute to the predictive diagnosis and the measurement of interventional prevention and therapy response
Conclusions | they cause significant morbidity through a compression of regional structures and an inappropriate expression of pituitary hormones that disturb the multiple hypothalamic - pituitary - target organ axis systems . unlike functional pituitary adenomas , nonfunctional ( nf ) pituitary adenomas are typically quite large , do not cause any clinical hormone hypersecretion , and result in hypopituitarism or blindness from regional compression . therefore , an early - stage diagnosis of nf pituitary adenomas is difficult because there is no hormone hypersecretion and there is no effective chemical therapy for nf pituitary adenomas . predictive diagnosis / prevention and early - stage diagnosis / clinical therapy are the crucial aspects needed to conquer a tumor because most of nf pituitary adenomas remain as late - stage diagnosed and neurosurgically / radiation treated . study of the proteomic variations will result in new concepts and models for the more - accurate prediction , prevention , and personalized patient therapy for nf pituitary adenomas . this review article addresses the contribution of tissue - proteomic variations to the interventional prevention and personalized patient treatment , and the contribution of body - fluid proteomic variations to the predictive diagnosis and the assessment of preventive and therapeutic responses . pituitary adenomas are monoclonal in the cell of origin [ 2 , 3 ] , and might arise from any one of five pituitary anterior cell types ( fig . acth oversecretion results in cushing s disease , with features of hypercortisolism ; gh hypersecretion leads to acral overgrowth and metabolic dysfunction associated with acromegaly ; and prl oversecretion leads to gonadal failure , secondary infertility , and galactorrhea . in contrast , tumors that arise from gonadotroph cells do not efficiently secrete their gene products , and they are usually clinically silent . they are commonly referred to as nf pituitary adenomas , which can not be diagnosed early because no hormone change is observed . however , nf pituitary adenomas are highly heterogeneous in the cell of origin ( table 1 ) . except for a gonadotroph - originated intact lh / fsh adenoma ( up to 79% of nf adnomas ) ,
immunocytochemical staining of hormones also reveals that some nf pituitary adenomas might arise from a silent corticotroph ( acth - positive ; 8% ) , silent somatotroph ( gh - positive ; 3% ) , no hormone - expressed oncocytoma ( 6% ) , and null cell ( 17% ) . those heterogeneous cells of origin of nf pituitary adenomas produce differences in a proteome relative to the control and among different nf pituitary adenoma patients , and that difference in the proteome dynamically changes with tumor progression . the proteomic difference among different types of nf pituitary adenomas , and the dynamic alteration with tumor progression ,
both contribute to the prediction , interventional prevention , and personalized patient treatment of nf pituitary adenoma patients . based on the new concept of prediction , prevention , and personalized patient treatment of an nf pituitary adenoma , pituitary tissues , cerebrospinal fluid ( csf ) , and blood plasma must be analyzed . any tissue - proteome variation directly reflects the pathophysiological changes of an nf pituitary adenoma . those tissue - proteome variations , and the corresponding changes in the biological system , can be developed into an accurate molecular classification , interventional prevention , and personalized patient treatment . in addition , in an nf pituitary adenoma patient , some secreted proteins and peptides will enter into the csf and blood circulation . those csf and blood plasma proteomic and peptidomic variations will lead to the development of an accurate predictive diagnosis and the measurement of interventional prevention and therapy response based on accurate molecular data . also , an nf pituitary adenoma is monoclonal in origin , and is generally benign . an nf pituitary adenoma tissue contains a relatively pure cell population , whereas control post - mortem pituitary tissues contain different types of cells , including at least corticotrophs , somatotrophs , lactotrophs , thyrotrophs , and gonadotrophs , etc . the proteins are visualized , and the image is scanned with a cydye 3 channel and cydye 5 channel to form two fluorescent images , which are processed with decyder dige analysis software or delta 2d software . although the gel methods are limited by the extremely acidic ( pi < 3.5 or 4)/basic ( pi > 7.5 or 8) proteins , extremely high - mass ( > 150 kda ) or low - mass ( < 10 kda ) proteins , and hydrophobic proteins , the non - gel techniques are complementary with the gel - based methods , and improve the proteomic coverage of an nf pituitary adenoma proteome . the recovery of the tumor - altered pathway system to the normal pathway system would contribute significantly to interventional prevention and effective chemical therapy . those proteomic variations include variations in protein expression and post - translational modifications ( ptms ) . those altered elements include protein differential expression ( deps ) , splicing isoforms ( such as hormone isoforms ) , and protein modifications ( such as tyrosine nitration and phosphorylation ) , etc . a large - scale comparative proteomics study was performed on a set of human pituitary samples [ 1 , 5 , 23 ] : controls ( n = 8) versus several different cell - types of nf pituitary adenomas ( nf- , n = 3 ; lh+ , n = 3 ; fsh+ , n = 3 ; fsh+/lh+ , n = 3 ) and hyperprolactinomas ( n = 4 ) . results indicated that : ( i ) neuroendocrine - related proteins , including somatotropin , secretagogin , and mu - crystallin homolog , were down - regulated in nf pituitary adenomas and the hyperprolactinoma ; ( ii ) somatotropin existed in at least 17 isoforms that were down - regulated in nf adenomas and the hyperprolactinomas ; ( iii ) prolactin existed in six isoforms that were down - regulated in nf adenomas , and no - change in the hyperprolactinomas ; ( iv ) immunologic regulation proteins and tumor - related antigen ( for example , immunoglobulin , tumor - rejection antigen 1 ) were down - regulated in nf adenomas ; ( v ) cell proliferation , differentiation , and apoptosis - related proteins were down - regulated in the nf - adenomas and the hyperprolactinomas ; ( vi ) some cell - defense and stress - resistance proteins ( for example , phospholipid hydroperoxide glutathione peroxidase , cd59 glycoprotein , and heat - shock 27 kda protein ) were down - regulated in the nf pituitary adenomas ; ( vii ) some metabolic enzyme - related proteins ( for example , cytoplasmic isocitrate dehydrogenase [ nadp ] , tryptophan 5-hydroxylase 2 , matrix metalloproteinase-9 , aldose reductase , lactoylglutathione lyase , acyl - coa - binding protein , etc . ) were up - regulated in the nf pituitary adenomas ; and ( viii ) for cell - signal proteins , some were down - regulated and some were up - regulated in nf adenomas ; those cell signals are involved in the complex biological roles in the cell growth , proliferation , differentiation , apoptosis , and death cycles . reproduced from zhan and desiderio , with permission from wiley - vch , copyright 2005 the heterogeneity of hormone expression is an important characteristic in an nf pituitary adenoma . somatotropin was significantly down - regulated at the protein and the mrna levels in the nf pituitary adenoma and in prolactinomas ; that finding is consistent with their monoclonal origin [ 2 , 3]an nf adenoma originated from gonadotrophs , and a prolactinoma from lactotrophs ; the gh receptor gene was unchanged . the down - regulated ratio of the different gh isoforms was different in each cell - type of nf pituitary adenoma relative to the controls . the proportional change of the different gh isoforms might have an important value in the clinical evaluation of nf adenomas . therefore , further detailed study of the different isoforms and ptms of gh and prolactin could lead to new insights into the clinical importance of pituitary hormones in the formation of an nf adenoma . dep analyses demonstrated that many reactive - oxygen species ( ros)-related proteins were changed in human pituitary adenomas , including heat shock protein , cytochrome p450 , gst , and phospholipid hydroperoxide glutathione peroxidase ( table 2 ) . those nf adenoma nitroproteins and their complexes are involved in the tumor necrosis factor ( tnf ) and interleukin 1 ( il1 ) signaling networks ( fig . reproduced from zhan and desiderio , with permission from elsevier , copyright 2006 four pathway - network systems , including mitochondrial dysfunction , oxidative stress , cell - cycle dysregulation , and mapk - signal abnormality for the nf adenoma , were generated from those dep and nitroprotein data . mitochondrial dysfunctions have been proposed as a cause of cancer , are a biomarker for the early detection of cancer , and are a therapeutic target for cancer . studies have demonstrated that the ectopic expression of cyclin d and the overexpresion of cyclins a , b , and e occur in an nf pituitary adenoma to regulate different phases of the cell cycle , and to accelerate the progression of the cell - cycle . the pathway analysis of nf pituitary adenoma nitroproteomic data and dep data clearly revealed the cell - cycle g2/m dna damage checkpoint - regulation pathway ( fig . recent studies demonstrate that an overexpression of b - raf mrna and protein is a feature of nf adenomas ; that overactivity highlights an overactivity of the ras - b - raf - map kinase pathway to promote pituitary tumorigenesis , and that the low levels of raf kinase inhibitory protein ( rkip ) in a gh - pituitary adenoma correlate with a poor clinical response to somatostatin analog therapy because rkip can bind to and inhibit raf1 kinase to attenuate mapk signaling . reproduced from zhan and desiderio , with permission from biomed central open access journal ; copyright remains with the authors those nf pituitary adenoma - related tissue proteome data demonstrate clearly that crucial elements in the proteome - network system occur as variations in the nf adenoma pathology , offer systematic concept to treat nf pituitary adenoma patients , and could improve patient healthcare . from a prospective viewpoint ,
those altered crucial elements in the nf pituitary adenoma tissue proteomic network system could have three main uses : ( i ) the accurate molecular classification of nf pituitary adenoma , which is necessary for an nf pituitary adenoma , because an nf pituitary adenoma is highly heterogeneous ( table 1 ) . ( ii ) interventional prevention of nf pituitary adenomas , which will be an important strategy to eliminate nf adenoma in the pre - clinical stage for population healthcare . tissue quantitative and comparative proteomics can identify the crucial protein elements unique to an nf pituitary adenoma in the proteome - network systems . from those crucial protein elements
the high - risk population that will be identified with body fluid ( serum ; csf ) biomarkers will benefit from those drugs to recover their proteome to the normal status , and to eliminate nf pituitary adenomas at the pre - clinical stage , which will significantly benefit the pituitary adenoma populations and reduce social economical load . the proteome differs among different type of nf pituitary adenomas , and dynamically alters with disease pathophysiological processes . with the development of proteomic techniques , computation biology , and system biology ,
the complete proteomic databases unique to each cell - type of nf pituitary adenomas , and different pathophysiological stages , will be accurately established . toward those personalized proteomic elements
, one could apply protein - engineering techniques to correct for those altered proteomic elements to the normal status in the proteome - network system in order to realize personalized patient treatment and to halt disease progression , which will provide a highly efficacious therapy , and will reduce the patient costs compared to traditional therapy strategy . therefore , blood serum contains accurate molecular information on the physiological state of all tissues / organs in the body , and changes with disease status . monitoring the changes in the peptide and protein composition of an nf pituitary adenoma patient s csf and serum can be used as a sensitive and accurate molecular indicator of tumor pathology . therefore , csf and blood serum / plasma are excellent targets to develop early - stage diagnostic markers , and to develop the assessment indices to measure the interventional prevention and therapy response for nf pituitary adenomas . we state here an important paradigm shift : in the future , multiple - parameter pattern - diagnostic markers and assessment indices of prevention and therapy response will replace the single - parameter biomarker for the prediction , prevention , and personalized treatment of nf pituitary adenomas . however , the reality is that nf pituitary adenomas are characterized by a heterogeneity among patients ; pituitary adenomas are initiated by numerous factors , and cause a range of different molecular changes . for the nf pituitary adenomas ,
no blood hormone levels are elevated , almost all nf pituitary adenomas are diagnosed at a late stage , and no molecular indices measure the therapy response and prognosis . multiple - parameter biomarkers from serum or csf will resolve these problems in the prediction , prevention , and personalized treatment assessment of an nf pituitary adenoma . the serum / csf protein / peptide pattern - recognition approach is emerging as a promising method for cancer prediction and diagnosis , and also offers the promise to measure the interventional prevention and therapeutic response . however , one should clearly realize that the standardized common serum / csf - sample handing procedure and the large - scale samples analyzed are essential , and that this pattern - recognition method would have more potential in combination with other genetic factor analysis , and with amino acid sequence data . it is another promising approach for prediction diagnosis and for the measurement of interventional prevention and therapeutic response . this microchip is incubated with a biological sample to detect any proteomic pattern variation in the disease relative to controls . with the prospective to identify the serum / csf protein / peptide patterns of nf pituitary adenoma ,
those signature protein / peptides or their antibodies could be immobilized onto a microchip for the predictive diagnosis and the measurement of preventive and therapeutic response for nf pituitary adenoma patients . the traditional assessment of the level of one serum - hormone is difficult to detect clinical nf pituitary adenomas ; furthermore , no effective chemical therapy exists to treat an nf pituitary adenoma . an nf pituitary adenoma contains highly heterogeneous cell types that are the distinguishing pathophysiological basis that causes the proteomic variation . in combination with multiple endogenous and exogenous factors ,
a more accurate multiple - parameter global systematic strategy will replace the limited single - factor strategy in the prediction , prevention , and personalized treatment of nf pituitary adenomas . the global - proteomic strategy offers the promise for the multiple - parameter assessment for the predictive diagnosis , measurement of preventive and therapeutic response , and personalized patient treatment . the diagram of the use of proteomic variations to predict , prevent , and personalize treatment of nf adenomas is shown ( fig . a tissue proteomic - variation study would lead to the accurate molecular classification of an nf adenoma , and the modality of proteomic variation for interventional prevention and halting the tumor progression . the serum / csf proteomic / peptidomic variation would lead to serum / csf protein / peptide pattern diagnostics , and the protein / antibody microarray for the predictive diagnosis and measurement of interventional prevention and personalized therapeutic response . briefly , a tumor - tissue proteomic - variation study would lead to an accurate molecular classification that will contribute to identification of personalized patients . modalities of the altered tissue - proteome of a personalized patient will realize the personalized treatment of nf pituitary adenomas . the high - risk population that might develop an nf pituitary adenoma will be determined with a predictive diagnosis strategy body - fluid ( serum and/or csf ) proteomic / peptidomic variations . modalities of the altered tissue - proteome of the high - risk population will realize the interventional prevention . in summary ,
the detection and modalities of proteomic variation will serve an effective role in the prediction , prevention , and personalized treatment of nf pituitary adenoma patients . 12diagram of the use of proteomic variations to predict , prevent , and personalize treatment for nf pituitary adenomas diagram of the use of proteomic variations to predict , prevent , and personalize treatment for nf pituitary adenomas proteomic variations offer numerous opportunities to predict , prevent , and provide a personalized treatment ( pppm ) for nf pituitary adenomas . currently , whole tissue - proteome variations ( protein expression ; hormone isoforms ; modifications ) are studied in nf pituitary adenomas , and the significant signal pathway networks associated with nf pituitary adenomas are mined from those whole tissue - proteome variation data . four important pathway - network systems , including mitochondrial dysfunction , oxidative stress , cell - cycle dysregulation , and mapk - signal abnormality , for the nf adenoma are identified . the follow - up studies of those tissue proteome data would contribute to the accurate molecular classification for the diagnosis of personalized patients and their personalized treatment . however , some problems remain to be resolved towards that pppm goal : ( i ) the complete tissue - proteomic databases unique to single - cell type pituitary adenomas , and different pathophysiological stages , must be accurately established towards the development of individualized unique proteomic elements for each patient . ( ii ) the complete body - fluid ( serum ; csf ) proteomic / peptidomic databases unique to single - cell type pituitary adenomas , and different pathophysiological stages , must be accurately established . ( iii ) the novel techniques such as protein - engineering techniques must be developed to correct for those altered personalized proteomic elements to the normal status in the proteome - network system towards an interventional prevention in the high - risk population and for the personalized treatment of nf pituitary adenomas . | [
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the discovery of x - rays by roentgen in the year 1895 and radioactivity by becquerel in the year 1896 can be considered as the turning point in human health care as the x - rays allowed to peep inside the human body [ 1 , 2 ] . although harmful effects of ionizing radiations were reported within a few months of discovery of x - rays , the real magnitude was not known .
study of occupational workers like physicians and scientists handling radioactivity gave a clear picture of the harmful effects of ionizing radiations , which was further strengthened after the study of japanese atomic bomb survivors of 1945 .
it is now fairly well established that radiation produces deleterious effects on the organisms and widespread use of radiation in diagnosis therapy , industry , energy sector and inadvertent exposure during air and space travel , nuclear accidents and nuclear terror attacks requires safeguard against human exposures .
lead shielding and other physical measures are cumbersome to use in such situations , therefore pharmacological intervention could be the most prudent strategy to protect humans against the harmful effect of ionizing radiations . the use of chemicals to protect against the harmful effects of radiation was attempted after world war ii with the realization of the need to safeguard humans against the military use of atomic weapons .
patt and his co - workers ( 1949 ) were the first to investigate the effect of amino - acid cysteine in rats exposed to lethal doses of x - rays .
thereafter , several chemical compounds and their analogues have been screened for their radioprotective ability however , their high toxicity at optimum protective doses precluded their clinical use [ 4 , 5 ] .
the other major drawback of these compounds was that they were unable to provide post - irradiation protection . with the recognition that normal tissue protection during radiotherapy is as important as the destruction of cancer cells ,
recent terror attacks throughout the world has strengthened the idea that it its necessary to devise appropriate measures against the nuclear terror attacks by using pharmacological agents that can protect against the ill effects of radiation .
the high toxicity of thiol compounds necessitated search for alternative agents , which could be less toxic and highly effective at non - toxic dose levels .
it was also thought that products / compounds isolated from natural sources could be of substantial use as non - toxic radioprotectors .
therefore , investigators diverted their attention towards the plant and natural products during the last two decades .
plants have been reported to play an important role in the discovery of new drugs for the treatment of human diseases , which indicated that natural products play a highly significant role in the drug discovery and development process .
this was particularly evident in the areas of cancer and infectious diseases , where over 60% and 75% of these drugs , respectively , were shown to be of natural origin .
a good chemical protector should be able to protect against the deleterious effect of ionizing radiation during therapeutic procedures as well as during nuclear accidents , space flight and background irradiation etc .
an ideal radioprotector should be cheap , does not have toxic implications in a wide dose range , orally administered , rapidly absorbed , possesses a reasonably good dose reduction factor and can act through multiple mechanisms .
they are usually non - toxic , relatively cheap , can be orally administered and could act through multiple mechanisms due to the presence of many chemicals .
the advantage of plants and natural products is that they are used in several traditional systems of medicines .
once this is done their use , as radioprotectors could be more successful than synthetic chemicals .
the most pragmatic approach to select the possible candidate to evaluate radioprotective effect is to look into the available properties of the substance .
whether a substance has anti - inflammatory , antioxidant , antimicrobial , immunomodulatory , free radical scavenging or anti - stress properties , if so , it may act as a potential radioprotector and could be the right candidate for evaluation of its radioprotective activity .
short - term in vitro tests can provide a basis for detailed evaluation of radioprotective activity .
assay of free radicals and antioxidant status of a pharmacological agent can also provide some leads regarding the radioprotective potential of such agents .
if a plant or a natural product is found to inhibit lipid peroxidation and scavenge free radicals , it may act as a possible radioprotector .
the next step is to evaluate its radioprotective potential in vitro using cell survival and micronuclei assays .
if it is found to elevate cell survival and reduce radiation - induced micronuclei formation , it certainly has a potential as a radioprotector .
there are other short - term tests like dna strand breaks , apoptosis and estimation of glutathione ( gsh ) and enzymes like catalase , glutathione peroxidase etc . that can also provide an inkling of the radioprotective activity of any pharmacological agent . however , the gold standard for radioprotective activity is the evaluation of 30-day survival in rodents , since the animal studies with death as the end point are the most confirmatory , because the 30-day survival after lethal whole body irradiation clearly indicates the capacity of the pharmacological agent in test to modulate the recovery and regeneration of the gastrointestinal epithelium and the hemopoietic progenitor cells in the bone marrow , the two most radiosensitive organs that are essential for sustenance of the life .
the most reliable procedures involve determination of a dose reduction factor ( drf ) . in animal studies ,
drfs are typically determined by irradiating mice with or without administering radioprotective agent at a range of radiation doses and then comparing the endpoint of interest .
for example , the drf for 30-day survival ( ld50/30 drug - treated divided by ld50/30 vehicle - treated ) quantifies protection of the hemopoietic system [ 8 , 9 ] . with sufficient loss of hemopoietic stem cells
the gi syndrome in mice can be assessed by determining survival up to ten days ( measure of gi death ) after exposure to comparatively high doses of whole - body radiation , whereas hemopoeitic syndrome can be assessed by monitoring the survival of irradiated animals up to 30 days post - irradiation [ 711 ] .
the intestinal crypt cell assay or functional changes also serve as indicators of gi damage .
the most informative and useful preclinical studies relate protective effects to the drug s toxicity in the same animal model .
the efficacy of radioprotectors in clinical practice requires different end points . among other endpoints amenable to the determination of beneficial effects of radioprotectors ,
the most readily evaluable is protection against mucositis and xerostomia resulting from head and neck radiotherapy and various side effects when the gi tract is in the radiation field .
an intravenous infusion of an ethanol extract of gingko biloba leaves , at a dose of 100 mg / person was found to be effective in patients with vasogenic edema observed after irradiation of the brain .
it has been reported to protect against the clastogenic factors from plasma of human subjects exposed to irradiation .
treatment of recovery workers from the chernobyl accident site was found to be effective when an oral dose of 40 mg / day of g. biloba was given 3 times daily for 2 months .
aqueous extract of centella asiatica reduced the adverse effect of low dose irradiation in sprague dawley rats by inhibiting radiation - induced body weight loss and conditioned taste aversion .
similarly , it has been found to protect against the radiation - induced weight loss in mice exposed to 8 gy -radiation .
oral administration of a hippophae rhamnoides fruit juice concentrate to rats before or after irradiation increased life span , restored the 11-oxycorticosteroid level in the blood and weight of isolated adrenals , and also normalized their basal activity and response to ( acth ) ( corticotropin ) under in vitro conditions .
hydroalcoholic extract of berries of h. rhamnoides also protected mice against -radiation - induced mortality , decline in endogenous colony forming unit ( cfu ) , micronuclei formation and various other hematological parameters [ 2022 ] .
the radioprotective property of osimum sanctum was first reported by jagetia et al . against the radiation - induced mortality , thereafter studies by uma devi and her coworkers established its radioprotective efficacy by evaluating mouse survival , spleen colony assay , and chromosome aberrations in mouse bone marrow cells .
apart from these osmium has been reported to protect against radiation - induced lipid peroxidation and reduction in glutathione concentration [ 24 , 25 ] .
the radioprotective efficacy of ginseng ( panax ginseng ) has been reported by several workers [ 2630 ] .
the whole extract of ginseng and the relative protective effects of various fractions ( carbohydrate , protein and saponins ) have been evaluated .
the results showed that the water - soluble whole extract of ginseng provided best protection against radiation induced damage in c3h mice , whereas isolated protein and carbohydrate fractions were less effective , the saponin fraction was ineffective .
similar results were obtained by kim and coworkers , who found that whole ginseng extract and its fractions increased endogenous spleen colony formation in irradiated mice and also reduced apoptosis in jejunal crypt cells .
the radioprotective effect of ginseng root extract on testicular enzymes ( acid and alkaline phosphatases and lipid peroxidation ) has also been reported .
hexandrum has been reported to protect against radiation - induced mortality , gastrointestinal damage and embryonic nervous system of developing mice [ 20 , 3335 ] .
it has also been reported to protect against radiation - induced decline in glutathione - s - transferase , superoxide dismutase in the liver and intestine of irradiated mice .
oral administration of an aqueous extract of guduchi , tinospora cordifolia has been reported to increase the survival of mice exposed to radiation .
treatment of mice with hydroalcoholic extract of tinospora cordifolia has been found to protect against the radiation - induced micronuclei formation and oxidative stress and decline in the mouse survival , spleen cfu and hematological parameters .
the fruit pulp of amala , emblica officinalis ( eo ) has been reported to increase the survival and inhibit radiation - induced weight loss in mice .
phyllanthus amarus has been reported to protect against the radiation - induced decline in white blood cells ( wbc ) , superoxide dismutase , catalase , glutathione - s - transferase , glutathione peroxidase , and glutathione reductase .
daily oral administration of 800 mg / kg body weight ( b. wt . ) of rajgira ( amaranthus paniculatus ) leaf extract for 15 consecutive days before whole body exposure to -radiation protected mice against the radiation - induced lethality with a dose reduction factor of 1.36 .
it increased endogenous spleen colony forming units and spleen weight without any side effects or toxicity .
rajgara extract also arrested radiation - induced lipid peroxidation and the decline in reduced glutathione in the liver and blood of mice .
the ethanolic extract of piper longum ( pippali ) fruits was found to protect mice against the radiation - induced decline in wbc , bone marrow cells -esterase positive cells and gsh .
pippali extract also reduced the elevated levels of glutathione pyruvate transaminase ( gpt ) , alkaline phosphatase ( alp ) , lipid peroxidation ( lpo ) in liver and serum of irradiated animals .
the dietary supplements , if found radioprotective may be of crucial importance , as they are in daily human use , nontoxic and have wide acceptability .
skeels also known as eugenia cumini ( family myrtaceae ) , and has been reported to posses several medicinal properties in the folklore system of medicine .
the micronucleus study of radioprotective effect of dichloromethane and methanol ( 1:1 ) extract of jamun ( sce ) in human peripheral blood lymphocytes ( hpbls ) ascertained its radioprotective potential , where 12.5 g / ml sce was found to reduce the micronuclei up to a maximum extent . in vivo evaluation
further established its radioprotective activity where it was found to reduce radiation - induced sickness , gastrointestinal and bone marrow deaths [ 7 , 44 ] .
not only leaf but the hydroalcoholic extract of jamun seeds ( jse ) also exhibited a greatest protective effect at 80 mg / kg jse .
the jse was more effective when administered through the intraperitoneal route at equimolar doses than the oral .
the jse treatment protected mice against the gastrointestinal as well as bone marrow deaths with a drf of 1.24 .
, family lamiaceae ) , a plant , native of japan , is used as a food seasoner , household remedy , and for industrial purposes .
extract of mint ( mentha arvensis linn ) protected against the radiation - induced sickness , gastrointestinal and bone marrow deaths with a drf of 1.2 .
further it was non - toxic up to a dose of 1000 mg / kg b. wt .
pre - treatment of mice with leaf extract of another species of pudina , i.e. mentha piperita has been reported to protect mice against the radiation - induced decline in hematological constituents , serum phosphatase , endogenous spleen colonies formation , spleen weight , goblet cells / villus section and chromosomal damage [ 4850 ] .
the rhizome of zingiber officinale , commonly known as ginger , is consumed daily worldwide as a spice and flavoring agent .
the rhizome of ginger has been reported to possess diverse medicinal properties in the traditional indian system of medicine , the ayurveda , and it is widely used in several medicinal preparations .
administration of 10 mg / kg ( i.p ) or 250 mg / kg ( orally ) hydroalcoholic extract once daily , consecutively for 5 days was found to protect mice against the radiation - sickness , gastrointestinal as well as bone marrow deaths with a drf of 1.15 .
ginger has been reported to increase glutathione , reduce lipid peroxidation in vivo and scavenging of various free radicals in vitro [ 52 , 53 ] .
ageratum conyzoides , ( family : asteraceae ) is commonly known as billy goat weed .
it has been used in various parts of africa , asia and south america for curing various diseases .
the study of various doses of alcoholic extract of ageratum conyzoides , linn . revealed that the best protective dose was 75 mg / kg and it reduced radiation - induced , sickness gastrointestinal as well as bone marrow deaths .
the radioprotective effect was due to scavenging of dpph ( 1,1-diphenyl-2-picrylhydrazyl ) , free radical .
aegle marmelos correa , commonly known as bael , is a spinous tree belonging to family rutaceae .
it is grown throughout the sub - continents as well as bangladesh , burma and srilanka .
the hydroalcoholic extract of aegle marmelos ( ame ) protected cultured hpbls against the radiation - induced micronuclei at a concentration of 5 g / ml . it was also reported to scavenge oh , o2 , dpph , abts and no ( nitric oxide ) radicals in vitro in a concentration dependent manner . the radioprotective efficacy of 15 or 250 mg / kg ame
was further confirmed in animal studies where its intraperitoneal as well as oral administration has been found to protect mice against the radiation - induced sickness , gastrointestinal and bone marrow deaths and mortality giving a drf of 1.2 .
it also protected mice against the radiation - induced lipid peroxidation and elevated gsh concentration in the liver , kidney , stomach and intestine at 31 days post - irradiation .
oral administration also protected mice against the gamma radiation - induced decline in erythrocytes , leukocytes , lymphocytes and clonogenicity of hemopoietic progenitor cells assessed by exogenous spleen colony forming assay .
pretreatment of mice with ame elevated the villus height and the crypt number accompanied by a decline in goblet and dead cell number [ 57 , 58 ] . not only leaf but also the hydroalcoholic extract of aegle marmelos fruit administered intraperitoneally at a dose of 20 mg / kg once daily , consecutively for five days found to protect mice against the radiation - induced sickness , gastrointestinal as well as bone marrow deaths with a drf of 1.1 .
parker [ amoora rohituka , amoora aphanamixis ( roxb . ) wight & arn . ] is a member of the family meliaceae .
the ethyl acetate fraction of aphanamixis polystachya at a dose of 7.5 mg / kg b. wt . before exposure to 15 gy of whole body gamma - radiation significantly reduced the frequencies of aberrant cells and chromosomal aberrations like acentric fragments , chromatid and chromosome breaks , centric rings , dicentrics , exchanges and total aberrations at all post - irradiation scoring times .
it also showed a concentration dependent scavenging of hydroxyl , superoxide , 2,2'-diphenyl-1-picryl hydrazyl ( dpph ) radicals and the 2,2-azino - bis-3-ethyl benzothiazoline-6-sulphonic acid ( abts ) cation radicals in vitro .
eap treatment also reduced lipid peroxidation in bone marrow cells in a concentration dependent manner .
ionizing radiations induce reactive oxygen species in the form of oh , h , singlet oxygen and peroxyl radicals that follows a cascade of events leading to dna damage such as single- or double - strand breaks ( dsb ) , base damage , and dna - dna or dna - protein cross - links , and these lesions cluster as complex local multiply damaged sites .
the dna - dsbs are considered the most lethal events following ionizing radiation and has been found to be the main target of cell killing by radiation .
the putative mechanisms of radioprotection by plant and herbal radioprotectors are shown in fig . 1 .
the radioprotective activity of plant and herbs may be mediated through several mechanisms , since they are complex mixtures of many chemicals .
the majority of plants and herbs contain polyphenols , scavenging of radiation - induced free radicals and elevation of cellular antioxidants by plants and herbs in irradiated systems could be leading mechanisms for radioprotection .
the polyphenols present in the plants and herbs may upregulate mrnas of antioxidant enzymes such as catalase , glutathione transferase , glutathione peroxidase , superoxide dismutase and thus may counteract the oxidative stress - induced by ionizing radiations .
upregulation of dna repair genes may also protect against radiation - induced damage by bringing error free repair of dna damage .
reduction in lipid peroxidation and elevation in non - protein sulphydryl groups may also contribute to some extent to their radioprotective activity .
the plants and herb may also inhibit activation of protein kinase c ( pkc ) , mitogen activated protein kinase ( mapk ) , cytochrome p-450 , nitric oxide and several other genes that may be responsible for inducing damage after irradiation .
the discovery of x - rays by roentgen in the year 1895 and radioactivity by becquerel in the year 1896 can be considered as the turning point in human health care as the x - rays allowed to peep inside the human body [ 1 , 2 ] . although harmful effects of ionizing radiations were reported within a few months of discovery of x - rays , the real magnitude was not known .
study of occupational workers like physicians and scientists handling radioactivity gave a clear picture of the harmful effects of ionizing radiations , which was further strengthened after the study of japanese atomic bomb survivors of 1945 .
it is now fairly well established that radiation produces deleterious effects on the organisms and widespread use of radiation in diagnosis therapy , industry , energy sector and inadvertent exposure during air and space travel , nuclear accidents and nuclear terror attacks requires safeguard against human exposures .
lead shielding and other physical measures are cumbersome to use in such situations , therefore pharmacological intervention could be the most prudent strategy to protect humans against the harmful effect of ionizing radiations .
the use of chemicals to protect against the harmful effects of radiation was attempted after world war ii with the realization of the need to safeguard humans against the military use of atomic weapons .
patt and his co - workers ( 1949 ) were the first to investigate the effect of amino - acid cysteine in rats exposed to lethal doses of x - rays .
thereafter , several chemical compounds and their analogues have been screened for their radioprotective ability however , their high toxicity at optimum protective doses precluded their clinical use [ 4 , 5 ] .
the other major drawback of these compounds was that they were unable to provide post - irradiation protection . with the recognition that normal tissue protection during radiotherapy is as important as the destruction of cancer cells ,
recent terror attacks throughout the world has strengthened the idea that it its necessary to devise appropriate measures against the nuclear terror attacks by using pharmacological agents that can protect against the ill effects of radiation .
the high toxicity of thiol compounds necessitated search for alternative agents , which could be less toxic and highly effective at non - toxic dose levels .
it was also thought that products / compounds isolated from natural sources could be of substantial use as non - toxic radioprotectors .
therefore , investigators diverted their attention towards the plant and natural products during the last two decades .
plants have been reported to play an important role in the discovery of new drugs for the treatment of human diseases , which indicated that natural products play a highly significant role in the drug discovery and development process .
this was particularly evident in the areas of cancer and infectious diseases , where over 60% and 75% of these drugs , respectively , were shown to be of natural origin .
a good chemical protector should be able to protect against the deleterious effect of ionizing radiation during therapeutic procedures as well as during nuclear accidents , space flight and background irradiation etc .
an ideal radioprotector should be cheap , does not have toxic implications in a wide dose range , orally administered , rapidly absorbed , possesses a reasonably good dose reduction factor and can act through multiple mechanisms .
they are usually non - toxic , relatively cheap , can be orally administered and could act through multiple mechanisms due to the presence of many chemicals . therefore , screening of plants and natural products is a useful paradigm for radioprotection .
the advantage of plants and natural products is that they are used in several traditional systems of medicines .
once this is done their use , as radioprotectors could be more successful than synthetic chemicals .
the most pragmatic approach to select the possible candidate to evaluate radioprotective effect is to look into the available properties of the substance .
whether a substance has anti - inflammatory , antioxidant , antimicrobial , immunomodulatory , free radical scavenging or anti - stress properties , if so , it may act as a potential radioprotector and could be the right candidate for evaluation of its radioprotective activity .
short - term in vitro tests can provide a basis for detailed evaluation of radioprotective activity .
assay of free radicals and antioxidant status of a pharmacological agent can also provide some leads regarding the radioprotective potential of such agents .
if a plant or a natural product is found to inhibit lipid peroxidation and scavenge free radicals , it may act as a possible radioprotector .
the next step is to evaluate its radioprotective potential in vitro using cell survival and micronuclei assays .
if it is found to elevate cell survival and reduce radiation - induced micronuclei formation , it certainly has a potential as a radioprotector .
there are other short - term tests like dna strand breaks , apoptosis and estimation of glutathione ( gsh ) and enzymes like catalase , glutathione peroxidase etc . that can also provide an inkling of the radioprotective activity of any pharmacological agent . however , the gold standard for radioprotective activity is the evaluation of 30-day survival in rodents , since the animal studies with death as the end point are the most confirmatory , because the 30-day survival after lethal whole body irradiation clearly indicates the capacity of the pharmacological agent in test to modulate the recovery and regeneration of the gastrointestinal epithelium and the hemopoietic progenitor cells in the bone marrow , the two most radiosensitive organs that are essential for sustenance of the life .
the most reliable procedures involve determination of a dose reduction factor ( drf ) . in animal studies ,
drfs are typically determined by irradiating mice with or without administering radioprotective agent at a range of radiation doses and then comparing the endpoint of interest .
for example , the drf for 30-day survival ( ld50/30 drug - treated divided by ld50/30 vehicle - treated ) quantifies protection of the hemopoietic system [ 8 , 9 ] . with sufficient loss of hemopoietic stem cells
the gi syndrome in mice can be assessed by determining survival up to ten days ( measure of gi death ) after exposure to comparatively high doses of whole - body radiation , whereas hemopoeitic syndrome can be assessed by monitoring the survival of irradiated animals up to 30 days post - irradiation [ 711 ] .
the intestinal crypt cell assay or functional changes also serve as indicators of gi damage .
the most informative and useful preclinical studies relate protective effects to the drug s toxicity in the same animal model .
the efficacy of radioprotectors in clinical practice requires different end points . among other endpoints amenable to the determination of beneficial effects of radioprotectors ,
the most readily evaluable is protection against mucositis and xerostomia resulting from head and neck radiotherapy and various side effects when the gi tract is in the radiation field .
an intravenous infusion of an ethanol extract of gingko biloba leaves , at a dose of 100 mg / person was found to be effective in patients with vasogenic edema observed after irradiation of the brain .
it has been reported to protect against the clastogenic factors from plasma of human subjects exposed to irradiation .
treatment of recovery workers from the chernobyl accident site was found to be effective when an oral dose of 40 mg / day of g. biloba was given 3 times daily for 2 months .
aqueous extract of centella asiatica reduced the adverse effect of low dose irradiation in sprague dawley rats by inhibiting radiation - induced body weight loss and conditioned taste aversion .
similarly , it has been found to protect against the radiation - induced weight loss in mice exposed to 8 gy -radiation .
oral administration of a hippophae rhamnoides fruit juice concentrate to rats before or after irradiation increased life span , restored the 11-oxycorticosteroid level in the blood and weight of isolated adrenals , and also normalized their basal activity and response to ( acth ) ( corticotropin ) under in vitro conditions .
hydroalcoholic extract of berries of h. rhamnoides also protected mice against -radiation - induced mortality , decline in endogenous colony forming unit ( cfu ) , micronuclei formation and various other hematological parameters [ 2022 ] .
the radioprotective property of osimum sanctum was first reported by jagetia et al . against the radiation - induced mortality , thereafter studies by uma devi and her coworkers established its radioprotective efficacy by evaluating mouse survival , spleen colony assay , and chromosome aberrations in mouse bone marrow cells .
apart from these osmium has been reported to protect against radiation - induced lipid peroxidation and reduction in glutathione concentration [ 24 , 25 ] .
the radioprotective efficacy of ginseng ( panax ginseng ) has been reported by several workers [ 2630 ] .
the whole extract of ginseng and the relative protective effects of various fractions ( carbohydrate , protein and saponins ) have been evaluated .
the results showed that the water - soluble whole extract of ginseng provided best protection against radiation induced damage in c3h mice , whereas isolated protein and carbohydrate fractions were less effective , the saponin fraction was ineffective .
similar results were obtained by kim and coworkers , who found that whole ginseng extract and its fractions increased endogenous spleen colony formation in irradiated mice and also reduced apoptosis in jejunal crypt cells .
the radioprotective effect of ginseng root extract on testicular enzymes ( acid and alkaline phosphatases and lipid peroxidation ) has also been reported .
hexandrum has been reported to protect against radiation - induced mortality , gastrointestinal damage and embryonic nervous system of developing mice [ 20 , 3335 ] .
it has also been reported to protect against radiation - induced decline in glutathione - s - transferase , superoxide dismutase in the liver and intestine of irradiated mice .
oral administration of an aqueous extract of guduchi , tinospora cordifolia has been reported to increase the survival of mice exposed to radiation .
treatment of mice with hydroalcoholic extract of tinospora cordifolia has been found to protect against the radiation - induced micronuclei formation and oxidative stress and decline in the mouse survival , spleen cfu and hematological parameters .
the fruit pulp of amala , emblica officinalis ( eo ) has been reported to increase the survival and inhibit radiation - induced weight loss in mice .
phyllanthus amarus has been reported to protect against the radiation - induced decline in white blood cells ( wbc ) , superoxide dismutase , catalase , glutathione - s - transferase , glutathione peroxidase , and glutathione reductase .
daily oral administration of 800 mg / kg body weight ( b. wt . ) of rajgira ( amaranthus paniculatus ) leaf extract for 15 consecutive days before whole body exposure to -radiation protected mice against the radiation - induced lethality with a dose reduction factor of 1.36 .
it increased endogenous spleen colony forming units and spleen weight without any side effects or toxicity .
rajgara extract also arrested radiation - induced lipid peroxidation and the decline in reduced glutathione in the liver and blood of mice .
the ethanolic extract of piper longum ( pippali ) fruits was found to protect mice against the radiation - induced decline in wbc , bone marrow cells -esterase positive cells and gsh .
pippali extract also reduced the elevated levels of glutathione pyruvate transaminase ( gpt ) , alkaline phosphatase ( alp ) , lipid peroxidation ( lpo ) in liver and serum of irradiated animals .
the dietary supplements , if found radioprotective may be of crucial importance , as they are in daily human use , nontoxic and have wide acceptability .
skeels also known as eugenia cumini ( family myrtaceae ) , and has been reported to posses several medicinal properties in the folklore system of medicine .
the micronucleus study of radioprotective effect of dichloromethane and methanol ( 1:1 ) extract of jamun ( sce ) in human peripheral blood lymphocytes ( hpbls ) ascertained its radioprotective potential , where 12.5 g / ml sce was found to reduce the micronuclei up to a maximum extent .
in vivo evaluation further established its radioprotective activity where it was found to reduce radiation - induced sickness , gastrointestinal and bone marrow deaths [ 7 , 44 ] .
not only leaf but the hydroalcoholic extract of jamun seeds ( jse ) also exhibited a greatest protective effect at 80 mg / kg jse .
the jse was more effective when administered through the intraperitoneal route at equimolar doses than the oral .
the jse treatment protected mice against the gastrointestinal as well as bone marrow deaths with a drf of 1.24 .
, family lamiaceae ) , a plant , native of japan , is used as a food seasoner , household remedy , and for industrial purposes .
extract of mint ( mentha arvensis linn ) protected against the radiation - induced sickness , gastrointestinal and bone marrow deaths with a drf of 1.2 .
further it was non - toxic up to a dose of 1000 mg / kg b. wt .
, the highest drug dose that could be tested for acute toxicity . pre - treatment of mice with leaf extract of another species of pudina , i.e. mentha piperita has been reported to protect mice against the radiation - induced decline in hematological constituents , serum phosphatase , endogenous spleen colonies formation , spleen weight , goblet cells / villus section and chromosomal damage [ 4850 ] .
the rhizome of zingiber officinale , commonly known as ginger , is consumed daily worldwide as a spice and flavoring agent .
the rhizome of ginger has been reported to possess diverse medicinal properties in the traditional indian system of medicine , the ayurveda , and it is widely used in several medicinal preparations .
administration of 10 mg / kg ( i.p ) or 250 mg / kg ( orally ) hydroalcoholic extract once daily , consecutively for 5 days was found to protect mice against the radiation - sickness , gastrointestinal as well as bone marrow deaths with a drf of 1.15 .
ginger has been reported to increase glutathione , reduce lipid peroxidation in vivo and scavenging of various free radicals in vitro [ 52 , 53 ] .
ageratum conyzoides , ( family : asteraceae ) is commonly known as billy goat weed .
it has been used in various parts of africa , asia and south america for curing various diseases .
the study of various doses of alcoholic extract of ageratum conyzoides , linn . revealed that the best protective dose was 75 mg / kg and it reduced radiation - induced , sickness gastrointestinal as well as bone marrow deaths .
the radioprotective effect was due to scavenging of dpph ( 1,1-diphenyl-2-picrylhydrazyl ) , free radical .
aegle marmelos correa , commonly known as bael , is a spinous tree belonging to family rutaceae .
it is grown throughout the sub - continents as well as bangladesh , burma and srilanka .
the hydroalcoholic extract of aegle marmelos ( ame ) protected cultured hpbls against the radiation - induced micronuclei at a concentration of 5 g / ml . it was also reported to scavenge oh , o2 , dpph , abts and no ( nitric oxide ) radicals in vitro in a concentration dependent manner .
the radioprotective efficacy of 15 or 250 mg / kg ame was further confirmed in animal studies where its intraperitoneal as well as oral administration has been found to protect mice against the radiation - induced sickness , gastrointestinal and bone marrow deaths and mortality giving a drf of 1.2 .
it also protected mice against the radiation - induced lipid peroxidation and elevated gsh concentration in the liver , kidney , stomach and intestine at 31 days post - irradiation .
oral administration also protected mice against the gamma radiation - induced decline in erythrocytes , leukocytes , lymphocytes and clonogenicity of hemopoietic progenitor cells assessed by exogenous spleen colony forming assay .
pretreatment of mice with ame elevated the villus height and the crypt number accompanied by a decline in goblet and dead cell number [ 57 , 58 ] . not only leaf but also the hydroalcoholic extract of aegle marmelos fruit administered intraperitoneally at a dose of 20 mg / kg once daily , consecutively for five days found to protect mice against the radiation - induced sickness , gastrointestinal as well as bone marrow deaths with a drf of 1.1 .
parker [ amoora rohituka , amoora aphanamixis ( roxb . ) wight & arn . ] is a member of the family meliaceae .
the ethyl acetate fraction of aphanamixis polystachya at a dose of 7.5 mg / kg b. wt . before exposure to 15 gy of whole body gamma - radiation significantly reduced the frequencies of aberrant cells and chromosomal aberrations like acentric fragments , chromatid and chromosome breaks , centric rings , dicentrics , exchanges and total aberrations at all post - irradiation scoring times .
it also showed a concentration dependent scavenging of hydroxyl , superoxide , 2,2'-diphenyl-1-picryl hydrazyl ( dpph ) radicals and the 2,2-azino - bis-3-ethyl benzothiazoline-6-sulphonic acid ( abts ) cation radicals in vitro .
eap treatment also reduced lipid peroxidation in bone marrow cells in a concentration dependent manner .
ionizing radiations induce reactive oxygen species in the form of oh , h , singlet oxygen and peroxyl radicals that follows a cascade of events leading to dna damage such as single- or double - strand breaks ( dsb ) , base damage , and dna - dna or dna - protein cross - links , and these lesions cluster as complex local multiply damaged sites .
the dna - dsbs are considered the most lethal events following ionizing radiation and has been found to be the main target of cell killing by radiation .
the putative mechanisms of radioprotection by plant and herbal radioprotectors are shown in fig . 1 .
the radioprotective activity of plant and herbs may be mediated through several mechanisms , since they are complex mixtures of many chemicals .
the majority of plants and herbs contain polyphenols , scavenging of radiation - induced free radicals and elevation of cellular antioxidants by plants and herbs in irradiated systems could be leading mechanisms for radioprotection .
the polyphenols present in the plants and herbs may upregulate mrnas of antioxidant enzymes such as catalase , glutathione transferase , glutathione peroxidase , superoxide dismutase and thus may counteract the oxidative stress - induced by ionizing radiations .
upregulation of dna repair genes may also protect against radiation - induced damage by bringing error free repair of dna damage .
reduction in lipid peroxidation and elevation in non - protein sulphydryl groups may also contribute to some extent to their radioprotective activity .
the plants and herb may also inhibit activation of protein kinase c ( pkc ) , mitogen activated protein kinase ( mapk ) , cytochrome p-450 , nitric oxide and several other genes that may be responsible for inducing damage after irradiation .
apart from these humans also get exposed to ionizing radiations during air and space travel , background radiation nuclear accidents , and use of electronic devices .
nuclear terror attacks are not distant possibility , therefore it is essential to protect humans from ionizing radiations by pharmacological intervention .
recently , focus of radiation protection has shifted to test the radioprotective potential of plants and herbs in the hope that one day it will be possible to find a suitable pharmacological agent / s that could protect humans against the deleterious effects of ionizing radiation in clinical and other conditions as well as during nuclear terror attack .
majority of plant and herbs described in this review have medicinal properties and are being used in traditional ayurvedic or chinese systems of medicine to treat various ailments in humans .
they protect against the radiation - induced damage by scavenging of free radicals and increasing antioxidant status .
fractionation guided evaluation may result in the development of ideal radioprotector / s in the near future . | ionizing radiations produce deleterious effects in the living organisms and the rapid technological advancement has increased human exposure to ionizing radiations enormously .
there is a need to protect humans against such effects of ionizing radiation .
attempts to protect against the deleterious effects of ionizing radiations by pharmacological intervention were made as early as 1949 and efforts are continued to search radioprotectors , which may be of great help for human application .
this review mainly dwells on the radioprotective potential of plant and herbal extracts .
the results obtained from in vitro and in vivo studies indicate that several botanicals such as gingko biloba , centella asiatica , hippophae rhamnoides , ocimum sanctum , panax ginseng , podophyllum hexandrum , amaranthus paniculatus , emblica officinalis , phyllanthus amarus , piper longum , tinospora cordifoila , mentha arvensis , mentha piperita , syzygium cumini , zingiber officinale , ageratum conyzoides , aegle marmelos and aphanamixis polystachya protect against radiation - induced lethality , lipid peroxidation and dna damage .
the fractionation - guided evaluation may help to develop new radioprotectors of desired activities . | Introduction
Need for chemical radioprotection
Chemical radioprotection
Assessment of radioprotective potential of plants and herbs
Plants and herbs as radioprotectors
Mechanism of action
Conclusions | although harmful effects of ionizing radiations were reported within a few months of discovery of x - rays , the real magnitude was not known . study of occupational workers like physicians and scientists handling radioactivity gave a clear picture of the harmful effects of ionizing radiations , which was further strengthened after the study of japanese atomic bomb survivors of 1945 . it is now fairly well established that radiation produces deleterious effects on the organisms and widespread use of radiation in diagnosis therapy , industry , energy sector and inadvertent exposure during air and space travel , nuclear accidents and nuclear terror attacks requires safeguard against human exposures . lead shielding and other physical measures are cumbersome to use in such situations , therefore pharmacological intervention could be the most prudent strategy to protect humans against the harmful effect of ionizing radiations . the use of chemicals to protect against the harmful effects of radiation was attempted after world war ii with the realization of the need to safeguard humans against the military use of atomic weapons . with the recognition that normal tissue protection during radiotherapy is as important as the destruction of cancer cells ,
recent terror attacks throughout the world has strengthened the idea that it its necessary to devise appropriate measures against the nuclear terror attacks by using pharmacological agents that can protect against the ill effects of radiation . this was particularly evident in the areas of cancer and infectious diseases , where over 60% and 75% of these drugs , respectively , were shown to be of natural origin . a good chemical protector should be able to protect against the deleterious effect of ionizing radiation during therapeutic procedures as well as during nuclear accidents , space flight and background irradiation etc . assay of free radicals and antioxidant status of a pharmacological agent can also provide some leads regarding the radioprotective potential of such agents . if a plant or a natural product is found to inhibit lipid peroxidation and scavenge free radicals , it may act as a possible radioprotector . the next step is to evaluate its radioprotective potential in vitro using cell survival and micronuclei assays . if it is found to elevate cell survival and reduce radiation - induced micronuclei formation , it certainly has a potential as a radioprotector . however , the gold standard for radioprotective activity is the evaluation of 30-day survival in rodents , since the animal studies with death as the end point are the most confirmatory , because the 30-day survival after lethal whole body irradiation clearly indicates the capacity of the pharmacological agent in test to modulate the recovery and regeneration of the gastrointestinal epithelium and the hemopoietic progenitor cells in the bone marrow , the two most radiosensitive organs that are essential for sustenance of the life . among other endpoints amenable to the determination of beneficial effects of radioprotectors ,
the most readily evaluable is protection against mucositis and xerostomia resulting from head and neck radiotherapy and various side effects when the gi tract is in the radiation field . it has been reported to protect against the clastogenic factors from plasma of human subjects exposed to irradiation . aqueous extract of centella asiatica reduced the adverse effect of low dose irradiation in sprague dawley rats by inhibiting radiation - induced body weight loss and conditioned taste aversion . similarly , it has been found to protect against the radiation - induced weight loss in mice exposed to 8 gy -radiation . oral administration of a hippophae rhamnoides fruit juice concentrate to rats before or after irradiation increased life span , restored the 11-oxycorticosteroid level in the blood and weight of isolated adrenals , and also normalized their basal activity and response to ( acth ) ( corticotropin ) under in vitro conditions . against the radiation - induced mortality , thereafter studies by uma devi and her coworkers established its radioprotective efficacy by evaluating mouse survival , spleen colony assay , and chromosome aberrations in mouse bone marrow cells . apart from these osmium has been reported to protect against radiation - induced lipid peroxidation and reduction in glutathione concentration [ 24 , 25 ] . the radioprotective effect of ginseng root extract on testicular enzymes ( acid and alkaline phosphatases and lipid peroxidation ) has also been reported . hexandrum has been reported to protect against radiation - induced mortality , gastrointestinal damage and embryonic nervous system of developing mice [ 20 , 3335 ] . it has also been reported to protect against radiation - induced decline in glutathione - s - transferase , superoxide dismutase in the liver and intestine of irradiated mice . treatment of mice with hydroalcoholic extract of tinospora cordifolia has been found to protect against the radiation - induced micronuclei formation and oxidative stress and decline in the mouse survival , spleen cfu and hematological parameters . the fruit pulp of amala , emblica officinalis ( eo ) has been reported to increase the survival and inhibit radiation - induced weight loss in mice . phyllanthus amarus has been reported to protect against the radiation - induced decline in white blood cells ( wbc ) , superoxide dismutase , catalase , glutathione - s - transferase , glutathione peroxidase , and glutathione reductase . of rajgira ( amaranthus paniculatus ) leaf extract for 15 consecutive days before whole body exposure to -radiation protected mice against the radiation - induced lethality with a dose reduction factor of 1.36 . rajgara extract also arrested radiation - induced lipid peroxidation and the decline in reduced glutathione in the liver and blood of mice . the ethanolic extract of piper longum ( pippali ) fruits was found to protect mice against the radiation - induced decline in wbc , bone marrow cells -esterase positive cells and gsh . pippali extract also reduced the elevated levels of glutathione pyruvate transaminase ( gpt ) , alkaline phosphatase ( alp ) , lipid peroxidation ( lpo ) in liver and serum of irradiated animals . the dietary supplements , if found radioprotective may be of crucial importance , as they are in daily human use , nontoxic and have wide acceptability . in vivo evaluation
further established its radioprotective activity where it was found to reduce radiation - induced sickness , gastrointestinal and bone marrow deaths [ 7 , 44 ] . extract of mint ( mentha arvensis linn ) protected against the radiation - induced sickness , gastrointestinal and bone marrow deaths with a drf of 1.2 . mentha piperita has been reported to protect mice against the radiation - induced decline in hematological constituents , serum phosphatase , endogenous spleen colonies formation , spleen weight , goblet cells / villus section and chromosomal damage [ 4850 ] . administration of 10 mg / kg ( i.p ) or 250 mg / kg ( orally ) hydroalcoholic extract once daily , consecutively for 5 days was found to protect mice against the radiation - sickness , gastrointestinal as well as bone marrow deaths with a drf of 1.15 . ginger has been reported to increase glutathione , reduce lipid peroxidation in vivo and scavenging of various free radicals in vitro [ 52 , 53 ] . aegle marmelos correa , commonly known as bael , is a spinous tree belonging to family rutaceae . the hydroalcoholic extract of aegle marmelos ( ame ) protected cultured hpbls against the radiation - induced micronuclei at a concentration of 5 g / ml . the radioprotective efficacy of 15 or 250 mg / kg ame
was further confirmed in animal studies where its intraperitoneal as well as oral administration has been found to protect mice against the radiation - induced sickness , gastrointestinal and bone marrow deaths and mortality giving a drf of 1.2 . it also protected mice against the radiation - induced lipid peroxidation and elevated gsh concentration in the liver , kidney , stomach and intestine at 31 days post - irradiation . oral administration also protected mice against the gamma radiation - induced decline in erythrocytes , leukocytes , lymphocytes and clonogenicity of hemopoietic progenitor cells assessed by exogenous spleen colony forming assay . not only leaf but also the hydroalcoholic extract of aegle marmelos fruit administered intraperitoneally at a dose of 20 mg / kg once daily , consecutively for five days found to protect mice against the radiation - induced sickness , gastrointestinal as well as bone marrow deaths with a drf of 1.1 . ionizing radiations induce reactive oxygen species in the form of oh , h , singlet oxygen and peroxyl radicals that follows a cascade of events leading to dna damage such as single- or double - strand breaks ( dsb ) , base damage , and dna - dna or dna - protein cross - links , and these lesions cluster as complex local multiply damaged sites . the radioprotective activity of plant and herbs may be mediated through several mechanisms , since they are complex mixtures of many chemicals . the majority of plants and herbs contain polyphenols , scavenging of radiation - induced free radicals and elevation of cellular antioxidants by plants and herbs in irradiated systems could be leading mechanisms for radioprotection . the polyphenols present in the plants and herbs may upregulate mrnas of antioxidant enzymes such as catalase , glutathione transferase , glutathione peroxidase , superoxide dismutase and thus may counteract the oxidative stress - induced by ionizing radiations . upregulation of dna repair genes may also protect against radiation - induced damage by bringing error free repair of dna damage . although harmful effects of ionizing radiations were reported within a few months of discovery of x - rays , the real magnitude was not known . study of occupational workers like physicians and scientists handling radioactivity gave a clear picture of the harmful effects of ionizing radiations , which was further strengthened after the study of japanese atomic bomb survivors of 1945 . it is now fairly well established that radiation produces deleterious effects on the organisms and widespread use of radiation in diagnosis therapy , industry , energy sector and inadvertent exposure during air and space travel , nuclear accidents and nuclear terror attacks requires safeguard against human exposures . lead shielding and other physical measures are cumbersome to use in such situations , therefore pharmacological intervention could be the most prudent strategy to protect humans against the harmful effect of ionizing radiations . the use of chemicals to protect against the harmful effects of radiation was attempted after world war ii with the realization of the need to safeguard humans against the military use of atomic weapons . with the recognition that normal tissue protection during radiotherapy is as important as the destruction of cancer cells ,
recent terror attacks throughout the world has strengthened the idea that it its necessary to devise appropriate measures against the nuclear terror attacks by using pharmacological agents that can protect against the ill effects of radiation . plants have been reported to play an important role in the discovery of new drugs for the treatment of human diseases , which indicated that natural products play a highly significant role in the drug discovery and development process . this was particularly evident in the areas of cancer and infectious diseases , where over 60% and 75% of these drugs , respectively , were shown to be of natural origin . a good chemical protector should be able to protect against the deleterious effect of ionizing radiation during therapeutic procedures as well as during nuclear accidents , space flight and background irradiation etc . assay of free radicals and antioxidant status of a pharmacological agent can also provide some leads regarding the radioprotective potential of such agents . if a plant or a natural product is found to inhibit lipid peroxidation and scavenge free radicals , it may act as a possible radioprotector . among other endpoints amenable to the determination of beneficial effects of radioprotectors ,
the most readily evaluable is protection against mucositis and xerostomia resulting from head and neck radiotherapy and various side effects when the gi tract is in the radiation field . it has been reported to protect against the clastogenic factors from plasma of human subjects exposed to irradiation . aqueous extract of centella asiatica reduced the adverse effect of low dose irradiation in sprague dawley rats by inhibiting radiation - induced body weight loss and conditioned taste aversion . similarly , it has been found to protect against the radiation - induced weight loss in mice exposed to 8 gy -radiation . oral administration of a hippophae rhamnoides fruit juice concentrate to rats before or after irradiation increased life span , restored the 11-oxycorticosteroid level in the blood and weight of isolated adrenals , and also normalized their basal activity and response to ( acth ) ( corticotropin ) under in vitro conditions . against the radiation - induced mortality , thereafter studies by uma devi and her coworkers established its radioprotective efficacy by evaluating mouse survival , spleen colony assay , and chromosome aberrations in mouse bone marrow cells . apart from these osmium has been reported to protect against radiation - induced lipid peroxidation and reduction in glutathione concentration [ 24 , 25 ] . the results showed that the water - soluble whole extract of ginseng provided best protection against radiation induced damage in c3h mice , whereas isolated protein and carbohydrate fractions were less effective , the saponin fraction was ineffective . the radioprotective effect of ginseng root extract on testicular enzymes ( acid and alkaline phosphatases and lipid peroxidation ) has also been reported . hexandrum has been reported to protect against radiation - induced mortality , gastrointestinal damage and embryonic nervous system of developing mice [ 20 , 3335 ] . it has also been reported to protect against radiation - induced decline in glutathione - s - transferase , superoxide dismutase in the liver and intestine of irradiated mice . treatment of mice with hydroalcoholic extract of tinospora cordifolia has been found to protect against the radiation - induced micronuclei formation and oxidative stress and decline in the mouse survival , spleen cfu and hematological parameters . the fruit pulp of amala , emblica officinalis ( eo ) has been reported to increase the survival and inhibit radiation - induced weight loss in mice . phyllanthus amarus has been reported to protect against the radiation - induced decline in white blood cells ( wbc ) , superoxide dismutase , catalase , glutathione - s - transferase , glutathione peroxidase , and glutathione reductase . of rajgira ( amaranthus paniculatus ) leaf extract for 15 consecutive days before whole body exposure to -radiation protected mice against the radiation - induced lethality with a dose reduction factor of 1.36 . rajgara extract also arrested radiation - induced lipid peroxidation and the decline in reduced glutathione in the liver and blood of mice . the ethanolic extract of piper longum ( pippali ) fruits was found to protect mice against the radiation - induced decline in wbc , bone marrow cells -esterase positive cells and gsh . pippali extract also reduced the elevated levels of glutathione pyruvate transaminase ( gpt ) , alkaline phosphatase ( alp ) , lipid peroxidation ( lpo ) in liver and serum of irradiated animals . in vivo evaluation further established its radioprotective activity where it was found to reduce radiation - induced sickness , gastrointestinal and bone marrow deaths [ 7 , 44 ] . extract of mint ( mentha arvensis linn ) protected against the radiation - induced sickness , gastrointestinal and bone marrow deaths with a drf of 1.2 . mentha piperita has been reported to protect mice against the radiation - induced decline in hematological constituents , serum phosphatase , endogenous spleen colonies formation , spleen weight , goblet cells / villus section and chromosomal damage [ 4850 ] . the rhizome of zingiber officinale , commonly known as ginger , is consumed daily worldwide as a spice and flavoring agent . administration of 10 mg / kg ( i.p ) or 250 mg / kg ( orally ) hydroalcoholic extract once daily , consecutively for 5 days was found to protect mice against the radiation - sickness , gastrointestinal as well as bone marrow deaths with a drf of 1.15 . ginger has been reported to increase glutathione , reduce lipid peroxidation in vivo and scavenging of various free radicals in vitro [ 52 , 53 ] . revealed that the best protective dose was 75 mg / kg and it reduced radiation - induced , sickness gastrointestinal as well as bone marrow deaths . the hydroalcoholic extract of aegle marmelos ( ame ) protected cultured hpbls against the radiation - induced micronuclei at a concentration of 5 g / ml . the radioprotective efficacy of 15 or 250 mg / kg ame was further confirmed in animal studies where its intraperitoneal as well as oral administration has been found to protect mice against the radiation - induced sickness , gastrointestinal and bone marrow deaths and mortality giving a drf of 1.2 . it also protected mice against the radiation - induced lipid peroxidation and elevated gsh concentration in the liver , kidney , stomach and intestine at 31 days post - irradiation . oral administration also protected mice against the gamma radiation - induced decline in erythrocytes , leukocytes , lymphocytes and clonogenicity of hemopoietic progenitor cells assessed by exogenous spleen colony forming assay . not only leaf but also the hydroalcoholic extract of aegle marmelos fruit administered intraperitoneally at a dose of 20 mg / kg once daily , consecutively for five days found to protect mice against the radiation - induced sickness , gastrointestinal as well as bone marrow deaths with a drf of 1.1 . ionizing radiations induce reactive oxygen species in the form of oh , h , singlet oxygen and peroxyl radicals that follows a cascade of events leading to dna damage such as single- or double - strand breaks ( dsb ) , base damage , and dna - dna or dna - protein cross - links , and these lesions cluster as complex local multiply damaged sites . the radioprotective activity of plant and herbs may be mediated through several mechanisms , since they are complex mixtures of many chemicals . the majority of plants and herbs contain polyphenols , scavenging of radiation - induced free radicals and elevation of cellular antioxidants by plants and herbs in irradiated systems could be leading mechanisms for radioprotection . the polyphenols present in the plants and herbs may upregulate mrnas of antioxidant enzymes such as catalase , glutathione transferase , glutathione peroxidase , superoxide dismutase and thus may counteract the oxidative stress - induced by ionizing radiations . upregulation of dna repair genes may also protect against radiation - induced damage by bringing error free repair of dna damage . nuclear terror attacks are not distant possibility , therefore it is essential to protect humans from ionizing radiations by pharmacological intervention . recently , focus of radiation protection has shifted to test the radioprotective potential of plants and herbs in the hope that one day it will be possible to find a suitable pharmacological agent / s that could protect humans against the deleterious effects of ionizing radiation in clinical and other conditions as well as during nuclear terror attack . majority of plant and herbs described in this review have medicinal properties and are being used in traditional ayurvedic or chinese systems of medicine to treat various ailments in humans . they protect against the radiation - induced damage by scavenging of free radicals and increasing antioxidant status . fractionation guided evaluation may result in the development of ideal radioprotector / s in the near future . | [
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] |
snare mediated liposome fusion is typically studied at physiological ionic strength , with nacl or kcl concentrations of 100150 mm where the debye charge screening length is only ~7 .
we studied snare - mediated fusion at low ionic strength by employing a fret - based lipid mixing assay ( without synaptotagmin-1 ; fig .
1a ) . the r - snare population of liposomes contained a 1:1,000 molar protein - to - lipid ratio of recombinant synaptobrevin-2 and was labeled with the fluorescent lipid analog oregon green - phosphatidylethanolamine ( og - pe ; donor fluorophore ) .
the q - snare population of liposomes contained 1:2,000 of a complex consisting of two copies of recombinant syntaxin-1a ( lacking its inhibitory n - terminal habc - domain ; residues 183288 ) and a single snap-25 ( 2:1-complex ) .
as already shown in numerous studies , robust , snare specific lipid mixing was observed at normal ionic strength ( 20 mm k - hepes ph 7.4 , 150 mm kcl ; fig .
surprisingly however , lipid mixing was almost completely blocked at low ionic strength ( 20 mm k - hepes ph 7.4 , 5 mm kcl , 300 mm sucrose ) where the debye screening length was ~25 .
lipid mixing at low ionic strength was also blocked with 1:8,000 molar protein - to - lipid ratio of the binary q - snare complex stabilized with a fragment of synaptobrevin-2 ( residues 4996 ; fig .
this stabilized complex ensures that all q - snares are available for fusion and displays a dramatically enhanced fusion efficiency compared to native q - snares .
these results indicate that the absence of lipid mixing at low ionic strength was not due to unavailability of the binary q - snare complex . reversing the fluorescent labels or increasing the snare density up to 1:500 showed similar results .
we were unable to incorporate snare proteins in the liposomes at salt concentrations below 150 mm and had to reduce the ionic strength by diluting the liposomes afterward .
membrane fusion was still blocked at low ionic strength when the sucrose was omitted , but the liposomes looked osmotically deformed by negative - staining electron microscopy ( em ; fig .
1b ) and the liposomes became leaky at osmotic differences above 100 mosm ( supp .
the presence of sucrose increased the viscosity 33% and this decreases the diffusion of both soluble and membrane associated molecules .
the liposomes contained ~10% anionic lipids , and we speculated that the electrostatic repulsion might be too high for snare - mediated membrane fusion at low ionic strength . to validate this hypothesis , we performed experiments with liposomes composed of pure zwitterionic phospatidylcholine ( pc ; fig .
d ) . indeed , without ps , lipid mixing at low ionic strength still occurred , albeit somewhat slower than at normal ionic strength because of the sucrose ( increased viscosity ) .
in contrast , lipid mixing was completely blocked with 10% ps at low but not at normal ionic strength .
thus , the electrostatic repulsion at low ionic strength increases the energy barrier of membrane fusion .
snare complex formation was directly followed by fret with liposomes containing a 1:8,000 protein - to - lipid ratio of the stabilized q - snare - complex with syntaxin-1a labeled with alexa fluor 488 ( s225c ; donor fluorophore ) . mixing these liposomes with 100 nm of the soluble synaptobrevin-2196 fragment labeled with texas red ( s61c ; acceptor fluorophore ) resulted in fast complex formation independent of ionic strength ( fig .
accordingly , mixing liposomes containing full - length synaptobrevin-2 with a soluble q - snare complex ( snap-25 , syntaxin-1a183262 and synaptobrevin-24986 ) also resulted in complex formation , albeit somewhat slower .
this decrease is probably due to increased electrostatic repulsion at low ionic strength ( the q - snare complex has an acidic surface ) .
in contrast to the soluble snares , snare complex formation was only observed at normal but not at low ionic strength when liposomes bearing membrane - anchored q- and r - snares were used ( fig .
these fret data were in perfect agreement with the dissociation of the synaptobrevin-24996 fragment from the stabilized acceptor complex ( supp .
thus , we conclude that although snare zippering can occur at low ionic strength , the high electrostatic repulsion between the liposomes blocks not only lipid mixing but also snare nucleation .
we reconstituted recombinant full - length synaptotagmin-1 at a protein - to - lipid ratio of 1:4,000 in the r - snare liposomes . at normal ionic strength ,
in contrast , lipid mixing was almost completely blocked at low ionic strength and 1 mm ca now dramatically increased lipid mixing .
this lipid mixing was strictly dependent on both synaptotagmin-1 and the snares . under those low ionic strength conditions ,
experiments where ca was mixed by controlled flow focusing in a microfluidics flow chamber showed that fusion could be readily triggered with ca ( fig . 2c
e ) . compared to cuvette - based experiments , flow cytometry allows for relatively fast and well controlled mixing . moreover , under our conditions , the synaptotagmin-1 mediated membrane fusion efficiency correlated with the ca concentration in a dose dependent manner from 10 m up to 1 mm ca and mg had only little effect ( fig .
this ca sensitivity depends on precise protein densities and compositions of the membranes and buffer . at normal ionic strength , purified synaptic vesicles from rat brain fuse independent of ca ( ref .
however , as these membranes contain about 15% anionic lipids , it seemed possible that ( similar to the liposomes ) membrane fusion of synaptic vesicles becomes ca - sensitive at low ionic strength . to test this hypothesis , we fused synaptic vesicles with q - snare liposomes containing both donor and acceptor fluorophores . in this case , membrane fusion dilutes the fluorescent lipid analogs with the vesicle membranes , resulting in a loss of fret and a decrease of acceptor fluorescence .
indeed , at low ionic strength , lipid mixing was largely ca - dependent ( fig .
thus , at low ionic strength ca - synaptotagmin-1 triggers lipid mixing not only of liposomes , but also of native synaptic vesicles .
because lipid mixing does not distinguish between hemifusion and full membrane fusion , it is conceivable that the membrane fusion did not progress beyond the hemifusion state . to resolve this issue , we employed a content mixing assay where liposomes with encapsulated calcein at self quenching concentrations were fused with empty ( calcein - free ) liposomes .
indeed , a snare and ca - synaptotagmin-1 specific increase in fluorescence was observed ( fig .
this content mixing was not caused by leakage of the calcein from the liposomes , as leakage was only 45% of total calcein ( supp .
in addition , fret and fluorescence anisotropy measurements indicated that there was no snare nucleation without ca ( fig .
1e , 3c ) , which we attribute to charge - shielding by the bivalent ca cations , which allows for a low degree of snare interaction and membrane fusion .
snare complex formation was inhibited by synaptobrevin-2196 even when added after the mixing of the r- and q - snare liposomes , and regardless of whether wild - type synaptotagmin-1 was used , or a mutant in which two lysines of the polybasic patch are converted to alanines ( k325a k326a ; kaka - mutant ; fig .
this indicates that no substantial snare nucleation occurred without ca . disrupting ca - binding of the c2a - domain ( d178a d230a d232a )
g ) . in contrast , the corresponding c2b - mutant ( d309a d363a d365a ) had little effect on the membrane fusion efficiency .
this has been reported before , but contradicts in vivo data where disruption of ca - binding to the c2b - domain impaired release much more severely than c2a disruption .
this discrepancy may be because unlike in the synapse both fusion partners are small liposomes , which bypasses the requirement for membrane bending by the c2b - domain . to evaluate whether membrane curvature influences the ability of the c2b - domain to stimulate membrane fusion at low ionic strength , we performed experiments with 1020 m giant unilamellar vesicles ( guvs ; fig .
these membranes are much less curved than the small 36 nm - sized liposomes used elsewhere in this study . fusing
the guvs with small synaptotagmin-1synaptobrevin-2 liposomes showed that fusion was abolished when ca - binding was disrupted to the c2b - mutant , similar to normal ionic strength .
importantly , fusion of the c2b - mutant could be rescued by disruption of the guvs by sonication ( supp .
, we conclude that at low ionic strength ca - synaptotagmin-1 triggers snare - mediated membrane fusion and both c2-domains are required .
synaptotagmin-1 binds to anionic lipids both with and without ca and this binding leads to membrane clustering .
indeed , negative - staining em showed strong instantaneous clustering of the liposomes when synaptotagmin-1 liposomes were mixed with empty liposomes ( no snares ; supp .
this clustering was pi(4,5)p2 and synaptotagmin-1 specific and was reduced when the kaka mutant of synaptotagmin-1 was used ( supp .
the clustering of liposomes by synaptotagmin-1 was confirmed with dynamic light scattering ( dls ) experiments ( fig .
in addition , we developed a microscale thermophoresis assay to assess liposome clustering without ca ( fig .
, did - labeled liposomes were inserted in a capillary that was heated ~5c locally with a focused infrared laser . because of the soret - effect , these liposomes thermodiffused away from the heated spot , hence creating a local drop in the concentration and fluorescence intensity (= positive thermophoresis ) .
the extent of this depletion depends on the surface properties of the liposomes ( solvation entropy ) .
contrary to the did - liposomes , liposomes labeled with sufficient ( > 5 mol% ) og - pe thermodiffuse towards the heated spot and are locally enriched (= negative thermophoresis ) .
thus , did - liposomes are separated from og - pe - liposomes by the heating laser , unless they are tethered by synaptotagmin-1 in which case they co - segregate resulting in intermediate thermophoresis .
this tethering was dependent on pi(4,5)p2 and was reduced when the kaka mutant of synaptotagmin-1 was used , all in agreement with the dls and em data .
density gradient flotation experiments with various mutants of the soluble c2ab - domain showed that without ca the polybasic lysine patch of synaptotagmin-1 bound to membranes , whereas binding with ca occurred primarily via the ca - binding sites ( supp .
, synaptotagmin-1 tethered the liposomes at very narrow distances even without ca ( squeezed together ; supp .
e ) suggesting that the vesicles are still separated by a distance too far for trans snare interactions ( fig .
4e ) . however , negative - staining em involves fixation and drying and is thus unsuitable for assessing membrane distances .
we could not perform cryo - em , because of the presence of sucrose to compensate for the osmolarity .
4e ) , based on the ~5% fret resulting from clustering of dii with did labeled liposomes at distances below ~5 nm . in the presence of synaptotagmin-1 ( no snares ) ,
d ) . accordingly , ca induced fret of both the wild - type and the kaka mutant and this increased substantially when 1% pi(4,5)p2 was present . disrupting ca - binding to the c2a- or c2b - domain largely impaired this fret signal .
the observed fret was caused by close membrane proximity rather than by residual lipid mixing , because the fret signal was reversed by addition of 1 mm edta .
lastly , dynamic light scattering experiments with the c2ab fragment ( fig . 4h ; supp . fig .
4f ) showed that this fragment was sufficient for liposome clustering in the presence of ca , similar to normal ionic strength .
together , we conclude that without ca the polybasic patch of synaptotagmin-1 tethers the liposomes too far for snare nucleation . ca binds to the c2ab - domain , which functions as a charge bridge bringing the membranes close enough for snare nucleation .
a comparison of wild - type synaptotagmin-1 and the kaka mutant ( with reduced membrane tethering ) indicates that this tethering can accelerate lipid mixing at low , rate - limiting concentrations of liposomes ( fig .
a specific interaction between synaptotagmin-1 and the neuronal snares does not seem required , because the snares of constitutive exocytosis ( snap-23 and syntaxin-4 without habc - domain ; residues 191298 ) can also mediate ca - synaptotagmin-1-triggered membrane fusion ( fig .
these findings are not surprising since snare interaction of ca - synaptotagmin-1 is not required to bring two membranes in close proximity ( fig .
however , this contradicts earlier data on the c2ab fragment , likely because : ( i ) these studies were performed with much higher snare densities and at normal ionic strength where synaptotagmin-1snare interactions may help tethering of the liposomes and/or may modulate the snares .
( ii ) we can not exclude that synaptotagmin-1 interacts with snap-23syntaxin-4 at low ionic strength .
lastly , lipid mixing experiments showed that liposome tethering at close distance by the ca - bound c2ab fragment is sufficient to trigger membrane fusion ( supp .
we estimated how far synaptotagmin-1 could tether two membranes in various conformations with coarse - grain molecular dynamics simulations .
the c2ab - domain was simulated based on the crystal structure ( pdb 1dqv ) .
each individual c2-domain was conformationally fixed , but had full translational and rotational mobility and was connected by a flexible linker ( residues 266273 ) , as supported by nmr data .
we then pulled pair - wise on the ca - binding patches , the polybasic patch and the n - terminus ( fig .
the distances between those sites reflect the maximum length that synaptotagmin-1 can connect two membranes and are determined by the interconnecting linker and the surface interactions between the c2-domains .
the distance from the polybasic patch to the transmembrane helix is ~28 nm ( including the ~23 nm linker ) .
however , the distance synaptotagmin-1 tethers liposomes without ca is likely shorter since the debye length is only ~25 , but higher than the ~5 nm from the fret experiments ( fig .
the maximum distances the ca - bound c2ab - domain could span two membranes is ~27.5 nm , close to the 4 nm from cryo - em . these distances would explain why decreasing the ionic strength and increasing the debye length from ~7 to 25 has such a dramatic effect on membrane fusion .
thus , at low ionic strength , ca changes the membrane distance from 528 nm to below that required for snare complex formation ( 8 nm at normal ionic strength ) such that membrane fusion can occur .
snare mediated liposome fusion is typically studied at physiological ionic strength , with nacl or kcl concentrations of 100150 mm where the debye charge screening length is only ~7 .
we studied snare - mediated fusion at low ionic strength by employing a fret - based lipid mixing assay ( without synaptotagmin-1 ; fig .
1a ) . the r - snare population of liposomes contained a 1:1,000 molar protein - to - lipid ratio of recombinant synaptobrevin-2 and was labeled with the fluorescent lipid analog oregon green - phosphatidylethanolamine ( og - pe ; donor fluorophore ) .
the q - snare population of liposomes contained 1:2,000 of a complex consisting of two copies of recombinant syntaxin-1a ( lacking its inhibitory n - terminal habc - domain ; residues 183288 ) and a single snap-25 ( 2:1-complex ) .
as already shown in numerous studies , robust , snare specific lipid mixing was observed at normal ionic strength ( 20 mm k - hepes ph 7.4 , 150 mm kcl ; fig .
surprisingly however , lipid mixing was almost completely blocked at low ionic strength ( 20 mm k - hepes ph 7.4 , 5 mm kcl , 300 mm sucrose ) where the debye screening length was ~25 .
lipid mixing at low ionic strength was also blocked with 1:8,000 molar protein - to - lipid ratio of the binary q - snare complex stabilized with a fragment of synaptobrevin-2 ( residues 4996 ; fig .
this stabilized complex ensures that all q - snares are available for fusion and displays a dramatically enhanced fusion efficiency compared to native q - snares .
these results indicate that the absence of lipid mixing at low ionic strength was not due to unavailability of the binary q - snare complex . reversing the fluorescent labels or increasing the snare density up to 1:500 showed similar results .
we were unable to incorporate snare proteins in the liposomes at salt concentrations below 150 mm and had to reduce the ionic strength by diluting the liposomes afterward .
membrane fusion was still blocked at low ionic strength when the sucrose was omitted , but the liposomes looked osmotically deformed by negative - staining electron microscopy ( em ; fig .
1b ) and the liposomes became leaky at osmotic differences above 100 mosm ( supp .
the presence of sucrose increased the viscosity 33% and this decreases the diffusion of both soluble and membrane associated molecules .
the liposomes contained ~10% anionic lipids , and we speculated that the electrostatic repulsion might be too high for snare - mediated membrane fusion at low ionic strength . to validate this hypothesis , we performed experiments with liposomes composed of pure zwitterionic phospatidylcholine ( pc ; fig .
d ) . indeed , without ps , lipid mixing at low ionic strength still occurred , albeit somewhat slower than at normal ionic strength because of the sucrose ( increased viscosity ) .
in contrast , lipid mixing was completely blocked with 10% ps at low but not at normal ionic strength .
thus , the electrostatic repulsion at low ionic strength increases the energy barrier of membrane fusion .
snare complex formation was directly followed by fret with liposomes containing a 1:8,000 protein - to - lipid ratio of the stabilized q - snare - complex with syntaxin-1a labeled with alexa fluor 488 ( s225c ; donor fluorophore ) . mixing these liposomes with 100 nm of the soluble synaptobrevin-2196 fragment labeled with texas red ( s61c ; acceptor fluorophore ) resulted in fast complex formation independent of ionic strength ( fig .
accordingly , mixing liposomes containing full - length synaptobrevin-2 with a soluble q - snare complex ( snap-25 , syntaxin-1a183262 and synaptobrevin-24986 ) also resulted in complex formation , albeit somewhat slower .
this decrease is probably due to increased electrostatic repulsion at low ionic strength ( the q - snare complex has an acidic surface ) .
in contrast to the soluble snares , snare complex formation was only observed at normal but not at low ionic strength when liposomes bearing membrane - anchored q- and r - snares were used ( fig .
these fret data were in perfect agreement with the dissociation of the synaptobrevin-24996 fragment from the stabilized acceptor complex ( supp .
thus , we conclude that although snare zippering can occur at low ionic strength , the high electrostatic repulsion between the liposomes blocks not only lipid mixing but also snare nucleation .
we reconstituted recombinant full - length synaptotagmin-1 at a protein - to - lipid ratio of 1:4,000 in the r - snare liposomes . at normal ionic strength ,
in contrast , lipid mixing was almost completely blocked at low ionic strength and 1 mm ca now dramatically increased lipid mixing .
this lipid mixing was strictly dependent on both synaptotagmin-1 and the snares . under those low ionic strength conditions ,
experiments where ca was mixed by controlled flow focusing in a microfluidics flow chamber showed that fusion could be readily triggered with ca ( fig . 2c e ) .
compared to cuvette - based experiments , flow cytometry allows for relatively fast and well controlled mixing . moreover , under our conditions , the synaptotagmin-1 mediated membrane fusion efficiency correlated with the ca concentration in a dose dependent manner from 10 m up to 1 mm ca and mg had only little effect ( fig .
this ca sensitivity depends on precise protein densities and compositions of the membranes and buffer . at normal ionic strength , purified synaptic vesicles from rat brain fuse independent of ca ( ref .
however , as these membranes contain about 15% anionic lipids , it seemed possible that ( similar to the liposomes ) membrane fusion of synaptic vesicles becomes ca - sensitive at low ionic strength . to test this hypothesis , we fused synaptic vesicles with q - snare liposomes containing both donor and acceptor fluorophores . in this case , membrane fusion dilutes the fluorescent lipid analogs with the vesicle membranes , resulting in a loss of fret and a decrease of acceptor fluorescence . indeed , at low ionic strength , lipid mixing was largely ca - dependent ( fig .
thus , at low ionic strength ca - synaptotagmin-1 triggers lipid mixing not only of liposomes , but also of native synaptic vesicles . because lipid mixing does not distinguish between hemifusion and full membrane fusion , it is conceivable that the membrane fusion did not progress beyond the hemifusion state . to resolve this issue , we employed a content mixing assay where liposomes with encapsulated calcein at self quenching concentrations were fused with empty ( calcein - free ) liposomes .
this content mixing was not caused by leakage of the calcein from the liposomes , as leakage was only 45% of total calcein ( supp .
in addition , fret and fluorescence anisotropy measurements indicated that there was no snare nucleation without ca ( fig .
1e f , 3b e ) . in the absence of synaptotagmin-1 , some residual snare complex formation was observed ( fig .
1e , 3c ) , which we attribute to charge - shielding by the bivalent ca cations , which allows for a low degree of snare interaction and membrane fusion .
snare complex formation was inhibited by synaptobrevin-2196 even when added after the mixing of the r- and q - snare liposomes , and regardless of whether wild - type synaptotagmin-1 was used , or a mutant in which two lysines of the polybasic patch are converted to alanines ( k325a k326a ; kaka - mutant ; fig .
disrupting ca - binding of the c2a - domain ( d178a d230a d232a ) reduced both the lipid - mixing and snare - complex formation substantially ( fig .
g ) . in contrast , the corresponding c2b - mutant ( d309a d363a d365a ) had little effect on the membrane fusion efficiency .
this has been reported before , but contradicts in vivo data where disruption of ca - binding to the c2b - domain impaired release much more severely than c2a disruption .
this discrepancy may be because unlike in the synapse both fusion partners are small liposomes , which bypasses the requirement for membrane bending by the c2b - domain . to evaluate whether membrane curvature influences the ability of the c2b - domain to stimulate membrane fusion at low ionic strength , we performed experiments with 1020 m giant unilamellar vesicles ( guvs ; fig .
these membranes are much less curved than the small 36 nm - sized liposomes used elsewhere in this study . fusing
the guvs with small synaptotagmin-1synaptobrevin-2 liposomes showed that fusion was abolished when ca - binding was disrupted to the c2b - mutant , similar to normal ionic strength .
importantly , fusion of the c2b - mutant could be rescued by disruption of the guvs by sonication ( supp .
3b ) . all together , we conclude that at low ionic strength ca - synaptotagmin-1 triggers snare - mediated membrane fusion and both c2-domains are required .
synaptotagmin-1 binds to anionic lipids both with and without ca and this binding leads to membrane clustering . indeed , negative - staining em showed strong instantaneous clustering of the liposomes when synaptotagmin-1 liposomes were mixed with empty liposomes ( no snares ; supp .
this clustering was pi(4,5)p2 and synaptotagmin-1 specific and was reduced when the kaka mutant of synaptotagmin-1 was used ( supp .
the clustering of liposomes by synaptotagmin-1 was confirmed with dynamic light scattering ( dls ) experiments ( fig .
, we developed a microscale thermophoresis assay to assess liposome clustering without ca ( fig .
, did - labeled liposomes were inserted in a capillary that was heated ~5c locally with a focused infrared laser . because of the soret - effect , these liposomes thermodiffused away from the heated spot , hence creating a local drop in the concentration and fluorescence intensity (= positive thermophoresis ) .
the extent of this depletion depends on the surface properties of the liposomes ( solvation entropy ) .
contrary to the did - liposomes , liposomes labeled with sufficient ( > 5 mol% ) og - pe thermodiffuse towards the heated spot and are locally enriched (= negative thermophoresis ) .
thus , did - liposomes are separated from og - pe - liposomes by the heating laser , unless they are tethered by synaptotagmin-1 in which case they co - segregate resulting in intermediate thermophoresis .
this tethering was dependent on pi(4,5)p2 and was reduced when the kaka mutant of synaptotagmin-1 was used , all in agreement with the dls and em data .
density gradient flotation experiments with various mutants of the soluble c2ab - domain showed that without ca the polybasic lysine patch of synaptotagmin-1 bound to membranes , whereas binding with ca occurred primarily via the ca - binding sites ( supp .
, synaptotagmin-1 tethered the liposomes at very narrow distances even without ca ( squeezed together ; supp .
e ) suggesting that the vesicles are still separated by a distance too far for trans snare interactions ( fig .
4e ) . however , negative - staining em involves fixation and drying and is thus unsuitable for assessing membrane distances .
we could not perform cryo - em , because of the presence of sucrose to compensate for the osmolarity . therefore , we used the fret approach of yoon et al .
4e ) , based on the ~5% fret resulting from clustering of dii with did labeled liposomes at distances below ~5 nm . in the presence of synaptotagmin-1 ( no snares ) ,
d ) . accordingly , ca induced fret of both the wild - type and the kaka mutant and this increased substantially when 1% pi(4,5)p2 was present . disrupting ca - binding to the c2a- or c2b - domain largely impaired this fret signal .
the observed fret was caused by close membrane proximity rather than by residual lipid mixing , because the fret signal was reversed by addition of 1 mm edta .
lastly , dynamic light scattering experiments with the c2ab fragment ( fig . 4h ; supp . fig .
4f ) showed that this fragment was sufficient for liposome clustering in the presence of ca , similar to normal ionic strength .
together , we conclude that without ca the polybasic patch of synaptotagmin-1 tethers the liposomes too far for snare nucleation .
ca binds to the c2ab - domain , which functions as a charge bridge bringing the membranes close enough for snare nucleation . a comparison of wild - type synaptotagmin-1 and
the kaka mutant ( with reduced membrane tethering ) indicates that this tethering can accelerate lipid mixing at low , rate - limiting concentrations of liposomes ( fig .
a specific interaction between synaptotagmin-1 and the neuronal snares does not seem required , because the snares of constitutive exocytosis ( snap-23 and syntaxin-4 without habc - domain ; residues 191298 ) can also mediate ca - synaptotagmin-1-triggered membrane fusion ( fig .
these findings are not surprising since snare interaction of ca - synaptotagmin-1 is not required to bring two membranes in close proximity ( fig .
however , this contradicts earlier data on the c2ab fragment , likely because : ( i ) these studies were performed with much higher snare densities and at normal ionic strength where synaptotagmin-1snare interactions may help tethering of the liposomes and/or may modulate the snares .
( ii ) we can not exclude that synaptotagmin-1 interacts with snap-23syntaxin-4 at low ionic strength .
lastly , lipid mixing experiments showed that liposome tethering at close distance by the ca - bound c2ab fragment is sufficient to trigger membrane fusion ( supp .
we estimated how far synaptotagmin-1 could tether two membranes in various conformations with coarse - grain molecular dynamics simulations .
the c2ab - domain was simulated based on the crystal structure ( pdb 1dqv ) .
each individual c2-domain was conformationally fixed , but had full translational and rotational mobility and was connected by a flexible linker ( residues 266273 ) , as supported by nmr data .
we then pulled pair - wise on the ca - binding patches , the polybasic patch and the n - terminus ( fig .
the distances between those sites reflect the maximum length that synaptotagmin-1 can connect two membranes and are determined by the interconnecting linker and the surface interactions between the c2-domains .
the distance from the polybasic patch to the transmembrane helix is ~28 nm ( including the ~23 nm linker ) .
however , the distance synaptotagmin-1 tethers liposomes without ca is likely shorter since the debye length is only ~25 , but higher than the ~5 nm from the fret experiments ( fig .
the maximum distances the ca - bound c2ab - domain could span two membranes is ~27.5 nm , close to the 4 nm from cryo - em .
these distances would explain why decreasing the ionic strength and increasing the debye length from ~7 to 25 has such a dramatic effect on membrane fusion .
thus , at low ionic strength , ca changes the membrane distance from 528 nm to below that required for snare complex formation ( 8 nm at normal ionic strength ) such that membrane fusion can occur .
at low ionic strength synaptotagmin-1 can regulate membrane fusion upstream of snare nucleation by acting as a distance regulator . in the absence of ca , the polybasic patch on the c2b - domain tethers the liposomes , but the electrostatic repulsion keeps them too far apart for snare complex formation to occur ( fig .
the linker connecting the c2ab - domain to the vesicle membrane is 61 residues long ( residues 81142 ) which means it can extend to maximum ~23 nm ( 0.38 nm residue ) ; a distance longer than the ~2 25 electrical double layers separating the membranes .
ca acts as a charge bridge , and the ca - bound c2ab - domain docks the vesicles at a closer distance where snare complex formation and membrane fusion can occur ( step b ) .
similar models where synaptotagmin-1 functions as a membrane tethering factor have been proposed previously and we now present direct evidence that , at least at low ionic strength , such a mechanism can indeed trigger membrane fusion . we needed low ionic strength to assure that the membrane repulsion was large enough to prevent snare nucleation .
although this condition does not reflect physiological conditions , an intermediate state was isolated at which vesicles can be tethered ( e.g. by synaptotagmin-1 ) but the distance between the membranes was too high for synaptotagmin-1 independent membrane fusion and synaptotagmin-1 mediated reworking of the snares and the membranes .
ca binding to synaptotagmin-1 reduced the distance between the docked membranes , bringing them close enough for snare nucleation and subsequent fusion .
although specific increases in membrane fusion efficiency have been reported previously for both the c2ab fragment and full - length synaptotagmin-1 , this increase is larger in our case because background fusion ( i.e. in the absence of synaptotagmin-1 or ca ) is essentially blocked by keeping the membranes apart at low ionic strength .
thus , our finding adds a new facet to the still controversially discussed mechanism of synaptotagmin-1 action in exocytosis and may help in interpreting many of the often controversial data on synaptotagmin-1 mediated fusion of liposomes .
the question is how this mechanism is integrated in the molecular steps governing vesicle docking and fusion in the synapse . in our opinion ,
two alternative scenarios are possible : one mainstream scenario in which synaptotagmin-1 acts downstream of snare nucleation ( which does not preclude an additional earlier role in docking ) , and an alternative scenario according to which snares are not in trans - contact with each other before synaptotagmin-1 receives the ca signal . in the first case , docked and primed synaptic vesicles are characterized by the snares being partially zippered in trans as depicted in virtually all current models .
further zippering is prevented by an energy barrier that may involve membrane straining and/or inhibitory proteins such as complexins .
indeed , mutants interfering with either n - terminal or c - terminal snare zippering had profoundly different effects on exocytosis in chromaffin cells , which can be best explained by kinetically separate states ( i.e. docking and fusion ) .
accordingly , synaptotagmins ability to pull membranes a bit closer together may contribute to the overcoming of the energy barrier by relieving strain , in addition to an increase in local curvature and/or an activation / disinhibition of c - terminal snare zippering .
in the second case , ca - synaptotagmin-1 would trigger membrane fusion by rapidly decreasing the distance between the vesicles and the plasma membrane , thus allowing snare nucleation to occur ( as proposed previously ) .
thus , snare nucleation takes place after ca - influx similar to our findings ( fig .
indeed , it is still not resolved whether docked vesicles have their snare complexes arrested in a partially assembled complex .
there are good reasons for challenging the view that a metastable trans snare - complex represents an energy minimum along the fusion pathway .
firstly , knocking out synaptobrevin-2 or cleaving it with tetanus neurotoxin does not affect vesicle docking , in opposition with a requirement of snare nucleation for docking .
secondly , it is difficult to understand how a strained and partially zippered snare complex can be arrested , especially considering that only 13 snare complexes are required for membrane fusion . at least in vitro ,
even the synaptobrevin-24996 fragment ( which binds with nm affinity to the q - snares ) can not block membrane fusion . whereas both candidates for such a clamping role , synaptotagmin-1 and complexin can reduce membrane fusion , they bind with much lower affinity to the q - snare complex than synaptobrevin-24996 .
while effective energies may be different at the fusion site , there is so far no strong and direct evidence for any of these proteins slowing or even preventing snare assembly . in summary ,
our data show that in a simple reconstituted system , key elements of synaptic transmission can be mimicked if snare nucleation is prevented by keeping the membranes apart before ca - triggering . under these conditions ,
unleashed as soon as the membranes are pulled a bit closer by ca - dependent membrane binding of the c2-domains ( fig . 6b ) . while we needed to use a non - physiological trick ( low ionic strength ) to keep the membranes apart
, it is conceivable that this separator role is fulfilled by proteins residing in the space between the vesicle and the plasma membrane .
considering that both synaptic vesicles and release sites are indeed crowded with membrane proteins , such a scenario is not unlikely
. however , further experiments are needed to determine whether under physiological conditions snare nucleation occurs before or after ca - triggering in regulated exocytosis of synaptic vesicles .
lipid mixing was performed as described , except that 1.5 mol% 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate ( did ; invitrogen ; emission 670 nm ) was used as acceptor fluorophore to reduce cross - talk .
the lipid composition of the r - snare liposomes was chosen to mimic the synaptic vesicle and ( unless stated otherwise ) consisted of a 5:2:1:1 ratio of pc , phosphatidylethanolamine ( pe ) , ps and cholesterol ( all lipids from avanti polar lipids ) .
the lipid composition of the did - labeled q - snare liposomes was identical , except that 1 mol% pc was replaced with the plasma membrane lipid pi(4,5)p2 . unless stated otherwise , membrane fusion was triggered with 1 mm ca .
if not stated otherwise , all experiments at high ionic strength were performed in 20 mm k - hepes ph 7.4 , 150 mm kcl . for low ionic strength
, the same buffer was used , but now with 5 mm kcl and 300 mm sucrose . in all cases ,
lipid mixing could be well inhibited by competitive inhibition with either 10 m synaptobrevin-2196 or with a combination of 10 m snap-25 and 10 m syntaxin-1a183263 .
protocols have been described for the synaptic vesicles from rat brain , calcein dequenching , complex formation by fret and fluorescence anisotropy . in all cases ,
protein labeling efficiencies were > 80% as assessed by uv - vis spectroscopy , except for syntaxin-1a183262 s225c which was only ~30% .
em was performed as described , except that an additional wash step with 0.1% ( w / v ) glutharaldehyde in water was performed to remove the sucrose . for the flow cytometry ,
the emission at 540 and 610 nm was recorded simultaneously on a two - camera inverted fluorescence microscope setup .
microscale capillary thermophoresis was measured with a nanotemper nt.015 and 4 nm of liposomes containing 5 mol% og - pe and 40 nm of did - labeled liposomes .
dls was measured on a dynapro ( wyatt technology ) with a total liposome concentration of 4 nm in case of full - length synaptotagmin-1 or 2 nm liposomes in combination with 10 nm of the soluble c2ab fragment . for the fret docking experiments ( fig .
4f g ) the dii ( 1,1'-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate ; invitrogen ; 1.5 mol% ; donor fluorophore ) population of liposomes contained 1:4,000 synaptotagmin-1 and the did ( 1.5 mol% ; acceptor fluorophore ) liposome population contained 1% pi(4,5)p2 . for the molecular dynamics simulations , the martini coarse grained forcefield was applied to model synaptotagmin-1 based on the crystal structure .
the simulation box of 7.36.215.0 nm contained a single c2ab protein and 5,376 solvent particles .
we performed simulations up to 3 s at 310 k while pulling pair - wise with a constant velocity of 10 nm ps on the n - terminus ( residue 157 ) , the polybasic lysine patch ( 234237 ) and the two ca - binding sites of the c2a- ( 172 , 178 , 230 , 232 and 238 ) and c2b - domains ( 303 , 309 , 363 , 365 and 371 ) .
we fixed the structure of the c2a- and c2b - domains with an elastic network that connected each site between 0.5 to 0.9 nm by an harmonic bond with a force constant of 500 kj mol nm .
distances were calculated shortly before overstretching of the linker , which was apparent from a strong and continuous increase in the bond energy . | synaptotagmin-1 triggers ca2 + -sensitive , rapid neurotransmitter release by promoting the interaction of snare proteins between the synaptic vesicles and the plasma membrane . how synaptotagmin-1 promotes this interaction is controversial , and
the massive increase in membrane fusion efficiency of ca2 + -synaptotagmin-1 has not been reproduced in vitro . however ,
previous experiments have been performed at relatively high salt concentrations , screening potentially important electrostatic interactions . using functional reconstitution in liposomes ,
we show here that at low ionic strength snare - mediated membrane fusion becomes strictly dependent on both ca2 + and synaptotagmin-1 . under these conditions , synaptotagmin-1 functions as a distance regulator : tethering the liposomes too far for snare nucleation in the absence of ca2 + , but brings the liposomes close enough for membrane fusion in the presence of ca2 + .
these results may explain how the relatively weak electrostatic interactions of synaptotagmin-1 with membranes substantially accelerate fusion . | Results
Membrane fusion is blocked at low ionic strength
Ca
Synaptotagmin-1 acts as a distance regulator
Discussion
Methods
Supplementary Material | we studied snare - mediated fusion at low ionic strength by employing a fret - based lipid mixing assay ( without synaptotagmin-1 ; fig . surprisingly however , lipid mixing was almost completely blocked at low ionic strength ( 20 mm k - hepes ph 7.4 , 5 mm kcl , 300 mm sucrose ) where the debye screening length was ~25 . lipid mixing at low ionic strength was also blocked with 1:8,000 molar protein - to - lipid ratio of the binary q - snare complex stabilized with a fragment of synaptobrevin-2 ( residues 4996 ; fig . these results indicate that the absence of lipid mixing at low ionic strength was not due to unavailability of the binary q - snare complex . we were unable to incorporate snare proteins in the liposomes at salt concentrations below 150 mm and had to reduce the ionic strength by diluting the liposomes afterward . membrane fusion was still blocked at low ionic strength when the sucrose was omitted , but the liposomes looked osmotically deformed by negative - staining electron microscopy ( em ; fig . 1b ) and the liposomes became leaky at osmotic differences above 100 mosm ( supp . the liposomes contained ~10% anionic lipids , and we speculated that the electrostatic repulsion might be too high for snare - mediated membrane fusion at low ionic strength . indeed , without ps , lipid mixing at low ionic strength still occurred , albeit somewhat slower than at normal ionic strength because of the sucrose ( increased viscosity ) . in contrast , lipid mixing was completely blocked with 10% ps at low but not at normal ionic strength . thus , the electrostatic repulsion at low ionic strength increases the energy barrier of membrane fusion . this decrease is probably due to increased electrostatic repulsion at low ionic strength ( the q - snare complex has an acidic surface ) . in contrast to the soluble snares , snare complex formation was only observed at normal but not at low ionic strength when liposomes bearing membrane - anchored q- and r - snares were used ( fig . thus , we conclude that although snare zippering can occur at low ionic strength , the high electrostatic repulsion between the liposomes blocks not only lipid mixing but also snare nucleation . at normal ionic strength ,
in contrast , lipid mixing was almost completely blocked at low ionic strength and 1 mm ca now dramatically increased lipid mixing . this lipid mixing was strictly dependent on both synaptotagmin-1 and the snares . under those low ionic strength conditions ,
experiments where ca was mixed by controlled flow focusing in a microfluidics flow chamber showed that fusion could be readily triggered with ca ( fig . moreover , under our conditions , the synaptotagmin-1 mediated membrane fusion efficiency correlated with the ca concentration in a dose dependent manner from 10 m up to 1 mm ca and mg had only little effect ( fig . at normal ionic strength , purified synaptic vesicles from rat brain fuse independent of ca ( ref . however , as these membranes contain about 15% anionic lipids , it seemed possible that ( similar to the liposomes ) membrane fusion of synaptic vesicles becomes ca - sensitive at low ionic strength . to test this hypothesis , we fused synaptic vesicles with q - snare liposomes containing both donor and acceptor fluorophores . indeed , at low ionic strength , lipid mixing was largely ca - dependent ( fig . thus , at low ionic strength ca - synaptotagmin-1 triggers lipid mixing not only of liposomes , but also of native synaptic vesicles . this content mixing was not caused by leakage of the calcein from the liposomes , as leakage was only 45% of total calcein ( supp . 1e , 3c ) , which we attribute to charge - shielding by the bivalent ca cations , which allows for a low degree of snare interaction and membrane fusion . snare complex formation was inhibited by synaptobrevin-2196 even when added after the mixing of the r- and q - snare liposomes , and regardless of whether wild - type synaptotagmin-1 was used , or a mutant in which two lysines of the polybasic patch are converted to alanines ( k325a k326a ; kaka - mutant ; fig . this discrepancy may be because unlike in the synapse both fusion partners are small liposomes , which bypasses the requirement for membrane bending by the c2b - domain . to evaluate whether membrane curvature influences the ability of the c2b - domain to stimulate membrane fusion at low ionic strength , we performed experiments with 1020 m giant unilamellar vesicles ( guvs ; fig . , we conclude that at low ionic strength ca - synaptotagmin-1 triggers snare - mediated membrane fusion and both c2-domains are required . this clustering was pi(4,5)p2 and synaptotagmin-1 specific and was reduced when the kaka mutant of synaptotagmin-1 was used ( supp . this tethering was dependent on pi(4,5)p2 and was reduced when the kaka mutant of synaptotagmin-1 was used , all in agreement with the dls and em data . e ) suggesting that the vesicles are still separated by a distance too far for trans snare interactions ( fig . we could not perform cryo - em , because of the presence of sucrose to compensate for the osmolarity . in the presence of synaptotagmin-1 ( no snares ) ,
d ) . 4f ) showed that this fragment was sufficient for liposome clustering in the presence of ca , similar to normal ionic strength . together , we conclude that without ca the polybasic patch of synaptotagmin-1 tethers the liposomes too far for snare nucleation . ca binds to the c2ab - domain , which functions as a charge bridge bringing the membranes close enough for snare nucleation . a comparison of wild - type synaptotagmin-1 and the kaka mutant ( with reduced membrane tethering ) indicates that this tethering can accelerate lipid mixing at low , rate - limiting concentrations of liposomes ( fig . a specific interaction between synaptotagmin-1 and the neuronal snares does not seem required , because the snares of constitutive exocytosis ( snap-23 and syntaxin-4 without habc - domain ; residues 191298 ) can also mediate ca - synaptotagmin-1-triggered membrane fusion ( fig . however , this contradicts earlier data on the c2ab fragment , likely because : ( i ) these studies were performed with much higher snare densities and at normal ionic strength where synaptotagmin-1snare interactions may help tethering of the liposomes and/or may modulate the snares . ( ii ) we can not exclude that synaptotagmin-1 interacts with snap-23syntaxin-4 at low ionic strength . the distances between those sites reflect the maximum length that synaptotagmin-1 can connect two membranes and are determined by the interconnecting linker and the surface interactions between the c2-domains . however , the distance synaptotagmin-1 tethers liposomes without ca is likely shorter since the debye length is only ~25 , but higher than the ~5 nm from the fret experiments ( fig . thus , at low ionic strength , ca changes the membrane distance from 528 nm to below that required for snare complex formation ( 8 nm at normal ionic strength ) such that membrane fusion can occur . we studied snare - mediated fusion at low ionic strength by employing a fret - based lipid mixing assay ( without synaptotagmin-1 ; fig . surprisingly however , lipid mixing was almost completely blocked at low ionic strength ( 20 mm k - hepes ph 7.4 , 5 mm kcl , 300 mm sucrose ) where the debye screening length was ~25 . lipid mixing at low ionic strength was also blocked with 1:8,000 molar protein - to - lipid ratio of the binary q - snare complex stabilized with a fragment of synaptobrevin-2 ( residues 4996 ; fig . these results indicate that the absence of lipid mixing at low ionic strength was not due to unavailability of the binary q - snare complex . we were unable to incorporate snare proteins in the liposomes at salt concentrations below 150 mm and had to reduce the ionic strength by diluting the liposomes afterward . membrane fusion was still blocked at low ionic strength when the sucrose was omitted , but the liposomes looked osmotically deformed by negative - staining electron microscopy ( em ; fig . 1b ) and the liposomes became leaky at osmotic differences above 100 mosm ( supp . the presence of sucrose increased the viscosity 33% and this decreases the diffusion of both soluble and membrane associated molecules . the liposomes contained ~10% anionic lipids , and we speculated that the electrostatic repulsion might be too high for snare - mediated membrane fusion at low ionic strength . indeed , without ps , lipid mixing at low ionic strength still occurred , albeit somewhat slower than at normal ionic strength because of the sucrose ( increased viscosity ) . thus , the electrostatic repulsion at low ionic strength increases the energy barrier of membrane fusion . this decrease is probably due to increased electrostatic repulsion at low ionic strength ( the q - snare complex has an acidic surface ) . in contrast to the soluble snares , snare complex formation was only observed at normal but not at low ionic strength when liposomes bearing membrane - anchored q- and r - snares were used ( fig . thus , we conclude that although snare zippering can occur at low ionic strength , the high electrostatic repulsion between the liposomes blocks not only lipid mixing but also snare nucleation . at normal ionic strength ,
in contrast , lipid mixing was almost completely blocked at low ionic strength and 1 mm ca now dramatically increased lipid mixing . this lipid mixing was strictly dependent on both synaptotagmin-1 and the snares . under those low ionic strength conditions ,
experiments where ca was mixed by controlled flow focusing in a microfluidics flow chamber showed that fusion could be readily triggered with ca ( fig . moreover , under our conditions , the synaptotagmin-1 mediated membrane fusion efficiency correlated with the ca concentration in a dose dependent manner from 10 m up to 1 mm ca and mg had only little effect ( fig . however , as these membranes contain about 15% anionic lipids , it seemed possible that ( similar to the liposomes ) membrane fusion of synaptic vesicles becomes ca - sensitive at low ionic strength . to test this hypothesis , we fused synaptic vesicles with q - snare liposomes containing both donor and acceptor fluorophores . indeed , at low ionic strength , lipid mixing was largely ca - dependent ( fig . thus , at low ionic strength ca - synaptotagmin-1 triggers lipid mixing not only of liposomes , but also of native synaptic vesicles . in the absence of synaptotagmin-1 , some residual snare complex formation was observed ( fig . 1e , 3c ) , which we attribute to charge - shielding by the bivalent ca cations , which allows for a low degree of snare interaction and membrane fusion . snare complex formation was inhibited by synaptobrevin-2196 even when added after the mixing of the r- and q - snare liposomes , and regardless of whether wild - type synaptotagmin-1 was used , or a mutant in which two lysines of the polybasic patch are converted to alanines ( k325a k326a ; kaka - mutant ; fig . this discrepancy may be because unlike in the synapse both fusion partners are small liposomes , which bypasses the requirement for membrane bending by the c2b - domain . to evaluate whether membrane curvature influences the ability of the c2b - domain to stimulate membrane fusion at low ionic strength , we performed experiments with 1020 m giant unilamellar vesicles ( guvs ; fig . all together , we conclude that at low ionic strength ca - synaptotagmin-1 triggers snare - mediated membrane fusion and both c2-domains are required . this clustering was pi(4,5)p2 and synaptotagmin-1 specific and was reduced when the kaka mutant of synaptotagmin-1 was used ( supp . , synaptotagmin-1 tethered the liposomes at very narrow distances even without ca ( squeezed together ; supp . e ) suggesting that the vesicles are still separated by a distance too far for trans snare interactions ( fig . we could not perform cryo - em , because of the presence of sucrose to compensate for the osmolarity . in the presence of synaptotagmin-1 ( no snares ) ,
d ) . 4f ) showed that this fragment was sufficient for liposome clustering in the presence of ca , similar to normal ionic strength . together , we conclude that without ca the polybasic patch of synaptotagmin-1 tethers the liposomes too far for snare nucleation . ca binds to the c2ab - domain , which functions as a charge bridge bringing the membranes close enough for snare nucleation . a comparison of wild - type synaptotagmin-1 and
the kaka mutant ( with reduced membrane tethering ) indicates that this tethering can accelerate lipid mixing at low , rate - limiting concentrations of liposomes ( fig . however , this contradicts earlier data on the c2ab fragment , likely because : ( i ) these studies were performed with much higher snare densities and at normal ionic strength where synaptotagmin-1snare interactions may help tethering of the liposomes and/or may modulate the snares . ( ii ) we can not exclude that synaptotagmin-1 interacts with snap-23syntaxin-4 at low ionic strength . however , the distance synaptotagmin-1 tethers liposomes without ca is likely shorter since the debye length is only ~25 , but higher than the ~5 nm from the fret experiments ( fig . these distances would explain why decreasing the ionic strength and increasing the debye length from ~7 to 25 has such a dramatic effect on membrane fusion . thus , at low ionic strength , ca changes the membrane distance from 528 nm to below that required for snare complex formation ( 8 nm at normal ionic strength ) such that membrane fusion can occur . at low ionic strength synaptotagmin-1 can regulate membrane fusion upstream of snare nucleation by acting as a distance regulator . in the absence of ca , the polybasic patch on the c2b - domain tethers the liposomes , but the electrostatic repulsion keeps them too far apart for snare complex formation to occur ( fig . ca acts as a charge bridge , and the ca - bound c2ab - domain docks the vesicles at a closer distance where snare complex formation and membrane fusion can occur ( step b ) . similar models where synaptotagmin-1 functions as a membrane tethering factor have been proposed previously and we now present direct evidence that , at least at low ionic strength , such a mechanism can indeed trigger membrane fusion . we needed low ionic strength to assure that the membrane repulsion was large enough to prevent snare nucleation . by synaptotagmin-1 ) but the distance between the membranes was too high for synaptotagmin-1 independent membrane fusion and synaptotagmin-1 mediated reworking of the snares and the membranes . ca binding to synaptotagmin-1 reduced the distance between the docked membranes , bringing them close enough for snare nucleation and subsequent fusion . although specific increases in membrane fusion efficiency have been reported previously for both the c2ab fragment and full - length synaptotagmin-1 , this increase is larger in our case because background fusion ( i.e. in the absence of synaptotagmin-1 or ca ) is essentially blocked by keeping the membranes apart at low ionic strength . the question is how this mechanism is integrated in the molecular steps governing vesicle docking and fusion in the synapse . in our opinion ,
two alternative scenarios are possible : one mainstream scenario in which synaptotagmin-1 acts downstream of snare nucleation ( which does not preclude an additional earlier role in docking ) , and an alternative scenario according to which snares are not in trans - contact with each other before synaptotagmin-1 receives the ca signal . in the first case , docked and primed synaptic vesicles are characterized by the snares being partially zippered in trans as depicted in virtually all current models . in the second case , ca - synaptotagmin-1 would trigger membrane fusion by rapidly decreasing the distance between the vesicles and the plasma membrane , thus allowing snare nucleation to occur ( as proposed previously ) . firstly , knocking out synaptobrevin-2 or cleaving it with tetanus neurotoxin does not affect vesicle docking , in opposition with a requirement of snare nucleation for docking . secondly , it is difficult to understand how a strained and partially zippered snare complex can be arrested , especially considering that only 13 snare complexes are required for membrane fusion . at least in vitro ,
even the synaptobrevin-24996 fragment ( which binds with nm affinity to the q - snares ) can not block membrane fusion . whereas both candidates for such a clamping role , synaptotagmin-1 and complexin can reduce membrane fusion , they bind with much lower affinity to the q - snare complex than synaptobrevin-24996 . under these conditions ,
unleashed as soon as the membranes are pulled a bit closer by ca - dependent membrane binding of the c2-domains ( fig . while we needed to use a non - physiological trick ( low ionic strength ) to keep the membranes apart
, it is conceivable that this separator role is fulfilled by proteins residing in the space between the vesicle and the plasma membrane . however , further experiments are needed to determine whether under physiological conditions snare nucleation occurs before or after ca - triggering in regulated exocytosis of synaptic vesicles . the lipid composition of the did - labeled q - snare liposomes was identical , except that 1 mol% pc was replaced with the plasma membrane lipid pi(4,5)p2 . for low ionic strength
, the same buffer was used , but now with 5 mm kcl and 300 mm sucrose . protocols have been described for the synaptic vesicles from rat brain , calcein dequenching , complex formation by fret and fluorescence anisotropy . distances were calculated shortly before overstretching of the linker , which was apparent from a strong and continuous increase in the bond energy . | [
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biominerals such as
bones , teeth and seashells provide a wonderful
demonstration of the remarkable control that organic molecules can
achieve over inorganic crystal growth . moreover , biomineralization is of considerable academic
interest since it provides inspiration for the design of new synthetic
organic / inorganic nanocomposite materials with superior toughness
and hardness . in this context ,
exploring the molecular interaction between additives
and inorganic hosts to gain a better understanding of the precise
biomineralization mechanism is extremely important .
inspired
by nature , various artificial biominerals have been prepared by incorporating soluble ( macro)molecules into inorganic crystals .
however , the efficient occlusion of nanoparticles into inorganic crystals is much more difficult to achieve .
for example ,
wooley and co - workers used poly(acrylic acid)-stabilized copolymer
nanoparticles as additives for the in situ crystallization of halite
but found that these nanoparticles were solely located on the crystal
surface .
recently , it has been demonstrated
that nanoparticles decorated with suitable anionic carboxylate , phosphonate
or sulfonate surface groups can be occluded within single crystals
of calcite .
unfortunately , such nanoparticles required either intensive purification
and/or derivatization prior to occlusion experiments . nevertheless ,
these studies are important , partly because incorporating significant
quantities of nanoparticles into single crystals is counter - intuitive ,
but also because this represents a new route for the preparation of
next - generation nanocomposite materials
. however , a robust set of design rules for efficient occlusion remains
elusive .
indeed , progress in this field to date has mainly relied
on empirical trial - and - error experiments .
clearly , the surface
character of the nanoparticles must play a
crucial role in occlusion because this dictates the nature of the
host
however , the influence of surface chemistry
on the extent of occlusion is poorly understood .
this is in part because
precise control over the nanoparticle surface chemistry is somewhat
problematic when using conventional synthetic routes .
fortunately , polymerization - induced self - assembly ( pisa ) mediated
by reversible addition fragmentation chain transfer ( raft )
polymerization is a highly versatile technique that enables facile
synthesis of a wide range of sterically stabilized diblock copolymer
nanoparticles with controllable size , tunable morphology and adjustable
surface functionality . using this approach , a range of non - ionic , anionic , cationic or zwitterionic nanoparticles can be readily prepared in polar solvents
( e.g. , water or ethanol or mixtures thereof ) . in this article
we seek to precisely tune the surface composition
of anionic diblock copolymer nanoparticles to extend our understanding
of the design rules that govern nanoparticle occlusion within a model
host crystal ( calcite ) .
more specifically , we show for the first time
that pisa can be used to prepare copolymer nanoparticles with differing
stabilizer surface densities .
this is achieved by preparing poly(ammonium
2-sulfatoethyl methacrylate)-poly(benzyl methacrylate ) [ psem pbzma ]
nanoparticles using either raft dispersion polymerization in a 2:1
v / v ethanol / water mixture or raft aqueous emulsion polymerization
( see scheme 1 ) .
this
approach offers an unprecedented opportunity to examine the relationship
between the stabilizer surface density and the extent of nanoparticle
occlusion . for the sake of brevity ,
a shorthand notation is utilized :
psem and pbzma denote the two blocks in the main text , while s
and b respectively are used in figures , tables and
captions .
thus , sx by ( emulsion ) denotes a psemx pbzmay diblock
copolymer prepared by raft emulsion polymerization , where x and y indicate the mean degrees of polymerization
( dp ) of each block .
synthesis of poly(ammonium
2-sulfatoethyl methacrylate)-poly(benzyl methacrylate ) [ psem - pbzma ]
diblock copolymer nanoparticles at 10 % w / w solids by chain extension
of a psem macro - cta via either raft dispersion polymerization or raft
aqueous emulsion polymerization of benzyl methacrylate ( bzma ) at 70
c for 24 h. the schematic cartoons indicate subtle differences
in the mean aggregation number and stabilizer surface density when
using these two pisa formulations , as discussed in the main text .
two psem macromolecular
chain transfer agents ( macro - ctas ) with mean dps of either 32 or 73
were prepared via raft aqueous solution polymerization of ammonium
2-sulfatoethyl methacrylate ( sem ) monomer .
gel permeation chromatography
( gpc ) studies confirmed that each macro - cta had a relatively narrow
molecular weight distribution ( mw / mn < 1.15 , see figure s1 , supporting information ) , which in principle enables the preparation
of sterically stabilized nanoparticles with uniform corona thicknesses .
instead , chain extension
experiments conducted using these psem macro - ctas indicated high blocking
efficiencies on addition of a further charge of sem monomer , which
suggests a high degree of raft end - group functionalization ( see figure s1a and s1b ) .
these observations are consistent
with uv visible spectroscopy analysis , which indicated degrees
of raft end - group functionalization of more than 99% , ( see figure s1c and s1d ) . as illustrated in scheme 1 , a series of anionic
psem pbzma diblock copolymer nanoparticles were prepared by
either raft dispersion polymerization in a 2:1 v / v ethanol / water mixture
or raft aqueous emulsion polymerization .
the bzma conversion was monitored
by h nmr spectroscopy , which indicated that high conversions
( > 99% ) were achieved after 24 h at 70 c in all cases .
such
pisa
formulations enable the particle size to be readily controlled by
systematic variation of the target dp of the core - forming block .
thus , in the present study choosing
a psem macro - cta should lead to anionic nanoparticles .
pbzma diblock copolymer
nanoparticles prepared via pisa using either psem32 or
psem73 macro - ctas . in each case , the target dp of the core - forming
pbzma block was fixed at 300
. however , the precise nature of the pisa
formulation determines the final nanoparticle diameter .
pbzma nanoparticles prepared via raft dispersion polymerization
are significantly larger than those obtained by raft aqueous emulsion
polymerization .
this suggests a higher mean aggregation number in
the former case ( see table 1 ) , which is presumably because the repulsive electrostatic
forces operating between neighboring copolymer chains are significantly
weaker in a 2:1 ethanol / water mixture ( r
43 at 298 k ) compared to pure water ( r = 79.5 at
the same temperature ) .
moreover , using
a shorter psem stabilizer block for raft dispersion polymerization
produces larger psem pbzma nanoparticles ( compare figure 1a and 1c ) .
representative
tem images obtained for various anionic diblock
copolymer nanoparticles produced via raft - mediated pisa .
( a ) s32b300 ( dispersion ) ; ( b ) s32b300 ( emulsion ) ; ( c ) s73b300 ( dispersion ) ;
( d ) s73b300 ( emulsion ) .
[ s2p / c1s ( particle)]/[s2p / c1s ( stabilizer ) ]
( % ) .
further details of
the calculation
of this parameter can be found in the supporting information ( see page s10 ) .
stabilizer surface density was calculated
using eq 1 , which is
derived in the supporting information .
there are two types of stabilizer
in this case ; the calculated value refers only to the psem73 chains to aid comparison .
figure 2 shows dynamic
light scattering ( dls ) and aqueous electrophoresis data for various
diblock copolymer nanoparticles as a function of either ph or ca concentration . according to figure 2a ,
the hydrodynamic diameter of the nanoparticles
is independent of the solution ph , which is indicative of good colloidal
stability .
such behavior differs qualitatively from that of poly(methacrylic
acid)-stabilized diblock copolymer nanoparticles , since in the latter
case the weak polyelectrolyte stabilizer chains become protonated
at low ph , resulting in aggregation.figure 2b shows that psemx pbzmay diblock
copolymer nanoparticles exhibit highly anionic ph - independent zeta
potentials , as expected for a strong polyelectrolyte stabilizer .
this ph - independent character
enables aggregation to be avoided during attempted occlusion , even
if crystallization involves some variation in solution ph .
pbzmay nanoparticles prepared by raft dispersion
polymerization and raft emulsion polymerization , the former exhibit
more negative zeta potentials than the latter .
the physical reason
for this difference in electrophoretic behavior is examined later .
although the zeta potential for the psemx pbzmay diblock copolymer nanoparticles
is ph - independent , this parameter is affected by the addition of ca ions , even at a relatively low concentration ( 0.1 mm ) as
shown in figure 2c .
indeed , all nanoparticles exhibit a significant reduction in zeta
potential in the presence of ca , although an overall
zeta potential of around 15 mv is maintained at 1.5 mm ca or higher .
in addition , the intensity - average diameters
recorded for s73b300 ( emulsion ) and
s73b300 ( dispersion ) nanoparticles in
the presence of 1.5 mm ca ions are slightly smaller than
those determined in the absence of ca ions ( see figure s2 ) .
this is most likely due to charge
screening caused by the presence of salt , although it is worth noting
that the divalent ca cations do also bind strongly to
the anionic sulfate groups on the psem stabilizer chains .
importantly ,
the presence of 1.5 mm ca ions does not cause any aggregation
or precipitation ( see figure s2 ) .
such
psem stabilizer - ca ion interactions are likely to be
important for occlusion during in situ crystallization , because they
should promote nanoparticle adsorption onto the crystal surface .
dynamic light scattering and aqueous electrophoresis
data obtained
for spherical diblock copolymer nanoparticles conducted at a copolymer
concentration of 0.1% w / w : ( a ) hydrodynamic diameter vs ph ;
( b ) zeta potential vs ph in the presence of 1 mm nacl as background
electrolyte ; and ( c ) zeta potential vs ca concentration ,
conducted at a copolymer nanoparticle concentration of 0.01% w / w ( which
corresponds to the occlusion conditions ) .
the inset in ( c ) shows the
zeta potentials observed for nanoparticles at ca concentrations
ranging from 0 to 0.15 mm .
calcium carbonate crystals
were precipitated at ph 89 by exposing an aqueous solution
containing [ ca ] = 1.5 mm and 0.00.10% w / w anionic
diblock copolymer nanoparticles to ammonium carbonate vapor at 20
c for 24 h. a mixture of calcite
and vaterite was precipitated at a copolymer concentration of 0.10%
w / w ( figure s3 ) , which is consistent with
previous reports .
thus ,
a lower copolymer concentration of 0.01% w / w was selected for more
detailed studies .
precipitation under the above conditions yielded
3050 m rhombohedral calcite crystals in either the
absence or presence of psem pbzma diblock copolymer nanoparticles
or psem73 homopolymer , see figure s4 .
characteristic raman bands were detected at 154 and 280 cm ( lattice modes ) , 712 cm ( 4 ) and 1086 cm ( 1 ) for
both calcite control and copolymer / calcite nanocomposite ( figure s5 ) .
particle
occlusion within calcite was investigated by imaging fractured crystals
with field emission electron scanning microscopy ( fe - sem , figure 3 ) .
it is worth noting
that no occlusion was observed for poly(glycerol monomethacrylate)70-poly(benzyl methacrylate)300 ( pgma70pbzma300 ) copolymer nanoparticles , as expected
( see scheme s1a and figure s6 ) .
this is
because the poly(glycerol monomethacrylate ) ( g70 ) stabilizer
is non - ionic , hence there is no favorable electrostatic interaction
with the growing crystal .
however , some degree of occlusion did occur
when 50% of the non - ionic pgma70 stabilizer chains were
replaced with anionic psem73 stabilizer for the synthesis
of [ 0.5 psem73 + 0.5 pgma70]b300 ( emulsion ) nanoparticles , see scheme s1b and figure s7 .
random occlusion was observed for psem32pbzma300 and psem73pbzma300 nanoparticles prepared via raft dispersion polymerization ,
but such incorporation appears to be rather inhomogeneous ( see figure 3a and 3b , and corresponding low magnification images in figure s8 ) . in contrast , the psem32pbzma300 and psem73pbzma300 nanoparticles obtained using raft aqueous emulsion polymerization
are much more uniformly incorporated within calcite ( see figure 3c g ) .
representative
fe - sem images obtained for fractured calcium carbonate
crystals prepared in the presence of ( a ) s32b300 ( dispersion ) and ( b ) s73b300 ( dispersion ) nanoparticles at a fixed copolymer concentration of
0.01% w / w . the insets in ( a ) and ( b ) are magnified images corresponding
to the labeled rectangular areas .
in both cases only relatively low
levels of occlusion are observed ( further corresponding low magnification
fe - sem images are provided in figure s8 ) .
fe - sem images for ( c ) s32b300 ( emulsion )
and ( d ) s73b300 ( emulsion ) show fractured
crystals at low magnification .
the inset in ( d ) shows an optical micrograph
obtained for intact rhombohedral calcite crystals prior to fracture .
( e ) and ( f ) depict magnified images of selected areas as indicated
in ( d ) , confirming that such nanoparticles are efficiently and uniformly
occluded within calcium carbonate crystals .
( g ) magnified image showing
the rectangular area indicated in ( f ) .
the inset in ( g ) is a tem image
obtained for the s73b300 ( emulsion )
nanoparticles prior to their occlusion .
clearly , the dimensions of
the occluded features observed in ( g ) are consistent with the diameter
of the original nanoparticles .
the calcite crystals precipitated in the presence of 0.01%
w / w
psem73pbzma300 ( emulsion ) were examined
in more detail .
the internal structure of a fractured crystal is shown
in figure 3d g :
these images demonstrate that the nanoparticles are both non - aggregated
and uniformly occluded throughout the crystal .
it is also noteworthy
that all nanoparticle cavities are spherical and comparable in size
to the original psem73pbzma300 ( emulsion )
nanoparticles ( figure 3 g ) .
kim et al . previously reported that relatively soft anionic diblock
copolymer spherical micelles deform and flatten during their occlusion
into calcite .
this phenomenon was recently
studied in detail by cho et al . , who used atomic force microscopy
( afm ) and micromechanical simulations to rationalize the in situ change
in copolymer morphology
presumably ,
the relatively high glass transition temperature of the pbzma core - forming
block prevents deformation of the psem73pbzma300 ( emulsion ) nanoparticles examined in the present work .
the extent of occlusion was quantified using thermogravimetric
analysis ( tga , figure 4 ) which showed that the psem73pbzma300 ( emulsion ) diblock copolymer nanoparticles were completely pyrolyzed
on heating in air up to 550 c .
pure calcite decomposed to give
a cao residue of 56.4% by mass , which is close to its theoretical
cao content of 56.0% .
based on these data , it is calculated that the
occlusion is almost negligible for psem73pbzma300 ( dispersion ) nanoparticles .
for the [ 0.5 psem73 + 0.5 pgma70]pbzma300 nanoparticles ,
the extent of occlusion is 2% w / w .
the tga curve obtained
for the psem73pbzma300 ( emulsion)/calcite
nanocomposite crystals exhibited three distinct features .
first , a
3.7% mass loss was observed up to 600 c which is assigned to
decomposition of psem73pbzma300 ( emulsion )
nanoparticles located in the outer regions of the host crystal .
second ,
the tga curve was shifted to a higher temperature , suggesting enhanced
thermal stability for the nanocomposite crystal . finally , the 52.2%
residue obtained at 850 c is significantly lower than that observed
for pure calcite ( 56.4% ) , which indicates successful occlusion .
the
degree of occlusion calculated for psem73pbzma300 nanoparticles is 7.5% w / w , or approximately 16% v / v ( assuming
a copolymer density of 1.18 g cm , as determined
by helium pycnometry ; see supporting information for the detailed calculation ) .
the occlusion of psem73pbzma300 ( emulsion ) diblock copolymer nanoparticles
within calcite was also confirmed by ft - ir spectroscopy , since the
ester carbonyl band assigned to psem73pbzma300 was detected as a weak feature in the ft - ir spectrum recorded
for the psem73pbzma300 ( emulsion)/calcite
nanocomposite crystals ( see figure s9 ) .
thermogravimetric
analysis ( tga ) data obtained for s73b300/calcite nanocomposite crystals and corresponding
control samples .
( a ) calcite control , showing the expected approximate
44% mass loss as a result of cao formation via loss of co2 .
( b ) s73b300 ( dispersion)/calcite
nanocomposite crystals exhibit a slightly greater weight loss compared
to the calcite control ; this indicates a relatively low level of copolymer
nanoparticle occlusion and is consistent with fe - sem studies ( see figure 3b ) .
( d ) s73b300 ( emulsion)/calcite
nanocomposite crystals exhibit a significantly greater weight loss .
( e ) original s73b300 diblock copolymer
nanoparticles , indicating complete pyrolysis of this purely organic
component . on
the basis of studies to date ,
anionic nanoparticles comprising carboxylate ,
phosphonate or sulfonate surface functional groups can be occluded
within inorganic crystals such as calcite or zno .
hence progress in
this field has been hitherto mainly based on empirical trial - and - error
experiments .
thus , elucidation of a robust set of design rules is
highly desirable , both for optimizing the occlusion efficiency and
also for extending occlusion to include alternative inorganic host
crystals .
these two sets of nanoparticles possess apparently
the same surface chemistry but exhibit substantial differences with
regard to their extents of occlusion within calcite .
initially , this
discrepancy was considered to be possibly a particle size effect .
however , control experiments ruled out this hypothesis . more specifically ,
a series of psemx pbzmay ( dispersion ) nanoparticles ranging in size from
18 to 100 nm diameter were prepared by varying the pbzma dp at 10%
w / w solids and evaluated for occlusion into calcite under the same
conditions . remarkably , all psemx pbzmay nanoparticles prepared by raft dispersion
polymerization are either not occluded or only weakly occluded within
calcite ( see figure s10 ) , whereas all psemx pbzmay nanoparticles prepared by raft emulsion polymerization can be uniformly
incorporated into calcite regardless of their particle size ( see figure 3c g and figure s11 ) .
thus , it seems that occlusion is
not sensitive to the nanoparticle dimensions , at least over the 18
to 100 nm size range studied here .
in fact , kim and co - workers reported
that anionic carboxylated latexes of approximately 220250
nm diameter could be occluded within calcite . moreover , occlusion does not appear to be particularly sensitive
to the stabilizer dp because both psem32pbzmay and psem73pbzmay nanoparticles prepared via raft aqueous emulsion
polymerization can be uniformly occluded within calcite , see figure 3c , d , and figure s11 . as discussed above ,
the zeta
potential for nanoparticles prepared
by raft dispersion polymerization is significantly more negative than
that for nanoparticles prepared via raft aqueous emulsion polymerization
( see figure 2b ) .
this
observation led us to determine the stabilizer surface densities for
these two types of nanoparticles , because we hypothesized that this
parameter might explain their differing occlusion behavior . assuming perfect blocking efficiency , full bzma conversion and
a relatively narrow particle size distribution , the stabilizer surface
density can be calculated using eq 1 ( see supporting information for a derivation):1here ds is the
stabilizer surface density expressed as the number of chains per nm and npsem , na , and r denote the number of
moles of psem macro - cta ( mol ) , avogadro s number ( mol ) , the solid - state density of the dried nanoparticles ( g cm ) and the mean nanoparticle radius ( nm ) , respectively .
the masses
( g ) of the psem macro - cta and pbzma are given by ms and mb , respectively .
using eq 1 , the stabilizer
surface densities of the diblock copolymer nanoparticles were determined
from the nanoparticle dimensions and these data are summarized in table 1 . in all cases ,
psemx pbzmay nanoparticles prepared via raft dispersion polymerization have significantly
higher surface stabilizer densities than the corresponding nanoparticles
prepared via raft emulsion polymerization .
direct experimental evidence
of this finding was also provided by x - ray photoelectron spectroscopy
( xps ) , which is an established analytical technique for determining
surface chemical compositions .
xps has excellent inter - element resolution
and is highly surface - specific , with a typical sampling depth of 210
nm .
for the current study , the psem stabilizer
chains provide a unique source of sulfur atoms .
hence higher sulfur
contents indicate higher stabilizer surface densities ( see figure 5 ) .
both the psem
stabilizer and the pbzma core - forming block contain carbon atoms ,
which give rise to c1s signals in xps .
all samples were run under
the same conditions and the s2p / c1s atomic ratios calculated from
xps analysis of the sx homopolymers are
very close to theoretical values ( see table 1 ) .
thus , the level of surface carbon contamination
is low ( and assumed to be negligible ) .
normalized atomic ratios [ i.e. , s2p / c1s ( particle)]/[s2p / c1s ( stabilizer ) ]
were calculated to compare stabilizer surface densities for the nanoparticles ,
as summarized in table 1 .
as expected , no sulfur signals were detected for the pbzma300 control and the psem73 control had the highest
sulfur content ( strongest s2p signal ) .
more importantly , psemx pbzmay nanoparticles prepared via raft dispersion polymerization exhibited
consistently higher normalized [ s2p / c1s ( particle)]/[s2p / c1s ( stabilizer ) ]
atomic ratios ( by a factor of approximately two ) compared to the equivalent
nanoparticles prepared via raft emulsion polymerization .
this indicates
that the former nanoparticles have a higher stabilizer surface density
than the latter .
this difference is consistent with the theoretical
stabilizer surface densities calculated using eq 1 and can be ascribed to two reasons .
first ,
the relatively low dielectric constant for the 2:1 v / v ethanol / water
mixture ( compared to pure water ) reduces electrostatic repulsion between
neighboring highly anionic psem stabilizers during nanoparticle formation
via pisa , which results in a more densely packed coronal layer .
second ,
the pbzma chains are likely to be slightly more solvated ( and hence
more stretched , leading to more closely packed copolymer chains in
the nanoparticle cores ) when grown in the 2:1 v / v ethanol / water mixture ,
compared to pisa syntheses conducted in pure water .
one reviewer of this manuscript suggested that the higher
stabilizer surface density observed for psem73pbzma300 ( dispersion ) might be the result of a significant change
in core solvation during dialysis againt water .
however , both literature
data and dls studies conducted before
and after dialysis do not support this hypothesis , because only a
rather small change in the intensity - average particle diameter is
observed ( see figure s12 ) .
x - ray photoelectron survey
spectra recorded for a s73 homopolymer control , s73b300 ( dispersion ) ,
s73b300 ( emulsion ) , and a b300 homopolymer control .
the red rectangle highlights the relative s2p
intensities , which are in the order : b300 homopolymer <
s73b300 ( emulsion ) < s73b300 ( dispersion ) < s73 homopolymer .
naively , a higher stabilizer surface
density should provide stronger
nanoparticle binding to the crystal surface and hence lead to higher
levels of occlusion .
however , less occlusion is actually
observed , which at first sight appears to be counterintuitive .
the
present study shows that efficient occlusion actually
requires an optimum ( rather than maximum ) stabilizer
surface density .
prior
to precipitation of calcium carbonate , ca ions bind to
the anionic stabilizer chains of psemx pbzmay nanoparticles in aqueous
solution , as indicated by their significantly less anionic zeta potential
compared to nanoparticles in the absence of ca ( see figure 2c ) . under otherwise
identical conditions ( i.e. , 1.5 mm ca and 0.01% w / w copolymer
at 20 c )
, the extent of ca ion binding to the anionic
sulfate groups on the psem73pbzma300 ( emulsion ) and psem73pbzma300 ( dispersion )
nanoparticles is estimated to be 5.9 1.2 mg g and 6.1 1.4 mg g respectively , as determined
using a calcium ion - selective electrode ( see figure s13 ) .
if the stabilizer chains
are closely packed , the relatively high stabilizer surface density
means that ca ions primarily act as ionic cross - linkers
between adjacent psem chains .
thus , the ensuing loss
of conformational entropy reduces the ability of these sterically
stabilized nanoparticles to interact with the growing crystal , see figure 6a .
however , such
ionic cross - linking is much less likely to occur if the stabilizer
surface density is relatively low , which allows the nanoparticles
prepared via raft emulsion polymerization to retain more conformational
entropy .
hence a greater proportion of the stabilizer chains are able
to relax , which enables these nanoparticles to bind more strongly
on the growing calcite surface ( see figure 6b ) .
this interpretation is consistent with
recent in situ afm studies , which demonstrate that anionic block copolymer
micelles uniquely bind at calcite step edges prior to their incorporation .
a further reduction in the psem stabilizer surface
density can be achieved by introducing non - ionic pgma stabilizer chains .
such [ 0.5 psem73 + 0.5 pgma70]b300 ( emulsion ) nanoparticles exhibit an extent of occlusion
of just 2% w / w .
one possible explanation is that the presence of the
non - ionic pgma stabilizer chains restricts the ability of the anionic
psem stabilizer chains to interact efficiently with the growing calcite
crystals .
alternatively , the ( diluted ) psem surface density may be
simply too low to ensure efficient interaction between such copolymer
nanoparticles and the growing calcite crystals .
( a ) crystallization in the presence of sx by ( dispersion ) nanoparticles .
the relatively high stabilizer surface density means that ca ions primarily act as ionic cross - linkers between adjacent stabilizer
chains and the ensuing loss of conformational entropy reduces nanoparticle
interactions with the growing crystal .
( b ) crystallization in the
presence of sx by ( emulsion ) nanoparticles . in this case
the relatively low
stabilizer density reduces the degree of ionic cross - linking between
stabilizer chains and enables the nanoparticles to interact more strongly
with the growing crystals , hence promoting efficient occlusion . to examine the effect of varying
stabilizer surface density for
other inorganic crystal hosts
, we also performed some preliminary
occlusion experiments using zno instead of calcite . psem73pbzma300
nanoparticles prepared by raft emulsion
polymerization exhibited a significantly higher extent of occlusion
( 13.9% w / w vs 7.2% w / w , see figure s14 )
compared to psem73pbzma100 ( dispersion )
nanoparticles of comparable size .
this is in good agreement with the
calcite system and further suggests that an optimum stabilizer surface density is required to maximize the extent of occlusion .
in
summary , a range of well - defined highly anionic diblock copolymer
nanoparticles with tunable surface chemistry have been prepared via
raft - mediated pisa .
the stabilizer surface density depends on the
solvent quality for the stabilizer chains , which offers an unprecedented
opportunity to examine the relationship between nanoparticle surface
functionality and the corresponding extent of occlusion within calcite
crystals .
up to 7.5% w / w ( or 16% v / v ) psem73pbzma300 ( emulsion ) nanoparticles can be incorporated within calcite
( caco3 ) even when using a relatively low copolymer concentration
( 0.01% w / w ) .
surprisingly , more anionic nanoparticles prepared via
raft dispersion polymerization using a 2:1 v / v ethanol / water mixture
are occluded much less efficiently into calcite under identical crystallization
conditions .
this suggests that there is an optimum ( rather than a maximum ) surface density of anionic stabilizer chains
for occlusion into this host crystal .
thus , this work provides important
new insights regarding the rather subtle role played by the nanoparticle
surface chemistry in determining occlusion efficiencies within calcite .
moreover , our findings are expected to inform the design of new organic / inorganic
nanocomposite crystals . | polymerization - induced
self - assembly ( pisa ) offers a highly versatile
and efficient route to a wide range of organic nanoparticles . in this
article
, we demonstrate for the first time that poly(ammonium 2-sulfatoethyl
methacrylate)-poly(benzyl methacrylate ) [ psem pbzma ] diblock
copolymer nanoparticles can be prepared with either a high or low
psem stabilizer surface density using either raft dispersion polymerization
in a 2:1 v / v ethanol / water mixture or raft aqueous emulsion polymerization ,
respectively .
we then use these model nanoparticles to gain new insight
into a key topic in materials chemistry : the occlusion of organic
additives into inorganic crystals .
substantial differences are observed
for the extent of occlusion of these two types of anionic nanoparticles
into calcite ( caco3 ) , which serves as a suitable model
host crystal .
a low psem stabilizer surface density leads to uniform
nanoparticle occlusion within calcite at up to 7.5% w / w ( 16% v / v ) ,
while minimal occlusion occurs when using nanoparticles with a high
psem stabilizer surface density .
this counter - intuitive observation
suggests that an optimum anionic surface density is required for efficient
occlusion , which provides a hitherto unexpected design rule for the
incorporation of nanoparticles within crystals . | Introduction
Results and Discussion
Conclusions | inspired
by nature , various artificial biominerals have been prepared by incorporating soluble ( macro)molecules into inorganic crystals . however , the efficient occlusion of nanoparticles into inorganic crystals is much more difficult to achieve . nevertheless ,
these studies are important , partly because incorporating significant
quantities of nanoparticles into single crystals is counter - intuitive ,
but also because this represents a new route for the preparation of
next - generation nanocomposite materials
. however , a robust set of design rules for efficient occlusion remains
elusive . clearly , the surface
character of the nanoparticles must play a
crucial role in occlusion because this dictates the nature of the
host
however , the influence of surface chemistry
on the extent of occlusion is poorly understood . fortunately , polymerization - induced self - assembly ( pisa ) mediated
by reversible addition fragmentation chain transfer ( raft )
polymerization is a highly versatile technique that enables facile
synthesis of a wide range of sterically stabilized diblock copolymer
nanoparticles with controllable size , tunable morphology and adjustable
surface functionality . using this approach , a range of non - ionic , anionic , cationic or zwitterionic nanoparticles can be readily prepared in polar solvents
( e.g. in this article
we seek to precisely tune the surface composition
of anionic diblock copolymer nanoparticles to extend our understanding
of the design rules that govern nanoparticle occlusion within a model
host crystal ( calcite ) . more specifically , we show for the first time
that pisa can be used to prepare copolymer nanoparticles with differing
stabilizer surface densities . this is achieved by preparing poly(ammonium
2-sulfatoethyl methacrylate)-poly(benzyl methacrylate ) [ psem pbzma ]
nanoparticles using either raft dispersion polymerization in a 2:1
v / v ethanol / water mixture or raft aqueous emulsion polymerization
( see scheme 1 ) . this
approach offers an unprecedented opportunity to examine the relationship
between the stabilizer surface density and the extent of nanoparticle
occlusion . thus , sx by ( emulsion ) denotes a psemx pbzmay diblock
copolymer prepared by raft emulsion polymerization , where x and y indicate the mean degrees of polymerization
( dp ) of each block . synthesis of poly(ammonium
2-sulfatoethyl methacrylate)-poly(benzyl methacrylate ) [ psem - pbzma ]
diblock copolymer nanoparticles at 10 % w / w solids by chain extension
of a psem macro - cta via either raft dispersion polymerization or raft
aqueous emulsion polymerization of benzyl methacrylate ( bzma ) at 70
c for 24 h. the schematic cartoons indicate subtle differences
in the mean aggregation number and stabilizer surface density when
using these two pisa formulations , as discussed in the main text . gel permeation chromatography
( gpc ) studies confirmed that each macro - cta had a relatively narrow
molecular weight distribution ( mw / mn < 1.15 , see figure s1 , supporting information ) , which in principle enables the preparation
of sterically stabilized nanoparticles with uniform corona thicknesses . as illustrated in scheme 1 , a series of anionic
psem pbzma diblock copolymer nanoparticles were prepared by
either raft dispersion polymerization in a 2:1 v / v ethanol / water mixture
or raft aqueous emulsion polymerization . pbzma diblock copolymer
nanoparticles prepared via pisa using either psem32 or
psem73 macro - ctas . pbzma nanoparticles prepared via raft dispersion polymerization
are significantly larger than those obtained by raft aqueous emulsion
polymerization . this suggests a higher mean aggregation number in
the former case ( see table 1 ) , which is presumably because the repulsive electrostatic
forces operating between neighboring copolymer chains are significantly
weaker in a 2:1 ethanol / water mixture ( r
43 at 298 k ) compared to pure water ( r = 79.5 at
the same temperature ) . moreover , using
a shorter psem stabilizer block for raft dispersion polymerization
produces larger psem pbzma nanoparticles ( compare figure 1a and 1c ) . stabilizer surface density was calculated
using eq 1 , which is
derived in the supporting information . there are two types of stabilizer
in this case ; the calculated value refers only to the psem73 chains to aid comparison . figure 2 shows dynamic
light scattering ( dls ) and aqueous electrophoresis data for various
diblock copolymer nanoparticles as a function of either ph or ca concentration . such behavior differs qualitatively from that of poly(methacrylic
acid)-stabilized diblock copolymer nanoparticles , since in the latter
case the weak polyelectrolyte stabilizer chains become protonated
at low ph , resulting in aggregation.figure 2b shows that psemx pbzmay diblock
copolymer nanoparticles exhibit highly anionic ph - independent zeta
potentials , as expected for a strong polyelectrolyte stabilizer . pbzmay nanoparticles prepared by raft dispersion
polymerization and raft emulsion polymerization , the former exhibit
more negative zeta potentials than the latter . although the zeta potential for the psemx pbzmay diblock copolymer nanoparticles
is ph - independent , this parameter is affected by the addition of ca ions , even at a relatively low concentration ( 0.1 mm ) as
shown in figure 2c . dynamic light scattering and aqueous electrophoresis
data obtained
for spherical diblock copolymer nanoparticles conducted at a copolymer
concentration of 0.1% w / w : ( a ) hydrodynamic diameter vs ph ;
( b ) zeta potential vs ph in the presence of 1 mm nacl as background
electrolyte ; and ( c ) zeta potential vs ca concentration ,
conducted at a copolymer nanoparticle concentration of 0.01% w / w ( which
corresponds to the occlusion conditions ) . calcium carbonate crystals
were precipitated at ph 89 by exposing an aqueous solution
containing [ ca ] = 1.5 mm and 0.00.10% w / w anionic
diblock copolymer nanoparticles to ammonium carbonate vapor at 20
c for 24 h. a mixture of calcite
and vaterite was precipitated at a copolymer concentration of 0.10%
w / w ( figure s3 ) , which is consistent with
previous reports . precipitation under the above conditions yielded
3050 m rhombohedral calcite crystals in either the
absence or presence of psem pbzma diblock copolymer nanoparticles
or psem73 homopolymer , see figure s4 . it is worth noting
that no occlusion was observed for poly(glycerol monomethacrylate)70-poly(benzyl methacrylate)300 ( pgma70pbzma300 ) copolymer nanoparticles , as expected
( see scheme s1a and figure s6 ) . random occlusion was observed for psem32pbzma300 and psem73pbzma300 nanoparticles prepared via raft dispersion polymerization ,
but such incorporation appears to be rather inhomogeneous ( see figure 3a and 3b , and corresponding low magnification images in figure s8 ) . in contrast , the psem32pbzma300 and psem73pbzma300 nanoparticles obtained using raft aqueous emulsion polymerization
are much more uniformly incorporated within calcite ( see figure 3c g ) . previously reported that relatively soft anionic diblock
copolymer spherical micelles deform and flatten during their occlusion
into calcite . the extent of occlusion was quantified using thermogravimetric
analysis ( tga , figure 4 ) which showed that the psem73pbzma300 ( emulsion ) diblock copolymer nanoparticles were completely pyrolyzed
on heating in air up to 550 c . for the [ 0.5 psem73 + 0.5 pgma70]pbzma300 nanoparticles ,
the extent of occlusion is 2% w / w . finally , the 52.2%
residue obtained at 850 c is significantly lower than that observed
for pure calcite ( 56.4% ) , which indicates successful occlusion . the
degree of occlusion calculated for psem73pbzma300 nanoparticles is 7.5% w / w , or approximately 16% v / v ( assuming
a copolymer density of 1.18 g cm , as determined
by helium pycnometry ; see supporting information for the detailed calculation ) . the occlusion of psem73pbzma300 ( emulsion ) diblock copolymer nanoparticles
within calcite was also confirmed by ft - ir spectroscopy , since the
ester carbonyl band assigned to psem73pbzma300 was detected as a weak feature in the ft - ir spectrum recorded
for the psem73pbzma300 ( emulsion)/calcite
nanocomposite crystals ( see figure s9 ) . on
the basis of studies to date ,
anionic nanoparticles comprising carboxylate ,
phosphonate or sulfonate surface functional groups can be occluded
within inorganic crystals such as calcite or zno . these two sets of nanoparticles possess apparently
the same surface chemistry but exhibit substantial differences with
regard to their extents of occlusion within calcite . more specifically ,
a series of psemx pbzmay ( dispersion ) nanoparticles ranging in size from
18 to 100 nm diameter were prepared by varying the pbzma dp at 10%
w / w solids and evaluated for occlusion into calcite under the same
conditions . remarkably , all psemx pbzmay nanoparticles prepared by raft dispersion
polymerization are either not occluded or only weakly occluded within
calcite ( see figure s10 ) , whereas all psemx pbzmay nanoparticles prepared by raft emulsion polymerization can be uniformly
incorporated into calcite regardless of their particle size ( see figure 3c g and figure s11 ) . moreover , occlusion does not appear to be particularly sensitive
to the stabilizer dp because both psem32pbzmay and psem73pbzmay nanoparticles prepared via raft aqueous emulsion
polymerization can be uniformly occluded within calcite , see figure 3c , d , and figure s11 . as discussed above ,
the zeta
potential for nanoparticles prepared
by raft dispersion polymerization is significantly more negative than
that for nanoparticles prepared via raft aqueous emulsion polymerization
( see figure 2b ) . this
observation led us to determine the stabilizer surface densities for
these two types of nanoparticles , because we hypothesized that this
parameter might explain their differing occlusion behavior . assuming perfect blocking efficiency , full bzma conversion and
a relatively narrow particle size distribution , the stabilizer surface
density can be calculated using eq 1 ( see supporting information for a derivation):1here ds is the
stabilizer surface density expressed as the number of chains per nm and npsem , na , and r denote the number of
moles of psem macro - cta ( mol ) , avogadro s number ( mol ) , the solid - state density of the dried nanoparticles ( g cm ) and the mean nanoparticle radius ( nm ) , respectively . using eq 1 , the stabilizer
surface densities of the diblock copolymer nanoparticles were determined
from the nanoparticle dimensions and these data are summarized in table 1 . in all cases ,
psemx pbzmay nanoparticles prepared via raft dispersion polymerization have significantly
higher surface stabilizer densities than the corresponding nanoparticles
prepared via raft emulsion polymerization . direct experimental evidence
of this finding was also provided by x - ray photoelectron spectroscopy
( xps ) , which is an established analytical technique for determining
surface chemical compositions . for the current study , the psem stabilizer
chains provide a unique source of sulfur atoms . both the psem
stabilizer and the pbzma core - forming block contain carbon atoms ,
which give rise to c1s signals in xps . , s2p / c1s ( particle)]/[s2p / c1s ( stabilizer ) ]
were calculated to compare stabilizer surface densities for the nanoparticles ,
as summarized in table 1 . more importantly , psemx pbzmay nanoparticles prepared via raft dispersion polymerization exhibited
consistently higher normalized [ s2p / c1s ( particle)]/[s2p / c1s ( stabilizer ) ]
atomic ratios ( by a factor of approximately two ) compared to the equivalent
nanoparticles prepared via raft emulsion polymerization . this indicates
that the former nanoparticles have a higher stabilizer surface density
than the latter . this difference is consistent with the theoretical
stabilizer surface densities calculated using eq 1 and can be ascribed to two reasons . first ,
the relatively low dielectric constant for the 2:1 v / v ethanol / water
mixture ( compared to pure water ) reduces electrostatic repulsion between
neighboring highly anionic psem stabilizers during nanoparticle formation
via pisa , which results in a more densely packed coronal layer . second ,
the pbzma chains are likely to be slightly more solvated ( and hence
more stretched , leading to more closely packed copolymer chains in
the nanoparticle cores ) when grown in the 2:1 v / v ethanol / water mixture ,
compared to pisa syntheses conducted in pure water . one reviewer of this manuscript suggested that the higher
stabilizer surface density observed for psem73pbzma300 ( dispersion ) might be the result of a significant change
in core solvation during dialysis againt water . naively , a higher stabilizer surface
density should provide stronger
nanoparticle binding to the crystal surface and hence lead to higher
levels of occlusion . the
present study shows that efficient occlusion actually
requires an optimum ( rather than maximum ) stabilizer
surface density . , 1.5 mm ca and 0.01% w / w copolymer
at 20 c )
, the extent of ca ion binding to the anionic
sulfate groups on the psem73pbzma300 ( emulsion ) and psem73pbzma300 ( dispersion )
nanoparticles is estimated to be 5.9 1.2 mg g and 6.1 1.4 mg g respectively , as determined
using a calcium ion - selective electrode ( see figure s13 ) . if the stabilizer chains
are closely packed , the relatively high stabilizer surface density
means that ca ions primarily act as ionic cross - linkers
between adjacent psem chains . thus , the ensuing loss
of conformational entropy reduces the ability of these sterically
stabilized nanoparticles to interact with the growing crystal , see figure 6a . however , such
ionic cross - linking is much less likely to occur if the stabilizer
surface density is relatively low , which allows the nanoparticles
prepared via raft emulsion polymerization to retain more conformational
entropy . a further reduction in the psem stabilizer surface
density can be achieved by introducing non - ionic pgma stabilizer chains . such [ 0.5 psem73 + 0.5 pgma70]b300 ( emulsion ) nanoparticles exhibit an extent of occlusion
of just 2% w / w . alternatively , the ( diluted ) psem surface density may be
simply too low to ensure efficient interaction between such copolymer
nanoparticles and the growing calcite crystals . in this case
the relatively low
stabilizer density reduces the degree of ionic cross - linking between
stabilizer chains and enables the nanoparticles to interact more strongly
with the growing crystals , hence promoting efficient occlusion . to examine the effect of varying
stabilizer surface density for
other inorganic crystal hosts
, we also performed some preliminary
occlusion experiments using zno instead of calcite . psem73pbzma300
nanoparticles prepared by raft emulsion
polymerization exhibited a significantly higher extent of occlusion
( 13.9% w / w vs 7.2% w / w , see figure s14 )
compared to psem73pbzma100 ( dispersion )
nanoparticles of comparable size . this is in good agreement with the
calcite system and further suggests that an optimum stabilizer surface density is required to maximize the extent of occlusion . in
summary , a range of well - defined highly anionic diblock copolymer
nanoparticles with tunable surface chemistry have been prepared via
raft - mediated pisa . the stabilizer surface density depends on the
solvent quality for the stabilizer chains , which offers an unprecedented
opportunity to examine the relationship between nanoparticle surface
functionality and the corresponding extent of occlusion within calcite
crystals . up to 7.5% w / w ( or 16% v / v ) psem73pbzma300 ( emulsion ) nanoparticles can be incorporated within calcite
( caco3 ) even when using a relatively low copolymer concentration
( 0.01% w / w ) . surprisingly , more anionic nanoparticles prepared via
raft dispersion polymerization using a 2:1 v / v ethanol / water mixture
are occluded much less efficiently into calcite under identical crystallization
conditions . this suggests that there is an optimum ( rather than a maximum ) surface density of anionic stabilizer chains
for occlusion into this host crystal . | [
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hyperpolarization - activated , cyclic nucleotide - gated nonselective ( hcn ) channels show a considerable conductance at the resting membrane potential and mediate a slowly inactivating and inwardly rectifying na current [ 1 , 2 ] .
hence , these channels appear to be ideal modulators of both the membrane potential and the membrane resistance of neurons and thus may play a significant role in synaptic transmission . in recent years , it has become evident that hcn channels are also relevant modulators of synaptic plasticity [ 35 ] .
hcn channels may have differential effects on different synapses and/or various types of synaptic plasticity , depending on , for example , the synapse type , pre- or postsynaptic expression patterns , and ontogenetic stage .
thus , while long - term potentiation ( ltp ) following electrical stimulation to the schaffer collateral input of ca1 pyramidal cells was shown to be unaltered in hcn1-deficient adult mice , a significant enhancement of ltp was observed when the perforant path input to ca1 was stimulated .
such a constraining role of hcn channels on synaptic plasticity in ca1 was also found with long - term depression ( ltd ) following pharmacological activation of group i metabotropic glutamate receptors .
however , ltd following electrical stimulation at the same synapse was also assessed in the same study and failed to be affected by hcn channel inhibition . since the expression pattern of hcn channels changes during development
presynaptic hcn channels are expressed on glutamatergic axon terminals of the medial perforant path ( mpp ) input to dentate gyrus granule cells only during the first 2 - 3 weeks of postnatal development and subsequently are lost during maturation comparing plasticity in immature and mature tissue allows to dissect the impact of pre- and postsynaptic hcn channel expression . in immature tissue
, hcn channel - mediated effects will be predominantly presynaptically driven , in adult tissue , postsynaptically .
interestingly , short - term depression ( std ) at the immature synapse was enhanced following pharmacological hcn channel inhibition , as a first indication of a constraining role of hcn channels on synaptic plasticity . in the present study , we assessed nmda receptor - dependent low - frequency stimulation- ( lfs- ) induced ltd at the mpp - granule cell synapse at different developmental stages and found an enhancement of ltd following hcn channel inhibition in tissue from early postnatal , but not in tissue from adult animals .
these findings emphasize the modulating role of hcn channels on synaptic plasticity and provide evidence for an important role of presynaptic hcn channels in activity - dependent changes of synaptic transmission .
they also suggest that nitric oxide may play a role as a retrograde messenger that links postsynaptic nmda - receptor activation to presynaptic hcn channel function .
all experiments were performed with either early postnatal ( p915 ) or adult ( p3060 ) male cd rats purchased from charles river ( sulzfeld , germany ) and conformed to national and international guidelines on the ethical use of animals ( european council directive 86/609/eec ) . after deep anesthesia with diethyl ether ( mallinckrodt baker , deventer , the netherlands ) , rats were decapitated and the brain was rapidly removed and submerged into oxygenated ice - cold dissection solution containing ( in mm ) 125 nacl , 26 nahco3 , 3 kcl , 1.25 nah2po4 , 0.2 cacl2 , 5 mgcl2 , and 13 d - glucose ( gassed with 95% o2 , 5% co2 ; ph 7.4 ) . subsequently ,
horizontal brain slices ( 400 m ) of the hippocampus were prepared in this ice - cold dissection solution with a vibratome ( integraslice 7550 mm , campden instruments , loughborough , uk ) .
the hippocampal formations of both hemispheres were carefully excised and then transferred into a holding chamber at room temperature filled with artificial cerebrospinal fluid ( acsf ) containing ( in mm ) 125 nacl , 26 nahco3 , 3 kcl , 1.25 nah2po4 , 2.5 cacl2 , 1.3 mgcl2 , and 13 d - glucose ( gassed with 95% o2 , 5% co2 ; ph 7.4 ) . for electrophysiological recordings , hippocampal slices were transferred into an interface chamber ( bsc - ht , harvard apparatus , holliston , usa ) maintained at 32c ( tc-10 , npi electronic gmbh , tamm , germany ) and superfused with acsf ( composition as above , perfusion rate of 2 - 3 ml / min ) .
field excitatory postsynaptic potentials ( fepsps ) were recorded using borosilicate glass pipettes ( gb150 - 8p , science products , hofheim am taunus , germany ) with a tip resistance of 2 - 3 m ( fabricated with pip5 puller from heka elektronik , lambrecht , germany ) and filled with acsf .
bipolar stimulating electrodes were made from teflon - insulated platinum wires ( pt-2 t , science products ) , and double - pulse stimulation ( interstimulus interval 40 ms ) was delivered using a master-8 stimulator ( a.m.p.i . , jerusalem , israel ) and a stimulus isolator ( a365 , world precision instruments , sarasota , usa ) at a baseline rate of 0.033 hz .
the baseline stimulation strength was adjusted to 3040% of the maximal fepsp amplitude and typically in the range of 50100 a .
the evoked potentials were amplified and filtered at 1 khz ( ext-10 - 2f , npi ) , digitized ( micro1401 , ced , cambridge electronic design , cambridge , uk ) and stored for off - line analysis ( signal 2.16 , ced ) .
to record from the medial perforant path - granule cell synapse , both the stimulating and the recording electrodes were placed into the middle molecular layer ( mml ) of the dentate gyrus .
since these synapses typically display paired - pulse depression [ 6 , 7 ] , double - pulse stimulation ( interstimulus interval of 40 ms ) was used during the initial 57 minutes of each experiment , and the paired - pulse ratio ( ppr ) was calculated as the ratio of the 2nd pulse - evoked fepsp to the 1st pulse - evoked fepsp . in addition , gabazine ( 1 m , tocris , bristol , uk ) was added to the acsf in order to reduce contaminations by stimulation of local gabaergic interneuronal circuits , and single stimulation was used for the remaining experiment .
short - term depression ( std ) and long - term depression ( ltd ) were induced by a low - frequency stimulation ( lfs ) protocol consisting of 900 pulses at 1 hz ( baseline stimulation strength , 1.5-fold pulse width ) .
std ( average of fepsp amplitudes at 02 min after lfs ) and ltd ( average of fepsp amplitudes at 5560 minutes after lfs ) values are given as fepsp amplitudes expressed as the percentage of the baseline response .
the influence of hcn channels on lfs - induced ltd was tested by bath application of zd7288 ( tocris ) which was freshly dissolved in acsf ( 10 m ) .
the specific n - methyl - d - aspartate ( nmda ) receptor blocker d-2-amino-5-phosphonopentanoate ( d - ap5 ) and the nitric oxide synthase ( nos ) blocker n - nitro - l - arginine methyl ester ( l - name ) were also obtained from tocris .
since zd7288 was shown to depress glutamatergic receptors [ 8 , 9 ] , we performed interleaved experiments without lfs that were used to normalize the std and ltd values .
this normalization was performed by calculating the ratio of the lfs - treated by the non - lfs - treated control value and expressed as the percentage of baseline response .
significant differences are indicated by asterisks ( * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 ) for comparisons between untreated and treated slices ( unpaired test ) and by diamonds ( p < 0.05 ; p < 0.01 ) for comparisons between different time points within the same group ( paired test ) .
long - term depression ( ltd ) in the hippocampus is commonly induced by low - frequency stimulation ( lfs ) . here
, we employed an lfs protocol of 900 pulses at 1 hz ( indicated by an open bar in figure 1(a ) ) .
such a paradigm applied to the medial perforant path ( mpp ) input onto dentate gyrus - granule cells caused an immediate synaptic depression to 70 6% of the baseline fepsp amplitude ( figure 1(a ) , closed symbols , n = 9 ) . during the followup of the experiment ,
this short - term depression ( std ) resulted in stable ltd of 80 4% of baseline at 60 minutes after lfs .
interleaved time - control experiments without lfs demonstrated that our recording conditions were stable for at least 90 minutes ( 100 1% at the end of the experiment ; open symbols in figure 1(a ) , n = 12 ) . since we were interested in the effect of 10 m zd7288 on lfs - induction of ltd , this compound was bath applied 10 minutes prior to lfs ( figure 1(b ) , closed symbols , n = 7 ) . in these experiments , fepsp
amplitudes both immediately after lfs ( std ) and at the end of the experiment ( ltd ) were more markedly depressed ( 51 5% and 48 3% , n = 7 ) .
however , this enhanced std and ltd could be biased by the synaptic depression following zd7288 [ 8 , 9 ] .
therefore , we performed again interleaved time - control experiments without lfs ( figure 1(b ) , open symbols , n = 7 ) and indeed obtained a synaptic depression when zd7288 was bath applied to the slices ( 80 2% at the end of the experiment ; open symbols in figure 1(b ) ) . in order to directly compare lfs - induced ltd with and without hcn channel inhibition
, we normalized lfs - treated slices ( closed symbols in figures 1(a ) and 1(b ) ) to non - lfs - treated slices ( open symbols in figures 1(a ) and 1(b ) ) .
this normalization indeed revealed that both std and ltd were significantly enhanced by zd7288 preapplication as compared to interleaved time - control experiments ( std with zd7288 : 53 5% , std control : 69 6% ; p < 0.05 ; ltd with zd7288 : 62 4% , ltd control : 80 4% , p < 0.01 ; figures 1(d ) and 1(e ) ) .
these results so far indicate that hcn channel activation during lfs appears to constrain both std and ltd .
we then asked the question whether zd7288 would also exert this effect when applied after lfs has been completed .
this is an important control experiment since std in this case is recorded before zd7288 could actually depress synaptic transmission and thus should not be altered by hcn channel inhibition ( figure 1(c ) ) .
in fact , std was unaltered by zd7288 treatment after lfs ( 68 5% , n = 9 , gray symbols in figure 1(c ) ) . but importantly , fepsp amplitudes at the end of the experiment were 65 4% ( n = 9 ) in lfs - treated slices and 81 3% in non - lfs - treated control experiments ( open symbols in figure 1(c ) ) .
following normalization , both std and ltd were no longer significantly different from control experiments without zd7288 application ( std : 68 5% , p <
0.05 versus preapplied zd7288 ; ltd : 81 5% , p < 0.01 versus preapplied zd7288 ; figures 1(d ) and 1(e ) ) . on the one hand
, this experiment suggests that the ltd enhancement is a specific zd7288-mediated effect and not an artifact due to normalization procedure . on the other hand
, these data also indicate that hcn channel activation modulate the induction but not the expression of lfs - induced ltd .
another important pitfall in recording mpp - evoked fepsps is the potential contamination by field potentials following stimulation of the adjacent fiber tract the lateral perforant path ( lpp ) .
the propensity of ltd to be obtained in the middle molecular layer may actually vary with the degree of contamination by lpp stimulation .
therefore , we analyzed the paired - pulse ratio ( ppr ) in all six experimental groups at the beginning of the experiment , that is , before lfs and/or zd7288 was applied .
as shown in figure 1(f ) , there were no significant differences in the ppr between all groups ( on average 76% ) , thus indicating a negligible contamination by lpp - evoked field potentials .
the results presented so far indicated a constraining effect of hcn channels on lfs - induced ltd in early postnatal mpp - dentate gyrus synapses .
we then asked the question whether this ltd enhancement following zd7288 treatment was due to an inhibition of presynaptic hcn channels that are abundant in immature mpp , but disappear with aging .
we therefore repeated these experiments with adult rats ( p30 - 60 ) . in slices taken from these animals ,
lfs applied to the mpp caused a short - term depression to 82 2% of the baseline fepsp amplitude ( figure 2(a ) , closed symbols , n = 6 ) and resulted in stable ltd of 81 3% of baseline at 60 minutes after lfs .
interleaved time - control experiments without lfs demonstrated that our recording conditions were again stable for at least 90 minutes ( 100 2% at the end of the experiment ; open symbols in figure 2(a ) , n = 8) . as in early postnatal rats
, we studied the effect of 10 m zd7288 on lfs - induced ltd and thus applied this compound 10 minutes prior to lfs ( figure 2(b ) , closed symbols , n = 9 ) . under hcn channel blocking conditions
, both std and ltd appeared to be more depressed as compared to slices without zd7288 treatment ( 63 4% and 65 4% , n = 9 ) .
however , this enhanced std and ltd could again be biased by zd7288-mediated synaptic depression , and interleaved time - control experiments without lfs ( figure 2(b ) , open symbols , n = 6 ) indeed revealed a significant synaptic depression to 77 4% at the end of the experiment ( open symbols in figure 2(b ) ) .
therefore , it was again necessary to normalize lfs - treated slices ( closed symbols in figures 2(a ) and 2(b ) ) to non - lfs - treated slices ( open symbols in figures 2(a ) and 2(b ) ) .
in contrast to slices from early postnatal rats , the normalized values for both std and ltd were no longer significantly different between zd7288-treated and zd7288-untreated conditions ( std with zd7288 : 70 4% , std control : 81 5% ; ltd with zd7288 : 85 5% , ltd control : 85 6% , figures 2(c ) and 2(d ) ) .
thus , hcn channel activation failed to have a significant effect on std and ltd in adult rats .
figure 2(e ) illustrates again that there were no significant differences in the ppr between all groups ( on average 80% ) , a contamination by lpp - evoked field potentials is therefore unlikely .
our results may suggest that hcn channel - mediated suppression of lfs - induced ltd is downregulated during developmental maturation .
however , one could argue that lfs - induced ltd in different ages may involve different induction mechanisms .
we therefore asked whether nmda receptors are required for ltd induction in both age groups and repeated our experiments in the presence of the nmda receptor blocker d - ap5 ( 50 m ) .
we in fact found that ltd was abolished under these conditions in both adult ( 95 7% , n = 5 ) and early postnatal rats ( 95 3% , n = 8 ; figures 3(a ) and 3(b ) ; both age groups p < 0.05 versus untreated slices ) .
hence , differential nmda receptor dependence of ltd induction can not explain the differences in immature and adult tissues . however , immature mpp axon terminals express presynaptic hcn channels that have been demonstrated to be downregulated upon maturation .
thus , we asked whether zd7288 might alter the paired - pulse ratio ( ppr ) in slices from early postnatal and adult rats ( figures 3(c ) , 3(d ) , 3(e ) , and 3(f ) ) .
initial experiments revealed that bath application of zd7288 occasionally led to an exacerbation of disinhibitory network activity , probably due to gabazine which was present in all our solutions .
therefore , we first recorded a baseline with paired - pulse stimulation , then incubated the slices with zd7288 ( 10 m ) while switching off the stimulation for 15 min , and finally delivered paired - pulse stimulation again for another 20 min ( figures 3(c ) and 3(d ) ) .
as shown previously ( figures 1(b ) and 2(b ) ) , zd7288 generally depressed mpp - evoked fepsps .
however , in slices from immature rats , the field potential evoked by the first stimulus ( fepsp1 , gray boxes in figure 3(c ) ) appeared to be more affected by this compound than the second response ( fepsp2 , gray diamonds in figure 3(c ) ) . as a consequence , zd7288 caused a significant increase of the normalized paired - pulse ratio ( 117 4% , n = 8 , gray symbols / bars in figures 3(e ) and 3(f ) ) . in adult rats , however , zd7288 depressed the first and second fepsp to the same degree resulting in no significant change of the ppr ( 98 2% , n = 11 , open symbols / bars in figures 3(e ) and 3(f ) ) .
these findings indicate that hcn channel blockade causes a reduction of fepsp amplitudes in both immature and adult tissues .
but in addition , it leads to an increase of the paired - pulse ratio in immature synapses only suggesting that presynaptic hcn channels facilitate glutamate release .
our findings so far indicate that lfs - induced ltd is nmda receptor dependent and in the case of early postnatal synapses is compromised by hcn channel activation .
if indeed presynaptic hcn channels might be responsible for this modulation in immature tissue , a retrograde signaling pathway has to be postulated to transfer the information of postsynaptic nmda receptor activation to presynaptic hcn channels .
one major retrograde messenger is nitric oxide ( no ) produced by postsynaptic no synthase ( nos ) which travels through the synaptic cleft to activate presynaptic guanylate cyclases [ 10 , 11 ] .
we tested this hypothesis by repeating our experiments in the presence of the nos blocker l - name ( 100 m , figure 4 ) .
l - name itself had no affect on mpp - evoked fepsps in both age groups ( early postnatal : open symbols in figure 4(a ) ; adult : open symbols in figure 4(b ) ) .
however , lfs - induced ltd was more pronounced in slices from immature rats ( closed symbols in figure 4(a ) ) compared to those from adult animals ( closed symbols in figure 4(b ) ) .
normalization to non - lfs - treated slices revealed that l - name significantly enhanced ltd in early postnatal tissue ( 66 6% , n = 7 ) , when compared to control conditions ( 80 4% , n = 9 ; p < 0.05 , figure 4(c ) ) .
this modulatory role of l - name was lost in adult tissue ( with l - name : 81 3% , n = 7 ; without l - name : 85 6% , n = 6 , figure 4(c ) ) .
the ppr analysis of all l - name - treated slices ( on average 78% ) did not detect any significant differences , thus arguing against lpp contamination ( figure 4(d ) ) .
our results obtained with l - name are reminiscent of the effect of hcn channel inhibition on lfs - induced ltd and suggest that retrograde signaling with no might play a major role in the linkage between postsynaptic nmda receptor activation and presynaptic hcn channel function .
in the present study , we found that bath application of the hcn channel blocker zd7288 depressed synaptic transmission at medial perforant path ( mpp ) synapses .
in addition , low - frequency stimulation ( lfs ) delivered to these fibers resulted in ltd which was significantly enhanced after zd7288 during the early postnatal age , but not in adult animals .
hence , hcn channel activation during neuronal activity may compromise the propensity of ltd obtained at these synapses .
unfortunately , the explanation of mechanisms mediated by zd7288 in synaptic function may be confounded by other effects such as its action on glutamate receptors [ 5 , 9 ] . while ltd was found to be nmda receptor dependent in both age groups , hcn channels
are predominantly expressed on mpp axon terminals at early postnatal ages , but this presynaptic location is lost during development .
albeit postsynaptic expression of hcn channels on granule cell dendrites can not entirely be excluded , there were no differences between slices from early postnatal and adult animals . in the present study
, we in fact found a significant increase of the paired - pulse ratio by hcn channel inhibition in early postnatal , but not in adult , tissue , confirming a facilitating role of presynaptic hcn channels in transmitter release .
therefore , the differential effect of zd7288 on lfs - induced ltd in these two age groups strongly supports the idea that presynaptic hcn channels contribute to the zd7288-mediated ltd enhancement .
how can presynaptic hcn channels modulate ltd ? in general , activity - dependent changes in synaptic strength may arise from both pre- and postsynaptic mechanisms . in hippocampal dendrites , it was proposed that hcn channels have at least two major effects , that is , ( i ) an active shunting conductance and ( ii ) a tonic depolarization leading to a reduced excitatory postsynaptic potential ( epsp ) amplitude and a compromised temporal summation [ 1217 ] . on the other side , presynaptic hcn channels
have been suggested to increase the reliability of neurotransmitter release via exerting a tonic depolarization on the presynaptic membrane .
the underlying mechanisms may involve interaction with t - type ca channels as shown recently in axons terminating on entorhinal layer iii pyramidal cells , where the hcn channels were found to influence cav3.2 activity .
reducing presynaptic hcn activity could thus result in an altered frequency [ 20 , 21 ] or failure rate of action potential - dependent transmitter release .
importantly , hcn channels are very sensitive to changes of the intracellular milieu , such as altered levels of pip2 or cyclic amp [ 2325 ] .
interestingly , cyclic gmp may also stimulate hcn channel function , and activation of guanylyl cyclases is the typical downstream effect of the no retrograde signaling pathway .
there is indeed a large body of evidence supporting the role of no in modulating synaptic transmission in the ca1 region of the hippocampus [ 10 , 27 ] , reviewed in , but the role of no in the molecular layer of the dentate gyrus has attracted less attention .
one study found that no synthase inhibition blocked both ltp and ltd at these synapses .
at least in our hands , however , l - name had no effect on medial perforant path - evoked ltd in slices from adult rats , and in immature tissue , we found a significant increase of ltd values .
albeit the reasons for this discrepancy remain unclear , the results of the present study suggest that postsynaptic nmda receptor activation leads to no synthesis acting as a retrograde messenger traveling from the postsynaptic site to the presynaptic terminal . within the presynapse
, we propose the activation of no - dependent guanylyl cyclases and thereby production of cgmp which facilitates hcn channel function .
both no production and hcn channel activation seem to compromise ltd at immature synapses , since inhibition of these pathways enhanced ltd . | hyperpolarization - activated , cyclic nucleotide - gated nonselective ( hcn ) channels modulate both membrane potential and resistance and play a significant role in synaptic plasticity .
we compared the influence of hcn channels on long - term depression ( ltd ) at the medial perforant path - granule cell synapse in early postnatal ( p915 ) and adult ( p3060 ) rats .
ltd was elicited in p915 slices using low - frequency stimulation ( lfs , 900 pulses , 1 hz ; 80 4% of baseline ) .
application of the specific hcn channel blocker zd7288 ( 10 m ) before lfs significantly enhanced ltd ( 62 4% ; p < 0.01 ) , showing hcn channels restrain ltd induction .
however , when zd7288 was applied after lfs , ltd was similar to control values and significantly different from the values obtained with zd7288 application before lfs ( 81 5% ; p < 0.01 ) , indicating that hcn channels do not modulate ltd expression .
ltd in slices from adult rats were only marginally lower compared to those in p915 slices ( 85 6% ) , but bath application of zd7288 prior to lfs resulted in the same amount of ltd ( 85 5% ) .
hcn channels in adult tissue hence lose their modulatory effect . in conclusion
, we found that hcn channels at the medial perforant path - granule cell synapse compromise lfs - associated induction , but not expression of ltd in early postnatal , but not in adult , rats . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion | hyperpolarization - activated , cyclic nucleotide - gated nonselective ( hcn ) channels show a considerable conductance at the resting membrane potential and mediate a slowly inactivating and inwardly rectifying na current [ 1 , 2 ] . hence , these channels appear to be ideal modulators of both the membrane potential and the membrane resistance of neurons and thus may play a significant role in synaptic transmission . in recent years , it has become evident that hcn channels are also relevant modulators of synaptic plasticity [ 35 ] . hcn channels may have differential effects on different synapses and/or various types of synaptic plasticity , depending on , for example , the synapse type , pre- or postsynaptic expression patterns , and ontogenetic stage . thus , while long - term potentiation ( ltp ) following electrical stimulation to the schaffer collateral input of ca1 pyramidal cells was shown to be unaltered in hcn1-deficient adult mice , a significant enhancement of ltp was observed when the perforant path input to ca1 was stimulated . such a constraining role of hcn channels on synaptic plasticity in ca1 was also found with long - term depression ( ltd ) following pharmacological activation of group i metabotropic glutamate receptors . however , ltd following electrical stimulation at the same synapse was also assessed in the same study and failed to be affected by hcn channel inhibition . since the expression pattern of hcn channels changes during development
presynaptic hcn channels are expressed on glutamatergic axon terminals of the medial perforant path ( mpp ) input to dentate gyrus granule cells only during the first 2 - 3 weeks of postnatal development and subsequently are lost during maturation comparing plasticity in immature and mature tissue allows to dissect the impact of pre- and postsynaptic hcn channel expression . in immature tissue
, hcn channel - mediated effects will be predominantly presynaptically driven , in adult tissue , postsynaptically . interestingly , short - term depression ( std ) at the immature synapse was enhanced following pharmacological hcn channel inhibition , as a first indication of a constraining role of hcn channels on synaptic plasticity . in the present study , we assessed nmda receptor - dependent low - frequency stimulation- ( lfs- ) induced ltd at the mpp - granule cell synapse at different developmental stages and found an enhancement of ltd following hcn channel inhibition in tissue from early postnatal , but not in tissue from adult animals . these findings emphasize the modulating role of hcn channels on synaptic plasticity and provide evidence for an important role of presynaptic hcn channels in activity - dependent changes of synaptic transmission . they also suggest that nitric oxide may play a role as a retrograde messenger that links postsynaptic nmda - receptor activation to presynaptic hcn channel function . all experiments were performed with either early postnatal ( p915 ) or adult ( p3060 ) male cd rats purchased from charles river ( sulzfeld , germany ) and conformed to national and international guidelines on the ethical use of animals ( european council directive 86/609/eec ) . after deep anesthesia with diethyl ether ( mallinckrodt baker , deventer , the netherlands ) , rats were decapitated and the brain was rapidly removed and submerged into oxygenated ice - cold dissection solution containing ( in mm ) 125 nacl , 26 nahco3 , 3 kcl , 1.25 nah2po4 , 0.2 cacl2 , 5 mgcl2 , and 13 d - glucose ( gassed with 95% o2 , 5% co2 ; ph 7.4 ) . subsequently ,
horizontal brain slices ( 400 m ) of the hippocampus were prepared in this ice - cold dissection solution with a vibratome ( integraslice 7550 mm , campden instruments , loughborough , uk ) . bipolar stimulating electrodes were made from teflon - insulated platinum wires ( pt-2 t , science products ) , and double - pulse stimulation ( interstimulus interval 40 ms ) was delivered using a master-8 stimulator ( a.m.p.i . , jerusalem , israel ) and a stimulus isolator ( a365 , world precision instruments , sarasota , usa ) at a baseline rate of 0.033 hz . the evoked potentials were amplified and filtered at 1 khz ( ext-10 - 2f , npi ) , digitized ( micro1401 , ced , cambridge electronic design , cambridge , uk ) and stored for off - line analysis ( signal 2.16 , ced ) . to record from the medial perforant path - granule cell synapse , both the stimulating and the recording electrodes were placed into the middle molecular layer ( mml ) of the dentate gyrus . short - term depression ( std ) and long - term depression ( ltd ) were induced by a low - frequency stimulation ( lfs ) protocol consisting of 900 pulses at 1 hz ( baseline stimulation strength , 1.5-fold pulse width ) . std ( average of fepsp amplitudes at 02 min after lfs ) and ltd ( average of fepsp amplitudes at 5560 minutes after lfs ) values are given as fepsp amplitudes expressed as the percentage of the baseline response . the influence of hcn channels on lfs - induced ltd was tested by bath application of zd7288 ( tocris ) which was freshly dissolved in acsf ( 10 m ) . since zd7288 was shown to depress glutamatergic receptors [ 8 , 9 ] , we performed interleaved experiments without lfs that were used to normalize the std and ltd values . this normalization was performed by calculating the ratio of the lfs - treated by the non - lfs - treated control value and expressed as the percentage of baseline response . significant differences are indicated by asterisks ( * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 ) for comparisons between untreated and treated slices ( unpaired test ) and by diamonds ( p < 0.05 ; p < 0.01 ) for comparisons between different time points within the same group ( paired test ) . long - term depression ( ltd ) in the hippocampus is commonly induced by low - frequency stimulation ( lfs ) . here
, we employed an lfs protocol of 900 pulses at 1 hz ( indicated by an open bar in figure 1(a ) ) . such a paradigm applied to the medial perforant path ( mpp ) input onto dentate gyrus - granule cells caused an immediate synaptic depression to 70 6% of the baseline fepsp amplitude ( figure 1(a ) , closed symbols , n = 9 ) . during the followup of the experiment ,
this short - term depression ( std ) resulted in stable ltd of 80 4% of baseline at 60 minutes after lfs . interleaved time - control experiments without lfs demonstrated that our recording conditions were stable for at least 90 minutes ( 100 1% at the end of the experiment ; open symbols in figure 1(a ) , n = 12 ) . since we were interested in the effect of 10 m zd7288 on lfs - induction of ltd , this compound was bath applied 10 minutes prior to lfs ( figure 1(b ) , closed symbols , n = 7 ) . in these experiments , fepsp
amplitudes both immediately after lfs ( std ) and at the end of the experiment ( ltd ) were more markedly depressed ( 51 5% and 48 3% , n = 7 ) . therefore , we performed again interleaved time - control experiments without lfs ( figure 1(b ) , open symbols , n = 7 ) and indeed obtained a synaptic depression when zd7288 was bath applied to the slices ( 80 2% at the end of the experiment ; open symbols in figure 1(b ) ) . in order to directly compare lfs - induced ltd with and without hcn channel inhibition
, we normalized lfs - treated slices ( closed symbols in figures 1(a ) and 1(b ) ) to non - lfs - treated slices ( open symbols in figures 1(a ) and 1(b ) ) . this normalization indeed revealed that both std and ltd were significantly enhanced by zd7288 preapplication as compared to interleaved time - control experiments ( std with zd7288 : 53 5% , std control : 69 6% ; p < 0.05 ; ltd with zd7288 : 62 4% , ltd control : 80 4% , p < 0.01 ; figures 1(d ) and 1(e ) ) . in fact , std was unaltered by zd7288 treatment after lfs ( 68 5% , n = 9 , gray symbols in figure 1(c ) ) . but importantly , fepsp amplitudes at the end of the experiment were 65 4% ( n = 9 ) in lfs - treated slices and 81 3% in non - lfs - treated control experiments ( open symbols in figure 1(c ) ) . following normalization , both std and ltd were no longer significantly different from control experiments without zd7288 application ( std : 68 5% , p <
0.05 versus preapplied zd7288 ; ltd : 81 5% , p < 0.01 versus preapplied zd7288 ; figures 1(d ) and 1(e ) ) . on the other hand
, these data also indicate that hcn channel activation modulate the induction but not the expression of lfs - induced ltd . another important pitfall in recording mpp - evoked fepsps is the potential contamination by field potentials following stimulation of the adjacent fiber tract the lateral perforant path ( lpp ) . the propensity of ltd to be obtained in the middle molecular layer may actually vary with the degree of contamination by lpp stimulation . therefore , we analyzed the paired - pulse ratio ( ppr ) in all six experimental groups at the beginning of the experiment , that is , before lfs and/or zd7288 was applied . as shown in figure 1(f ) , there were no significant differences in the ppr between all groups ( on average 76% ) , thus indicating a negligible contamination by lpp - evoked field potentials . the results presented so far indicated a constraining effect of hcn channels on lfs - induced ltd in early postnatal mpp - dentate gyrus synapses . we then asked the question whether this ltd enhancement following zd7288 treatment was due to an inhibition of presynaptic hcn channels that are abundant in immature mpp , but disappear with aging . in slices taken from these animals ,
lfs applied to the mpp caused a short - term depression to 82 2% of the baseline fepsp amplitude ( figure 2(a ) , closed symbols , n = 6 ) and resulted in stable ltd of 81 3% of baseline at 60 minutes after lfs . interleaved time - control experiments without lfs demonstrated that our recording conditions were again stable for at least 90 minutes ( 100 2% at the end of the experiment ; open symbols in figure 2(a ) , n = 8) . as in early postnatal rats
, we studied the effect of 10 m zd7288 on lfs - induced ltd and thus applied this compound 10 minutes prior to lfs ( figure 2(b ) , closed symbols , n = 9 ) . under hcn channel blocking conditions
, both std and ltd appeared to be more depressed as compared to slices without zd7288 treatment ( 63 4% and 65 4% , n = 9 ) . however , this enhanced std and ltd could again be biased by zd7288-mediated synaptic depression , and interleaved time - control experiments without lfs ( figure 2(b ) , open symbols , n = 6 ) indeed revealed a significant synaptic depression to 77 4% at the end of the experiment ( open symbols in figure 2(b ) ) . therefore , it was again necessary to normalize lfs - treated slices ( closed symbols in figures 2(a ) and 2(b ) ) to non - lfs - treated slices ( open symbols in figures 2(a ) and 2(b ) ) . in contrast to slices from early postnatal rats , the normalized values for both std and ltd were no longer significantly different between zd7288-treated and zd7288-untreated conditions ( std with zd7288 : 70 4% , std control : 81 5% ; ltd with zd7288 : 85 5% , ltd control : 85 6% , figures 2(c ) and 2(d ) ) . thus , hcn channel activation failed to have a significant effect on std and ltd in adult rats . our results may suggest that hcn channel - mediated suppression of lfs - induced ltd is downregulated during developmental maturation . however , one could argue that lfs - induced ltd in different ages may involve different induction mechanisms . we therefore asked whether nmda receptors are required for ltd induction in both age groups and repeated our experiments in the presence of the nmda receptor blocker d - ap5 ( 50 m ) . we in fact found that ltd was abolished under these conditions in both adult ( 95 7% , n = 5 ) and early postnatal rats ( 95 3% , n = 8 ; figures 3(a ) and 3(b ) ; both age groups p < 0.05 versus untreated slices ) . hence , differential nmda receptor dependence of ltd induction can not explain the differences in immature and adult tissues . thus , we asked whether zd7288 might alter the paired - pulse ratio ( ppr ) in slices from early postnatal and adult rats ( figures 3(c ) , 3(d ) , 3(e ) , and 3(f ) ) . initial experiments revealed that bath application of zd7288 occasionally led to an exacerbation of disinhibitory network activity , probably due to gabazine which was present in all our solutions . therefore , we first recorded a baseline with paired - pulse stimulation , then incubated the slices with zd7288 ( 10 m ) while switching off the stimulation for 15 min , and finally delivered paired - pulse stimulation again for another 20 min ( figures 3(c ) and 3(d ) ) . however , in slices from immature rats , the field potential evoked by the first stimulus ( fepsp1 , gray boxes in figure 3(c ) ) appeared to be more affected by this compound than the second response ( fepsp2 , gray diamonds in figure 3(c ) ) . as a consequence , zd7288 caused a significant increase of the normalized paired - pulse ratio ( 117 4% , n = 8 , gray symbols / bars in figures 3(e ) and 3(f ) ) . in adult rats , however , zd7288 depressed the first and second fepsp to the same degree resulting in no significant change of the ppr ( 98 2% , n = 11 , open symbols / bars in figures 3(e ) and 3(f ) ) . these findings indicate that hcn channel blockade causes a reduction of fepsp amplitudes in both immature and adult tissues . our findings so far indicate that lfs - induced ltd is nmda receptor dependent and in the case of early postnatal synapses is compromised by hcn channel activation . however , lfs - induced ltd was more pronounced in slices from immature rats ( closed symbols in figure 4(a ) ) compared to those from adult animals ( closed symbols in figure 4(b ) ) . normalization to non - lfs - treated slices revealed that l - name significantly enhanced ltd in early postnatal tissue ( 66 6% , n = 7 ) , when compared to control conditions ( 80 4% , n = 9 ; p < 0.05 , figure 4(c ) ) . this modulatory role of l - name was lost in adult tissue ( with l - name : 81 3% , n = 7 ; without l - name : 85 6% , n = 6 , figure 4(c ) ) . our results obtained with l - name are reminiscent of the effect of hcn channel inhibition on lfs - induced ltd and suggest that retrograde signaling with no might play a major role in the linkage between postsynaptic nmda receptor activation and presynaptic hcn channel function . in the present study , we found that bath application of the hcn channel blocker zd7288 depressed synaptic transmission at medial perforant path ( mpp ) synapses . in addition , low - frequency stimulation ( lfs ) delivered to these fibers resulted in ltd which was significantly enhanced after zd7288 during the early postnatal age , but not in adult animals . hence , hcn channel activation during neuronal activity may compromise the propensity of ltd obtained at these synapses . while ltd was found to be nmda receptor dependent in both age groups , hcn channels
are predominantly expressed on mpp axon terminals at early postnatal ages , but this presynaptic location is lost during development . albeit postsynaptic expression of hcn channels on granule cell dendrites can not entirely be excluded , there were no differences between slices from early postnatal and adult animals . in the present study
, we in fact found a significant increase of the paired - pulse ratio by hcn channel inhibition in early postnatal , but not in adult , tissue , confirming a facilitating role of presynaptic hcn channels in transmitter release . therefore , the differential effect of zd7288 on lfs - induced ltd in these two age groups strongly supports the idea that presynaptic hcn channels contribute to the zd7288-mediated ltd enhancement . how can presynaptic hcn channels modulate ltd ? in hippocampal dendrites , it was proposed that hcn channels have at least two major effects , that is , ( i ) an active shunting conductance and ( ii ) a tonic depolarization leading to a reduced excitatory postsynaptic potential ( epsp ) amplitude and a compromised temporal summation [ 1217 ] . importantly , hcn channels are very sensitive to changes of the intracellular milieu , such as altered levels of pip2 or cyclic amp [ 2325 ] . interestingly , cyclic gmp may also stimulate hcn channel function , and activation of guanylyl cyclases is the typical downstream effect of the no retrograde signaling pathway . there is indeed a large body of evidence supporting the role of no in modulating synaptic transmission in the ca1 region of the hippocampus [ 10 , 27 ] , reviewed in , but the role of no in the molecular layer of the dentate gyrus has attracted less attention . at least in our hands , however , l - name had no effect on medial perforant path - evoked ltd in slices from adult rats , and in immature tissue , we found a significant increase of ltd values . albeit the reasons for this discrepancy remain unclear , the results of the present study suggest that postsynaptic nmda receptor activation leads to no synthesis acting as a retrograde messenger traveling from the postsynaptic site to the presynaptic terminal . both no production and hcn channel activation seem to compromise ltd at immature synapses , since inhibition of these pathways enhanced ltd . | [
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during the past few decades quality of life assessment has become one central outcome in treatment of patients with chronic lower limb ischaemia . different generic quality of life instruments such as the nottingham health profile ( nhp ) and the short form 36 health survey ( sf-36 ) have previously been tested , revealing conflicting results in these patients according to psychometric attributes in short - term evaluations .
the strengths and weakness of the nhp and the sf-36 scales are not extensively examined and further research is needed to establish which is the more appropriate and responsive quality of life instrument for patients with chronic lower limb ischaemia in the long term .
the main goal of vascular surgical treatment is the relief of symptoms and improvement in patients quality of life .
a majority of the patients are elderly and have generally widespread arterial disease with numbers of symptoms due to the chronic lower limb ischaemia , which may affect the patients ' quality of life [ 3 - 5 ] .
intermittent claudication ( ic ) means leg pain constantly produced by walking or muscular activity and is relieved by rest , while critical leg ischaemia ( cli ) means pain even at rest and problems with non - healing ulcers or gangrene .
it is important to identify dimensions which are influenced by the severity and nature of the disease when selecting a suitable quality of life instrument .
physical and psychological health , social relationship perceptions , function and well - being which should be included in a generic quality of life instrument . generic instruments cover a broad range of dimensions and allow comparisons between different groups of patients .
disease - specific instruments , on the other hand , are specially designed for a particular disease , patient group or areas of function .
the functional scale , walking impairment questionnaire ( wiq ) and quality of life instruments such as intermittent claudication questionnaire ( icq ) and claudication scale ( clau - s ) are examples of disease - specific instruments which have been developed in recent years for patients with ic
. however , at present there is no accepted disease - specific questionnaire for quality of life assessment in patients with cli .
nevertheless , the transatlantic inter - society consensus ( tasc ) recommended that quality of life instruments should be used in all clinical trials and preferably include both generic and disease - specific quality of life measures .
outcome measures need to satisfy different criteria to be useful as a suitable health outcome instrument in clinical practice .
construct validity is one of the most important characteristics and is a lengthy and ongoing process .
an essential consideration is the instrument 's ability to discriminate between different levels of the disease ; another consideration is its reliability , which means the degree to which the instrument is free from random error and all items measure the same underlying attribute .
further , the requirement for a useful outcome measure is the responsiveness in detecting small but important clinical changes of quality of life in patients following vascular interventions .
finally the ideal quality of life instrument must also be acceptable to patients , simple and easy to use and preferably short .
comparisons among quality of life instruments and their psychometric characteristics and performance are needed to provide recommendations about their usefulness as outcome measures for these particular groups of patients .
the aim of this study was to compare two generic quality of life questionnaires , the nottingham health profile ( nhp ) and the short form 36 health survey ( sf-36 ) regarding the internal consistency reliability , validity , responsiveness and suitability as outcome measures in patients with lower limb ischaemia in a longitudinal perspective .
ninety consecutive patients from a swedish vascular unit in southern sweden were invited to participate in this study .
the assessment took place before and 12 months after revascularization . out of 90 patients , 24 ( 27% ) dropped out during the follow - up period , of whom 14 suffered from cli .
six patients ( 7% ) died , 15 ( 17% ) did not wish to participate and 3 ( 3% ) had other concurrent diseases .
the inclusion criteria were patients admitted for active treatment of documented lower limb ischaemia , having no communication problems and having no other disease restricting their walking capacity .
the severity of ischaemia was graded according to suggested standards for grading lower limb ischaemia .
sixty - two ( 68.8% ) patients were treated with a surgical bypass , 24 ( 26.6% ) had a percutaneous angioplasty ( pta ) and 4 ( 4.6% ) had a surgical thromboendatherectomy ( tea ) ( table 1 ) .
routine medical history , risk factors and clinical examinations , which included ankle blood pressure ( abp ) and ankle - brachial pressure index ( abpi ) , were obtained before and one year after revascularization in accordance with the swedish vascular registry ( swedvasc ) .
mann - whitney u - test chi - square test * include the patients ( n = 66 ) who completed the study one year postoperatively .
p - value = < 0.05 the nottingham health profile ( nhp ) was developed to be used in epidemiological studies of health and disease .
i contains 38 yes / no items in 6 dimensions : pain , physical mobility , emotional reactions , energy , social isolation and sleep .
the two parts may be used independently and part ii is not analysed in this study .
part i is scored using weighted values which give a range of possible scores from zero ( no problems at all ) to 100 ( presence of all problems within a dimension ) .
the swedish version has proved to be valid and reliable , for example , in patients with arthrosis of the hip joint and in patients suffering from grave ventricular arrhythmias .
the nhp scale has also proved capable of measuring changes in perceived health following different treatments such as radical surgery for colorectal cancer and after vascular interventions in lower limb ischaemia patients .
the short form 36 health survey ( sf-36 ) was developed by ware et al and designed to provide assessments involving generic health concepts that are not specific to age , disease or treatment group .
it includes 36 items covering eight health concepts : bodily pain , physical functioning , role limitations due to physical problems , mental health , vitality , social functioning , role limitations due to emotional problems and general health .
the response format is yes or no or in a three - to - six response scale . for each health concept questions scores
are coded , summed and transformed on a scale from zero ( worst health ) to 100 ( best health ) . in this study ,
the sf-36 has shown acceptable validity and reliability in population studies and in various groups of patients , for example after stroke and in patients with rheumatoid arthritis .
the sf-36 scale has also shown responsiveness to changes in health status over time in patients with critical ischaemia [ 30 - 32 ] and in patients with intermittent claudication following a revascularization [ 33 - 35 ] .
the patients were asked by the head nurse to fill out the nhp and the sf-36 questionnaire during their admission before treatment . at the one - year follow - up , the questionnaire was sent home to the patients with a covering letter and a prepaid envelope .
the ethics committee of lund university approved the study ( lu 470 - 98 , gbg m 098 - 98 ) .
differences in characteristics between patients with ic and with cli before revascularization were analysed using chi - squared test and mann - whitney u - test .
the prevalence of the lowest ( " floor " effect ) and highest ( " ceiling " effect ) possible quality of life score in nhp and sf-36 was also calculated .
construct validity was evaluated for aspects of convergent and discriminant validity by the multitrait - multimethod matrix ( mtmm ) based on five comparable domains , including pain , physical mobility , emotional reactions , energy and social isolation for the nhp and bodily pain , physical functioning , mental health , vitality and social functioning for the sf-36 ( table 2 ) .
further , the mann - whitney u - test was used to examine the relative ability of the two instruments to discriminate among the degrees of severity of the peripheral vascular disease .
spearman 's rank correlation coefficient was used to express the correlation between quality of life scores , abpi , type of intervention and age . the internal consistency based on correlations between items for each scale
the recommended reliability standard for group - level comparisons is a reliability coefficient of 0.70 , while comparisons between individuals demands a reliability coefficient of 0.90 .
comparable domains between the nottingham health profile ( nhp ) and the short form 36 ( sf-36 ) and number of items .
the wilcoxon signed ranks test was used to detect the responsiveness of within - subjects changes over time , before and one year after revascularization , in patients with ic and cli .
data analysis was performed for overall comparisons using the statistical package spss 11.0 and a p value of < .05 was taken as statistically significant .
ninety consecutive patients from a swedish vascular unit in southern sweden were invited to participate in this study .
the assessment took place before and 12 months after revascularization . out of 90 patients , 24 ( 27% ) dropped out during the follow - up period , of whom 14 suffered from cli .
six patients ( 7% ) died , 15 ( 17% ) did not wish to participate and 3 ( 3% ) had other concurrent diseases .
the inclusion criteria were patients admitted for active treatment of documented lower limb ischaemia , having no communication problems and having no other disease restricting their walking capacity .
the severity of ischaemia was graded according to suggested standards for grading lower limb ischaemia .
sixty - two ( 68.8% ) patients were treated with a surgical bypass , 24 ( 26.6% ) had a percutaneous angioplasty ( pta ) and 4 ( 4.6% ) had a surgical thromboendatherectomy ( tea ) ( table 1 ) .
routine medical history , risk factors and clinical examinations , which included ankle blood pressure ( abp ) and ankle - brachial pressure index ( abpi ) , were obtained before and one year after revascularization in accordance with the swedish vascular registry ( swedvasc ) .
mann - whitney u - test chi - square test * include the patients ( n = 66 ) who completed the study one year postoperatively .
the nottingham health profile ( nhp ) was developed to be used in epidemiological studies of health and disease .
part i contains 38 yes / no items in 6 dimensions : pain , physical mobility , emotional reactions , energy , social isolation and sleep .
the two parts may be used independently and part ii is not analysed in this study .
part i is scored using weighted values which give a range of possible scores from zero ( no problems at all ) to 100 ( presence of all problems within a dimension ) .
the swedish version has proved to be valid and reliable , for example , in patients with arthrosis of the hip joint and in patients suffering from grave ventricular arrhythmias .
the nhp scale has also proved capable of measuring changes in perceived health following different treatments such as radical surgery for colorectal cancer and after vascular interventions in lower limb ischaemia patients .
the short form 36 health survey ( sf-36 ) was developed by ware et al and designed to provide assessments involving generic health concepts that are not specific to age , disease or treatment group .
it includes 36 items covering eight health concepts : bodily pain , physical functioning , role limitations due to physical problems , mental health , vitality , social functioning , role limitations due to emotional problems and general health .
the response format is yes or no or in a three - to - six response scale . for each health concept questions scores
are coded , summed and transformed on a scale from zero ( worst health ) to 100 ( best health ) . in this study ,
the sf-36 has shown acceptable validity and reliability in population studies and in various groups of patients , for example after stroke and in patients with rheumatoid arthritis .
the sf-36 scale has also shown responsiveness to changes in health status over time in patients with critical ischaemia [ 30 - 32 ] and in patients with intermittent claudication following a revascularization [ 33 - 35 ] .
the patients were asked by the head nurse to fill out the nhp and the sf-36 questionnaire during their admission before treatment . at the one - year follow - up , the questionnaire was sent home to the patients with a covering letter and a prepaid envelope .
the ethics committee of lund university approved the study ( lu 470 - 98 , gbg m 098 - 98 ) .
differences in characteristics between patients with ic and with cli before revascularization were analysed using chi - squared test and mann - whitney u - test .
the prevalence of the lowest ( " floor " effect ) and highest ( " ceiling " effect ) possible quality of life score in nhp and sf-36 was also calculated .
construct validity was evaluated for aspects of convergent and discriminant validity by the multitrait - multimethod matrix ( mtmm ) based on five comparable domains , including pain , physical mobility , emotional reactions , energy and social isolation for the nhp and bodily pain , physical functioning , mental health , vitality and social functioning for the sf-36 ( table 2 ) .
further , the mann - whitney u - test was used to examine the relative ability of the two instruments to discriminate among the degrees of severity of the peripheral vascular disease .
spearman 's rank correlation coefficient was used to express the correlation between quality of life scores , abpi , type of intervention and age . the internal consistency based on correlations between items for each scale
the recommended reliability standard for group - level comparisons is a reliability coefficient of 0.70 , while comparisons between individuals demands a reliability coefficient of 0.90 .
comparable domains between the nottingham health profile ( nhp ) and the short form 36 ( sf-36 ) and number of items .
the wilcoxon signed ranks test was used to detect the responsiveness of within - subjects changes over time , before and one year after revascularization , in patients with ic and cli .
data analysis was performed for overall comparisons using the statistical package spss 11.0 and a p value of < .05 was taken as statistically significant .
forty - eight ( 53.3% ) patients had ic of whom 26 ( 54% ) were men .
the remaining 42 ( 46.7% ) suffered from cli and 22 ( 52% ) of them were men .
there was a significant difference in age between the two groups with a mean age of 67 and 71 respectively ( table 1 ) .
one year postoperatively , sixty - six ( 73% ) patients ( 38 with ic and 28 with cli ) remained in the study and secondary reconstructions were made on 7 ( 10% ) patients during the follow - up .
there were no significant differences at baseline in sex , age , abpi and quality of life scores between the drop - out patients and the patients who completed the study .
further , there were no significant differences between the drop - outs and the remaining patients regarding the method of treatment or severity of ischaemia .
analyses between the comparable domains showed that the nhp scores were more skewed than the sf-36 scores , especially in emotional reactions , energy and social isolation ( figure 1 ) . the " floor effect " , the proportion of individuals having the lowest possible scores ( sf-36 = 0 , nhp = 100 ) , was larger for the nhp scale in energy one year ( 19.7% ) after revascularization than for the sf-36 . the " ceiling effect " , the proportion of individuals having the best possible scores ( sf-36 = 100 , nhp = 0 ) , was also larger for the nhp scale in emotional reactions ( 50.0% ) , energy ( 42.4% ) and social isolation ( 71.2% ) one year after revascularization ( table 3 ) .
frequency distribution of scores on the nhp ( left side ) and comparable dimensions on the sf-36 ( right side ) one year after revascularization .
nhp scores had 100 subtracted for consistency with sf-36 reliability and " floor " and " ceiling " effects in comparable nhp and sf-36 scales before and one year after revascularization .
cronbach 's the average convergent validity coefficients exceeded 0.5 one year postoperatively except for physical mobility and physical functioning ( r = -0.46 ) and for social isolation and social functioning ( r = -0.32 ) , indicating a considerable convergence of the sf-36 and nhp ( table 4 ) .
one year postoperatively significant correlations between abpi and physical functioning ( r = 0.29 ) ( sf-36 ) , physical mobility ( r = 0.42 ) and pain ( r = 0.42 ) ( nhp ) were found .
the severity of the ischaemia had a significant influence in the nhp - measured domain of pain ( p < .003 ) and physical mobility ( p < .03 ) , indicating lower quality of life scores in patients with critical ischaemia . in the ability to discriminate between levels of ischaemia in the other comparable quality of life domains ,
multitrait - multimethod matrix of correlation coefficient for the comparable scales of the sf-36 and nhp in patients with varying degrees of lower limb ischaemia one year after revascularization .
correlation coefficients for comparable domains of the two questionnaires are shown in bold type correlation coefficients are negative because the two scales run in opposite directions .
differences in comparable domains in the nhp and the sf-36 between claudicants and patients with critical ischaemia before revascularization . *
a higher score ( 100 ) indicates more perceived problems . * * a higher score ( 100 ) indicates fewer perceived problems .
p - value , claudicants versus critical ischaemia patients before revascularization . tested by mann - whitney u - test .
p - value = < 0.05 physical functioning ( = 0.82 ) , mental health ( = 0.76 ) and vitality ( = 0.70 ) for the sf-36 and pain ( = 0.71 ) , emotional reactions ( = 0.76 ) and energy ( = 0.71 ) for the nhp scale were reliable , with coefficients > 0.70 before revascularization . for the sf-36 , all of the comparable domains except for social functioning ( = 0.64 ) exceeded the cronbach 's alpha value of 0.8 at the one - year follow - up . for the nhp the internal consistency coefficient was less than 0.8 but still exceeded 0.70 ( table 3 ) . the nhp scale and sf-36 were not equally good at detecting within - patient changes over time . in patients with ic
the sf-36 scale showed significant improvements in bodily pain ( p < .01 ) and in physical functioning ( p < .001 ) and for the patients with cli there were significant improvements in bodily pain ( p < .004 ) at the one - year follow - up ( figure 2 ) .
the nhp scale showed no significant improvements in patients with ic , while in patients with cli , significant improvements in pain ( p < .001 ) and physical mobility ( p <
changes in median score for the sf-36 in claudicants and critical ischaemia patients before and one year after revascularization . tested by wilcoxon signed ranks test .
bp = bodily pain , pf = physical functioning , mh = mental health , vt = vitality and sf = social functioning .
changes in median score for the nhp in claudicants and critical ischaemia patients before and one year after revascularization . tested by wilcoxon signed ranks test .
p = pain , pm = physical mobility , em = emotional reactions , e = energy and so = social isolation .
the average convergent validity coefficients exceeded 0.5 one year postoperatively except for physical mobility and physical functioning ( r = -0.46 ) and for social isolation and social functioning ( r = -0.32 ) , indicating a considerable convergence of the sf-36 and nhp ( table 4 ) .
one year postoperatively significant correlations between abpi and physical functioning ( r = 0.29 ) ( sf-36 ) , physical mobility ( r = 0.42 ) and pain ( r = 0.42 ) ( nhp ) were found .
the severity of the ischaemia had a significant influence in the nhp - measured domain of pain ( p < .003 ) and physical mobility ( p < .03 ) , indicating lower quality of life scores in patients with critical ischaemia . in the ability to discriminate between levels of ischaemia in the other comparable quality of life domains ,
multitrait - multimethod matrix of correlation coefficient for the comparable scales of the sf-36 and nhp in patients with varying degrees of lower limb ischaemia one year after revascularization .
correlation coefficients for comparable domains of the two questionnaires are shown in bold type correlation coefficients are negative because the two scales run in opposite directions .
differences in comparable domains in the nhp and the sf-36 between claudicants and patients with critical ischaemia before revascularization . *
p - value , claudicants versus critical ischaemia patients before revascularization . tested by mann - whitney u - test .
physical functioning ( = 0.82 ) , mental health ( = 0.76 ) and vitality ( = 0.70 ) for the sf-36 and pain ( = 0.71 ) , emotional reactions ( = 0.76 ) and energy ( = 0.71 ) for the nhp scale were reliable , with coefficients > 0.70 before revascularization .
for the sf-36 , all of the comparable domains except for social functioning ( = 0.64 ) exceeded the cronbach 's alpha value of 0.8 at the one - year follow - up .
for the nhp the internal consistency coefficient was less than 0.8 but still exceeded 0.70 ( table 3 ) .
the nhp scale and sf-36 were not equally good at detecting within - patient changes over time .
in patients with ic the sf-36 scale showed significant improvements in bodily pain ( p < .01 ) and in physical functioning ( p < .001 ) and for the patients with cli there were significant improvements in bodily pain ( p < .004 ) at the one - year follow - up ( figure 2 ) .
the nhp scale showed no significant improvements in patients with ic , while in patients with cli , significant improvements in pain ( p < .001 ) and physical mobility ( p <
changes in median score for the sf-36 in claudicants and critical ischaemia patients before and one year after revascularization . tested by wilcoxon signed ranks test .
bp = bodily pain , pf = physical functioning , mh = mental health , vt = vitality and sf = social functioning .
changes in median score for the nhp in claudicants and critical ischaemia patients before and one year after revascularization . tested by wilcoxon signed ranks test .
p = pain , pm = physical mobility , em = emotional reactions , e = energy and so = social isolation .
the result showed that the sf-36 was less skewed and more homogeneous with lower " floor " and " ceiling " effects than the nhp .
a considerable convergence in three of the five comparable domains one year postoperatively indicates an average convergent validity .
the sf-36 showed a higher internal consistency except for social functioning one - year postoperatively and was more responsive in detecting changes over time in the ic group .
the nhp was more sensitive in discriminating among levels of ischaemia regarding pain and more able to detect changes in the cli group .
the attrition or loss of subjects ( 27% ) in this study could have affected the outcome .
further analysis showed that there were no significant differences in quality of life , sex , age , method of treatment and severity of disease between the attrition group and those who completed the study .
the fact that the nhp and sf-36 differ in their nature and content may limit the study design . therefore the analyses in this study focused only on the comparable domains of the two instruments , including the basic domains of physical , mental and social health identified by the whoqol group . a suitable quality of life instrument for patients with chronic lower limb ischaemia should not only be valid , reliable and responsive but also simple for elderly people to understand and complete . in this study
there was no difference in response rate between the two instruments and both seemed to be user - friendly and took about 510 minutes to complete .
the findings strengthen earlier results suggesting that both scales are practical and acceptable to use in elderly patients .
a generic quality of life instrument , designed for a variety of populations and measuring a comprehensive set of health concepts , is likely to have problems with " ceiling " and " floor " effects . in this
study the nhp showed higher " ceiling " effects in all dimensions than the sf-36 .
there were minor " floor " effects in both the nhp and sf-36 , indicating the lowest possible quality of life .
this is in accordance to klevsgrd et al , who also showed higher " ceiling " effects in social isolation , emotional reactions and energy for the nhp than the sf-36 .
other studies have also reported fewer " ceiling " and " floor " effects in the sf-36 than in the nhp in patients with chronic obstructive pulmonary disease and after a myocardial infarction .
the advantage of the sf-36 may be due to its use of a likert - type response format with a number of possible different scores and its ability to detect positive as well negative states of health , whereas the nhp items are dichotomous and state more extreme ends of ill health .
this could mean that a patient with acceptable initial scores fails to improve even if the improvement is obvious .
furthermore , a false negative response will be more likely when a patient perceives to having perfect functioning on a measure that only assesses severe dysfunction .
the result confirms the importance of finding a quality of life instrument that does have a spectrum of dimensions which match the patients with chronic lower limb ischaemia related to the presence of numerous and often severe comorbid conditions . in this
study the internal consistency was not as high as desirable for any of the instruments before revascularization , but both instruments exceeded the minimum internal consistency value of 0.7 , except for social functioning in the sf-36 one year postoperatively .
several studies have previously reported that the sf-36 has higher cronbach 's values than the nhp , but the domains in which the highest and lowest values were estimated differ [ 37 - 40 ] .
the findings suggest that it is not only the magnitude of the correlation among items , but also the number of items in the scale that affects the internal consistency .
for example , the domains of pain and social functioning in the nhp contain 8 and 5 items respectively , while bodily pain and social functioning in the sf-36 contain only 2 items .
this is further strengthened by the fact that both the scales were not sensitive enough to identify significant within - patients changes in social isolation and social functioning .
another explanation could be that the patients were a more homogeneous group before treatment , with similar problems which affected the quality of life , but one year postoperatively they have become more heterogeneous and represent different states of recovery .
both instruments meet the reliability standards for group - level application in most respects , although none of them achieved the degree of reliability that be would be desirable in individual - based assessment . the result in this study showed significant convergent correlation coefficients between scores of the comparable dimensions except for physical activity and social activity , indicating a considerable convergence of the nhp and sf-36 scale .
prieto et al and meyer - rosberg et al demonstrated similar results with an average convergent validity .
thus the nhp and sf-36 are relatively equal in the validity and corroborate that the subscales probably reflect similar impacts of chronic lower limb ischaemia . however , social isolation in the nhp showed a higher correlation with mental health in the sf-36 and might measure more psychological aspects of social life , whilst social functioning in the sf-36 tends to assess social activities according to the higher correlation with energy in nhp .
similarly the physical functioning in the sf-36 showed a higher correlation with energy and may reflect physical activities of daily living rather than physical mobility .
this suggests that the sf-36 and nhp measure different aspects of physical and social activities .
validity in terms of the instruments ' relative ability to discriminate among different levels of the ischaemia could only demonstrate that patients with cli had significantly more problems with pain and physical mobility before treatment than patients with ic measured by the nhp .
klevsgrd et al showed similar results , that the nhp was more sensitive in discriminating deterioration in pain and physical mobility than the sf-36 .
in contrast , brown et al demonstrated that the sf-36 was more sensitive than the nhp for identifying people still troubled with angina or breathlessness after a myocardial infarction . despite the lack of significant differences between patients with ic and patients with cli
, the nhp scale tends to be more sensitive in explaining the quality of life in this group of patients with regard to the dimension of pain and physical mobility .
the important issue thus is to consider how well the measurement method explains health - related phenomena which are significant for the particular targeted disease or group of patients .
the sf-36 was the more responsive instrument in detecting changes in quality of life over time in patients with ic , including bodily pain and physical functioning one year postoperatively .
however , in patients with cli , the nhp was the more responsive instrument , with significant changes in pain and physical mobility , while the sf-36 showed a significant change only in bodily pain .
falcoz et al also demonstrated that the sf-36 was more responsive than the nhp in detecting changes five weeks after cardiac surgery .
in contrast , klevsgrd et al showed that the nhp was more responsive in patients with chronic lower limb ischaemia one month after revascularization .
the result of the present study supports the transatlantic inter - society consensus ( tasc ) recommendation that sf-36 should be used as a generic health outcome measure in patients with chronic lower limb ischaemia .
it seems to be more sensitive for detecting changes in quality of life than the nhp in patients with ic . in the group of cli patients who have more problems with mobility and pain ,
however , it is harder to evaluate whether the one questionnaire is superior to the other , the nhp could be a preferable instrument in this group of patients .
the findings indicate that both the sf-36 and the nhp have acceptable degrees of reliability for group - level comparisons , convergent and construct validity one year after revascularization .
nevertheless , the sf-36 seems generally to have more superior psychometric properties and was more suitable than the nhp for evaluating quality of life in patients with intermittent claudication .
the nhp , however , discriminated better among severity of ischaemia and was more responsive in detecting changes over time in patients with critical leg ischaemia .
this study was supported by the helge axon johnson foundation and the department of nursing science , lund university , the vrdal institute , the swedish institute for health sciences , department of surgery , sahlgrenska university hospital , gteborg , sweden . | backgrounddifferent generic quality of life instruments such as the nottingham health profile ( nhp ) and the short form 36 health survey ( sf-36 ) have revealed conflicting results in patients with chronic lower limb ischaemia in psychometric attributes in short - term evaluations .
the aim of this study was to compare the nhp and the sf-36 regarding internal consistency reliability , validity , responsiveness and suitability as outcome measures in patients with lower limb ischaemia in a longitudinal perspective.methods48 patients with intermittent claudication and 42 with critical ischaemia were included .
assessment was made before and one year after revascularization using comparable domains of the nhp and the sf-36 questionnaires.resultsthe sf-36 was less skewed and more homogeneous than the nhp .
there was an average convergent validity in three of the five comparable domains one year postoperatively .
the sf-36 showed a higher internal consistency except for social functioning one - year postoperatively and was more responsive in detecting changes over time in patients with intermittent claudication .
the nhp was more sensitive in discriminating among levels of ischaemia regarding pain and more able to detect changes in the critical ischaemia group.conclusionboth sf-36 and nhp have acceptable degrees of reliability for group - level comparisons , convergent and construct validity one year postoperatively .
nevertheless , the sf-36 has superior psychometric properties and was more suitable in patients with intermittent claudication .
the nhp however , discriminated better among severity of ischaemia and was more responsive in patients with critical ischaemia . | Background
Methods
Patients
Nottingham Health Profile
Short Form 36 Health Survey
Procedure
Statistical analysis
Results
Validity
Internal consistency
Responsiveness
Discussion
Conclusion
Acknowledgements | during the past few decades quality of life assessment has become one central outcome in treatment of patients with chronic lower limb ischaemia . different generic quality of life instruments such as the nottingham health profile ( nhp ) and the short form 36 health survey ( sf-36 ) have previously been tested , revealing conflicting results in these patients according to psychometric attributes in short - term evaluations . the strengths and weakness of the nhp and the sf-36 scales are not extensively examined and further research is needed to establish which is the more appropriate and responsive quality of life instrument for patients with chronic lower limb ischaemia in the long term . a majority of the patients are elderly and have generally widespread arterial disease with numbers of symptoms due to the chronic lower limb ischaemia , which may affect the patients ' quality of life [ 3 - 5 ] . physical and psychological health , social relationship perceptions , function and well - being which should be included in a generic quality of life instrument . the functional scale , walking impairment questionnaire ( wiq ) and quality of life instruments such as intermittent claudication questionnaire ( icq ) and claudication scale ( clau - s ) are examples of disease - specific instruments which have been developed in recent years for patients with ic
. however , at present there is no accepted disease - specific questionnaire for quality of life assessment in patients with cli . nevertheless , the transatlantic inter - society consensus ( tasc ) recommended that quality of life instruments should be used in all clinical trials and preferably include both generic and disease - specific quality of life measures . further , the requirement for a useful outcome measure is the responsiveness in detecting small but important clinical changes of quality of life in patients following vascular interventions . comparisons among quality of life instruments and their psychometric characteristics and performance are needed to provide recommendations about their usefulness as outcome measures for these particular groups of patients . the aim of this study was to compare two generic quality of life questionnaires , the nottingham health profile ( nhp ) and the short form 36 health survey ( sf-36 ) regarding the internal consistency reliability , validity , responsiveness and suitability as outcome measures in patients with lower limb ischaemia in a longitudinal perspective . the severity of ischaemia was graded according to suggested standards for grading lower limb ischaemia . routine medical history , risk factors and clinical examinations , which included ankle blood pressure ( abp ) and ankle - brachial pressure index ( abpi ) , were obtained before and one year after revascularization in accordance with the swedish vascular registry ( swedvasc ) . p - value = < 0.05 the nottingham health profile ( nhp ) was developed to be used in epidemiological studies of health and disease . the nhp scale has also proved capable of measuring changes in perceived health following different treatments such as radical surgery for colorectal cancer and after vascular interventions in lower limb ischaemia patients . the short form 36 health survey ( sf-36 ) was developed by ware et al and designed to provide assessments involving generic health concepts that are not specific to age , disease or treatment group . in this study ,
the sf-36 has shown acceptable validity and reliability in population studies and in various groups of patients , for example after stroke and in patients with rheumatoid arthritis . the sf-36 scale has also shown responsiveness to changes in health status over time in patients with critical ischaemia [ 30 - 32 ] and in patients with intermittent claudication following a revascularization [ 33 - 35 ] . the patients were asked by the head nurse to fill out the nhp and the sf-36 questionnaire during their admission before treatment . at the one - year follow - up , the questionnaire was sent home to the patients with a covering letter and a prepaid envelope . the prevalence of the lowest ( " floor " effect ) and highest ( " ceiling " effect ) possible quality of life score in nhp and sf-36 was also calculated . construct validity was evaluated for aspects of convergent and discriminant validity by the multitrait - multimethod matrix ( mtmm ) based on five comparable domains , including pain , physical mobility , emotional reactions , energy and social isolation for the nhp and bodily pain , physical functioning , mental health , vitality and social functioning for the sf-36 ( table 2 ) . further , the mann - whitney u - test was used to examine the relative ability of the two instruments to discriminate among the degrees of severity of the peripheral vascular disease . the internal consistency based on correlations between items for each scale
the recommended reliability standard for group - level comparisons is a reliability coefficient of 0.70 , while comparisons between individuals demands a reliability coefficient of 0.90 . comparable domains between the nottingham health profile ( nhp ) and the short form 36 ( sf-36 ) and number of items . the wilcoxon signed ranks test was used to detect the responsiveness of within - subjects changes over time , before and one year after revascularization , in patients with ic and cli . the severity of ischaemia was graded according to suggested standards for grading lower limb ischaemia . routine medical history , risk factors and clinical examinations , which included ankle blood pressure ( abp ) and ankle - brachial pressure index ( abpi ) , were obtained before and one year after revascularization in accordance with the swedish vascular registry ( swedvasc ) . the nottingham health profile ( nhp ) was developed to be used in epidemiological studies of health and disease . the nhp scale has also proved capable of measuring changes in perceived health following different treatments such as radical surgery for colorectal cancer and after vascular interventions in lower limb ischaemia patients . the short form 36 health survey ( sf-36 ) was developed by ware et al and designed to provide assessments involving generic health concepts that are not specific to age , disease or treatment group . in this study ,
the sf-36 has shown acceptable validity and reliability in population studies and in various groups of patients , for example after stroke and in patients with rheumatoid arthritis . the sf-36 scale has also shown responsiveness to changes in health status over time in patients with critical ischaemia [ 30 - 32 ] and in patients with intermittent claudication following a revascularization [ 33 - 35 ] . the prevalence of the lowest ( " floor " effect ) and highest ( " ceiling " effect ) possible quality of life score in nhp and sf-36 was also calculated . construct validity was evaluated for aspects of convergent and discriminant validity by the multitrait - multimethod matrix ( mtmm ) based on five comparable domains , including pain , physical mobility , emotional reactions , energy and social isolation for the nhp and bodily pain , physical functioning , mental health , vitality and social functioning for the sf-36 ( table 2 ) . the internal consistency based on correlations between items for each scale
the recommended reliability standard for group - level comparisons is a reliability coefficient of 0.70 , while comparisons between individuals demands a reliability coefficient of 0.90 . comparable domains between the nottingham health profile ( nhp ) and the short form 36 ( sf-36 ) and number of items . the wilcoxon signed ranks test was used to detect the responsiveness of within - subjects changes over time , before and one year after revascularization , in patients with ic and cli . analyses between the comparable domains showed that the nhp scores were more skewed than the sf-36 scores , especially in emotional reactions , energy and social isolation ( figure 1 ) . the " floor effect " , the proportion of individuals having the lowest possible scores ( sf-36 = 0 , nhp = 100 ) , was larger for the nhp scale in energy one year ( 19.7% ) after revascularization than for the sf-36 . the " ceiling effect " , the proportion of individuals having the best possible scores ( sf-36 = 100 , nhp = 0 ) , was also larger for the nhp scale in emotional reactions ( 50.0% ) , energy ( 42.4% ) and social isolation ( 71.2% ) one year after revascularization ( table 3 ) . frequency distribution of scores on the nhp ( left side ) and comparable dimensions on the sf-36 ( right side ) one year after revascularization . nhp scores had 100 subtracted for consistency with sf-36 reliability and " floor " and " ceiling " effects in comparable nhp and sf-36 scales before and one year after revascularization . cronbach 's the average convergent validity coefficients exceeded 0.5 one year postoperatively except for physical mobility and physical functioning ( r = -0.46 ) and for social isolation and social functioning ( r = -0.32 ) , indicating a considerable convergence of the sf-36 and nhp ( table 4 ) . one year postoperatively significant correlations between abpi and physical functioning ( r = 0.29 ) ( sf-36 ) , physical mobility ( r = 0.42 ) and pain ( r = 0.42 ) ( nhp ) were found . the severity of the ischaemia had a significant influence in the nhp - measured domain of pain ( p < .003 ) and physical mobility ( p < .03 ) , indicating lower quality of life scores in patients with critical ischaemia . in the ability to discriminate between levels of ischaemia in the other comparable quality of life domains ,
multitrait - multimethod matrix of correlation coefficient for the comparable scales of the sf-36 and nhp in patients with varying degrees of lower limb ischaemia one year after revascularization . differences in comparable domains in the nhp and the sf-36 between claudicants and patients with critical ischaemia before revascularization . p - value = < 0.05 physical functioning ( = 0.82 ) , mental health ( = 0.76 ) and vitality ( = 0.70 ) for the sf-36 and pain ( = 0.71 ) , emotional reactions ( = 0.76 ) and energy ( = 0.71 ) for the nhp scale were reliable , with coefficients > 0.70 before revascularization . for the sf-36 , all of the comparable domains except for social functioning ( = 0.64 ) exceeded the cronbach 's alpha value of 0.8 at the one - year follow - up . in patients with ic
the sf-36 scale showed significant improvements in bodily pain ( p < .01 ) and in physical functioning ( p < .001 ) and for the patients with cli there were significant improvements in bodily pain ( p < .004 ) at the one - year follow - up ( figure 2 ) . the nhp scale showed no significant improvements in patients with ic , while in patients with cli , significant improvements in pain ( p < .001 ) and physical mobility ( p <
changes in median score for the sf-36 in claudicants and critical ischaemia patients before and one year after revascularization . changes in median score for the nhp in claudicants and critical ischaemia patients before and one year after revascularization . the average convergent validity coefficients exceeded 0.5 one year postoperatively except for physical mobility and physical functioning ( r = -0.46 ) and for social isolation and social functioning ( r = -0.32 ) , indicating a considerable convergence of the sf-36 and nhp ( table 4 ) . one year postoperatively significant correlations between abpi and physical functioning ( r = 0.29 ) ( sf-36 ) , physical mobility ( r = 0.42 ) and pain ( r = 0.42 ) ( nhp ) were found . the severity of the ischaemia had a significant influence in the nhp - measured domain of pain ( p < .003 ) and physical mobility ( p < .03 ) , indicating lower quality of life scores in patients with critical ischaemia . in the ability to discriminate between levels of ischaemia in the other comparable quality of life domains ,
multitrait - multimethod matrix of correlation coefficient for the comparable scales of the sf-36 and nhp in patients with varying degrees of lower limb ischaemia one year after revascularization . differences in comparable domains in the nhp and the sf-36 between claudicants and patients with critical ischaemia before revascularization . physical functioning ( = 0.82 ) , mental health ( = 0.76 ) and vitality ( = 0.70 ) for the sf-36 and pain ( = 0.71 ) , emotional reactions ( = 0.76 ) and energy ( = 0.71 ) for the nhp scale were reliable , with coefficients > 0.70 before revascularization . for the sf-36 , all of the comparable domains except for social functioning ( = 0.64 ) exceeded the cronbach 's alpha value of 0.8 at the one - year follow - up . in patients with ic the sf-36 scale showed significant improvements in bodily pain ( p < .01 ) and in physical functioning ( p < .001 ) and for the patients with cli there were significant improvements in bodily pain ( p < .004 ) at the one - year follow - up ( figure 2 ) . the nhp scale showed no significant improvements in patients with ic , while in patients with cli , significant improvements in pain ( p < .001 ) and physical mobility ( p <
changes in median score for the sf-36 in claudicants and critical ischaemia patients before and one year after revascularization . changes in median score for the nhp in claudicants and critical ischaemia patients before and one year after revascularization . the result showed that the sf-36 was less skewed and more homogeneous with lower " floor " and " ceiling " effects than the nhp . a considerable convergence in three of the five comparable domains one year postoperatively indicates an average convergent validity . the sf-36 showed a higher internal consistency except for social functioning one - year postoperatively and was more responsive in detecting changes over time in the ic group . the nhp was more sensitive in discriminating among levels of ischaemia regarding pain and more able to detect changes in the cli group . therefore the analyses in this study focused only on the comparable domains of the two instruments , including the basic domains of physical , mental and social health identified by the whoqol group . a suitable quality of life instrument for patients with chronic lower limb ischaemia should not only be valid , reliable and responsive but also simple for elderly people to understand and complete . in this
study the nhp showed higher " ceiling " effects in all dimensions than the sf-36 . there were minor " floor " effects in both the nhp and sf-36 , indicating the lowest possible quality of life . other studies have also reported fewer " ceiling " and " floor " effects in the sf-36 than in the nhp in patients with chronic obstructive pulmonary disease and after a myocardial infarction . the result confirms the importance of finding a quality of life instrument that does have a spectrum of dimensions which match the patients with chronic lower limb ischaemia related to the presence of numerous and often severe comorbid conditions . in this
study the internal consistency was not as high as desirable for any of the instruments before revascularization , but both instruments exceeded the minimum internal consistency value of 0.7 , except for social functioning in the sf-36 one year postoperatively . for example , the domains of pain and social functioning in the nhp contain 8 and 5 items respectively , while bodily pain and social functioning in the sf-36 contain only 2 items . another explanation could be that the patients were a more homogeneous group before treatment , with similar problems which affected the quality of life , but one year postoperatively they have become more heterogeneous and represent different states of recovery . both instruments meet the reliability standards for group - level application in most respects , although none of them achieved the degree of reliability that be would be desirable in individual - based assessment . the result in this study showed significant convergent correlation coefficients between scores of the comparable dimensions except for physical activity and social activity , indicating a considerable convergence of the nhp and sf-36 scale . thus the nhp and sf-36 are relatively equal in the validity and corroborate that the subscales probably reflect similar impacts of chronic lower limb ischaemia . however , social isolation in the nhp showed a higher correlation with mental health in the sf-36 and might measure more psychological aspects of social life , whilst social functioning in the sf-36 tends to assess social activities according to the higher correlation with energy in nhp . similarly the physical functioning in the sf-36 showed a higher correlation with energy and may reflect physical activities of daily living rather than physical mobility . validity in terms of the instruments ' relative ability to discriminate among different levels of the ischaemia could only demonstrate that patients with cli had significantly more problems with pain and physical mobility before treatment than patients with ic measured by the nhp . klevsgrd et al showed similar results , that the nhp was more sensitive in discriminating deterioration in pain and physical mobility than the sf-36 . in contrast , brown et al demonstrated that the sf-36 was more sensitive than the nhp for identifying people still troubled with angina or breathlessness after a myocardial infarction . despite the lack of significant differences between patients with ic and patients with cli
, the nhp scale tends to be more sensitive in explaining the quality of life in this group of patients with regard to the dimension of pain and physical mobility . the sf-36 was the more responsive instrument in detecting changes in quality of life over time in patients with ic , including bodily pain and physical functioning one year postoperatively . however , in patients with cli , the nhp was the more responsive instrument , with significant changes in pain and physical mobility , while the sf-36 showed a significant change only in bodily pain . falcoz et al also demonstrated that the sf-36 was more responsive than the nhp in detecting changes five weeks after cardiac surgery . in contrast , klevsgrd et al showed that the nhp was more responsive in patients with chronic lower limb ischaemia one month after revascularization . the result of the present study supports the transatlantic inter - society consensus ( tasc ) recommendation that sf-36 should be used as a generic health outcome measure in patients with chronic lower limb ischaemia . it seems to be more sensitive for detecting changes in quality of life than the nhp in patients with ic . the findings indicate that both the sf-36 and the nhp have acceptable degrees of reliability for group - level comparisons , convergent and construct validity one year after revascularization . nevertheless , the sf-36 seems generally to have more superior psychometric properties and was more suitable than the nhp for evaluating quality of life in patients with intermittent claudication . the nhp , however , discriminated better among severity of ischaemia and was more responsive in detecting changes over time in patients with critical leg ischaemia . this study was supported by the helge axon johnson foundation and the department of nursing science , lund university , the vrdal institute , the swedish institute for health sciences , department of surgery , sahlgrenska university hospital , gteborg , sweden . | [
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] |
thyroid cancer , although being a relatively rare tumour , represents the most frequent endocrine malignancy .
according to globocan 2012 estimates , it accounts for 2.1% of total new cancer cases and 0.5% of total cancer related death . in india too , there is a significant burden of thyroid diseases with an estimated incidence of thyroid cancer as 1.4% of all new cancer diagnosed with 0.5% mortality rate .
it has been estimated that about forty - two million people in india suffer from thyroid diseases .
the major types of follicular cell derived thyroid cancer include papillary thyroid cancer ( ptc ) , follicular thyroid cancer ( ftc ) , and anaplastic thyroid cancer ( atc ) , while medullary thyroid cancer ( mtc ) is the parafollicular cell derived thyroid cancer . amongst the four histological types of thyroid cancer , ptc and ftc are the differentiated thyroid carcinomas arising from the follicular cells . during the last decades ,
a rising incidence of thyroid cancer has been noted specifically for ptc , which is the most frequent type , accounting for about 85% of all types of thyroid cancer [ 2 , 3 ] .
the literature repeatedly reports the association between the thyroid cancer and a history of benign diseases . also accumulating evidences
indicate that follicular cell derived thyroid cancer constitutes a biological continuum progressing from the highly curable well - differentiated thyroid cancer to the universally fatal anaplastic thyroid cancer .
although thyroid problems can be readily diagnosed using histologic criteria , very often the pathologist has to face up to thyroid lesions in which the distinction between benign and malignant can be quite subtle . as a result , the decision favouring one or another has clinical consequences and implies different treatment modalities .
it implies that , on one hand , there is a need to avoid excessive treatment and psychological discomfort to the patient who has benign thyroid disease or is in the initial stage of differentiated thyroid cancer and , on the other hand , patients with aggressive disease need to be guaranteed effective management right at the initial stage of the disease when it is still curable .
hence , in order to differentiate benign from malignant tumours and in the latter group to distinguish indolent / low risk tumours from aggressive high risk tumours , it is important to decipher the molecular mechanisms underlying thyroid tumourigenesis .
cytokines are the key mediators of inflammation , which is now being recognized as one of the hallmarks of cancer .
tumour necrosis factor - alpha ( tnf- ) is a 17 kda cytokine identified in the late 1970s and is primarily produced by activated macrophages , t lymphocytes , and nk cells .
extensive research so far has revealed various roles of tnf- such as in body development and immunity and in pathological responses such as inflammation , tumour growth , transplant rejection , rheumatoid arthritis , and septic shock .
although tnf- was first identified as a soluble factor capable of inducing tumour necrosis , various mechanisms have been described by which tnf- may promote cancer growth , invasion , and metastasis .
collective evidence has shown that tnf- is a key mediator of inflammation and cancer [ 8 , 9 ] .
constitutive production of tnf- from the tumour microenvironment is a feature of many malignant tumours and its presence is associated with poor prognosis . at cellular level , tnf- exerts its effects through its receptors to activate distinct signalling pathways that regulate cell survival , proliferation , or death .
, it exerts its anticancer property mainly through inducing cancer cell death , a process that could be used for cancer therapy while , on the other hand , it stimulates proliferation , survival , migration , and angiogenesis in most cancer cells that are resistant to tnf- induced cytotoxicity , resulting in tumour promotion .
it also activates vascular endothelial cells and causes endothelial cells to express adhesion molecules for neutrophils , monocytes , and lymphocytes .
have stated that changes in the expression or function of the cell adhesion molecules have been implicated in all steps of tumour progression , including detachment of tumour cells from primary site , intravasation into the blood stream , extravasation into distant target organs , and formation of secondary lesions . amongst the five families of adhesion molecules including the cadherins , integrins , selectins , immunoglobulins , and cd44 molecules , leukocyte- ( l- ) selectin is a member of the selectin family and vascular cell adhesion molecule-1 ( vcam-1 ) belongs to the immunoglobulin superfamily .
l - selectin , also called cd62l , promotes trafficking through binding interactions with carbohydrate ligands on high endothelial venules in lymph nodes or on activated endothelium at sites of inflammation . in a similar way
as they facilitate leukocyte arrest in the vasculature and migration to inflamed tissues , the selectins can also mediate tumour cell extravasation and metastasis .
in fact , the tumour cells express functional ligands of selectins and interact with selectins expressed on blood vessel walls [ 14 , 15 ] .
vcam-1 , also called cd106 , is a 90 kda glycoprotein , characterized by the presence of seven immunoglobulin domains .
the presence of vcam-1 has been demonstrated on endothelial cells , macrophages , dendritic cells , and surface of cancer cells .
the cell - cell adhesion leads to clustering of vcam-1 and the cytoplasmic domain of this clustered vcam-1 particularly activates ezrin which in turn activates the pi3k / akt signaling that suppresses apoptosis and promotes survival signal in the tumour cells .
further , the oncomine database , consisting of datasets derived from various microarray studies , provides evidence that vcam-1 is significantly upregulated in various cancer types including brain , breast , ovarian , and esophageal carcinomas .
hence , this study aimed to determine the circulating and tumoural protein expression of tnf- and the adhesion molecules : l - selectin and vcam-1 in primary tumours of ptc patients in relation to their expression in patients with benign thyroid diseases .
total 150 untreated patients with pathologically confirmed benign thyroid diseases ( n = 67 ) and papillary thyroid cancer ( n = 83 ) , enrolled between 2008 and 2012 at our institute , were included in this study .
none of the patients suffered from any autoimmune disease previously nor were they taking any immunosuppressive or immunomodulant drugs .
the patients with benign thyroid diseases comprised those diagnosed with having nodular goitre , multinodular goitre , colloid goitre , adenomatoid goitre , follicular adenoma , hrthle cell adenoma , and adenohyperplasia . amongst the 67 benign thyroid disease patients , 45 patients underwent surgery at our institute .
the thyroid cancer patients were staged according to the ajcc / uicc tnm staging system . as in this staging system , patients are staged on the basis of their age ( < 45/45 years ) ; the patients were grouped into younger ( < 45 years ) and elder ( 45 years ) age groups .
the ptc patients were followed for a period of 4 years or until death within that period . for overall survival ( os ) analysis ,
complete follow - up details were obtained in 92% ( 76/83 ) ptc patients . amongst them , 9% ( 7/76 ) patients had persistent disease and hence were not included for the disease - free survival ( dfs ) analysis .
informed consent was obtained from all patients prior to sample collection and the study was approved by institutional scientific and ethical committees .
pretherapeutic blood samples were collected from all patients as well as from 67 healthy individuals to detect the circulating levels of tnf- , l - selectin , and vcam-1 .
paraffin embedded tissue blocks of all the patients ( who underwent surgery ) were retrieved from the histopathology department of our institute .
the clinical and histopathological details of the patients were noted from the case files maintained at the medical record department of the institute .
the circulating levels of the tnf- , vcam-1 , and l - selectin were estimated from the serum samples using specific commercially available elisa kits ( tnf- : krishgen biosystems , l - selectin : abcam , and vcam-1 : invitrogen ) .
all standards and samples were run in duplicate and samples with concentration above the highest standard concentration were run after dilution .
the mean absorbance of each set of duplicate standards and samples was determined and absorbance of zero standard was subtracted from it .
a standard curve was plotted in graphpad prism 5 software with concentration of standards on x - axis and absorbance on y - axis .
the concentrations of the diluted samples were multiplied by the dilution factor to determine the actual concentration .
immunohistochemical staining was performed for detection of tumoural expression tnf- , l - selectin , and vcam-1 in primary tumours of ptc patients and in patients with benign thyroid diseases . briefly
, 35 m thick sections were cut from the formalin fixed paraffin embedded tissue blocks using leica microtome and mounted on apes coated glass slides .
the immunohistochemical staining was carried out using mach4 universal hrp - polymer detection system from biocare medicals , usa , as per the manufacturer 's protocol recommendations .
rat monoclonal antibody for tnf- ( abd serotec , mca1560 ; 1 : 20 ) , rabbit polyclonal antibody for l - selectin ( abcam , ab135792 ; 1 : 50 ) , and mouse monoclonal antibody for vcam-1 ( santa cruz biotechnology , inc .
antigenicity was retrieved by heating the sections in 10 mm sodium citrate buffer ( ph , 6.0 ) for 20 mins in a pressure cooker prior to application of the respective primary antibodies .
a semiquantitative immunoreactive score ( irs ) method of remmele and stegner based on staining positivity and staining intensity was implemented .
staining positivity was scored as 0 for no stained cells , 1 for staining in 1% to 10% of cells , 2 for staining in 11% to 50% of cells , 3 for staining in 50% to 80% of cells , and 4 for staining in > 80% of cells .
the staining intensity was scored as 0 for no staining , 1 for weak / faint staining , 2 for moderate staining , and 3 for intense / dark staining .
the irs score was then obtained by multiplying the staining positivity and the staining intensity and , therefore , theoretically the scores could range from 0 to 12 . for statistical evaluation , the median irs of each molecule in the two subgroups of patients was used as cut - off value to divide the patients into low ( median irs ) and high ( > median irs ) expression groups , respectively .
the data were analyzed statistically using the statistical package for social sciences ( spss ) software version 16 ( spss inc .
independent samples t - test was used to compare the means of circulating levels of analytes between two groups of subjects and also to assess the association of the analytes with the clinicopathological parameters of thyroid cancer patients .
receiver 's operating characteristic ( roc ) curves were constructed to determine the discriminating efficacy of the circulating markers between healthy individuals and patients .
two - tailed test was used to compare the tumoural protein expressions in benign and carcinoma patients and also to determine the association between protein expression and clinicopathological parameters of carcinoma patients . in case of less than five patients in the cells of 2 2 tables ,
yate 's continuity correction value along with its two - tailed significance was taken into consideration .
correlation between two parameters was calculated using spearman 's correlation coefficient ( r ) method .
univariate survival analysis was evaluated using kaplan - meier method and log rank test was used to analyze difference in survival curves and to assess the prognostic significance of dfs and os .
the circulating levels ( mean standard error , m se ) of tnf- , l - selectin , and vcam-1 in healthy individuals and patients with benign thyroid diseases and ptc are depicted in table 2 . it was observed that the circulating levels of tnf- , l - selectin , and vcam-1 were significantly higher in patients with benign thyroid diseases , as compared to that in healthy individuals ( p < 0.001 for each ) .
further , the levels of tnf- and l - selectin , but not vcam-1 , were found to be significantly higher in ptc patients as compared to those in patients with benign thyroid diseases ( tnf- : p = 0.009 , l - selectin : p < 0.001 , and vcam-1 : p = 0.912 ) .
further , the roc curves were generated to reveal the efficacy of these significantly elevated serum cytokine levels in order to differentiate the healthy individuals and patients with different thyroid diseases .
the results demonstrated that tnf- , l - selectin , and vcam-1 exhibited a good discriminatory efficacy between healthy individuals and patients with benign thyroid diseases ( figure 1 ) as well as between healthy individuals and ptc patients ( figure 2 ) .
moreover , tnf- ( auc = 0.598 , p = 0.040 ) and l - selectin ( auc = 0.692 , p
< 0.001 ) but not vcam-1 ( auc = 0.513 , p = 0.788 ) showed good power to discriminate between patients with benign thyroid diseases and ptc ( figure 3 ) .
the correlation of circulating levels of tnf- , l - selectin , and vcam-1 with the clinicopathological parameters of ptc patients has been depicted in table 3 .
although the circulating tnf- levels did not show significant correlation with any of the clinicopathological parameters , a trend of correlation was observed with calcification and extrathyroidal extension of tumours .
the circulating levels of l - selectin were significantly higher in ptc patients whose tumours showed haemorrhagic area as compared to those in whom haemorrhagic area was absent ( p = 0.010 ) and vcam-1 levels were significantly higher in patients having bilateral tumours than those with unilateral tumours ( p = 0.037 ) .
further , no substantial interaction of serum levels of the two adhesion molecules was observed with the remaining clinicopathological parameters ( table 3 ) .
the expression of tnf- and vcam-1 was observed in the cytoplasm of the thyroid follicles , while cytoplasmic and membranous staining was observed for l - selectin .
tnf- expression was observed in 67% ( 30/45 ) of patients with benign thyroid diseases with median irs-3 and in 89% ( 74/83 ) of ptc patients and irs-3 emerged as the median score .
l - selectin immunoreactivity was observed in 82% ( 37/45 ) of benign thyroid disease patients , and the median score was irs-6 .
further , 51% ( 23/45 ) of patients with benign thyroid diseases showed expression for vcam-1 having irs-1 as the median score . on the other hand , 99% ( 82/83 ) of the ptc patients
exhibited l - selectin expression and the irs-9 was observed as the median score and vcam-1 expression was positive in 84% ( 70/83 ) of the tumours with median score as irs-2 .
the immunoreactivity of tnf- and l - selectin was found to be significantly high in ptc patients as compared to the benign thyroid disease patients ( tnf- : = 7.657 , r = + 0.245 , p = 0.005 and l - selectin : = 5.276 , r = + 0.203 , p = 0.022 ) ( table 4 , figure 5 ) . on the other hand ,
the incidence of expression of vcam-1 in ptc patients was not significantly different from that in the benign thyroid disease patients ( = 1.831 , r = + 0.120 , p = 0.179 ) ( table 4 ) .
moreover , when correlated with the clinicopathological parameters of ptc patients , the tnf- expression was significantly positively correlated only with presence of calcification ( = 5.706 , r = + 0.262 , p = 0.017 ) .
further , significant positive correlation of l - selectin expression in ptc patients with larger tumour size ( t3 + t4 ) ( = 7.955 , r = + 0.310 , p = 0.004 ) and presence of extrathyroidal extension of tumours ( = 12.120 , r = + 0.382 ,
vcam-1 expression showed a significantly positive correlation with larger tumour size ( t3 + t4 ) ( = 6.219 , r = + 0.274 , p = 0.012 ) , presence of lymph node metastasis ( = 3.971 , r = + 0.219 , p = 0.047 ) , and extrathyroidal extension of tumours ( = 11.350 , r = + 0.370 , p = 0.001 ) as compared to their respective counterparts . on the other hand ,
a significant inverse correlation of vcam-1 expression was seen with the presence of multifocality of tumours ( = 4.040 , r = 0.221 , p = 0.045 ) .
apart from these , expression of none of the two adhesion molecules exhibited a significant association with the rest of the clinicopathological parameters ( table 5 ) .
a significant inverse correlation was observed in the circulating tnf- levels and tnf- protein expression in the primary tumour tissues of ptc patients ( r = 0.254 , p = 0.021 ) . however
, statistical data analysis did not express any significant correlation between the circulating adhesion molecules and their respective protein expressions in ptc patients .
further , the circulating levels of tnf- showed a significant positive correlation with the circulating levels of l - selectin ( r = + 0.303 , p = 0.005 ) , while the protein expression of tnf- ( r = + 0.347 , p = 0.001 ) showed a significant positive correlation with vcam-1 expression in the primary tumours of ptc patients ( table 6 ) . in univariate survival analysis ,
the circulating levels of none of the studied parameters could predict dfs in ptc patients ( table 7 ) .
further , log rank test indicated that besides age , gender , metastasis , stage , and multifocality , serum tnf- ( log rank = 5.12 , df = 1 , p = 0.024 ) was a significant prognosticator for os in ptc patients ( table 8) .
the kaplan - meier survival curve indicated significantly reduced os in ptc patients having higher levels of serum tnf- than those having lower circulating tnf- levels .
the event of death was significantly higher in ptc patients with high serum tnf- levels ( 18% , 7/38 ) than in patients with low serum tnf- levels ( 3% , 1/38 ) ( log rank = 5.129 , df = 1 , p = 0.024 ) ( figure 6 ) .
moreover , the circulating levels of l - selectin and vcam-1 were not able to predict os in the ptc patients .
the tumoral expression of none of the studied proteins was able to predict dfs or os , in the ptc patients .
the kaplan - meier survival analysis demonstrated higher incidence of disease relapse in patients with high vcam-1 expression ( 18% , 5/27 ) as compared to those with low vcam-1 expression ( 5% , 2/42 ) .
however , this difference was not statistically significant ( log rank = 3.415 , df = 1 , p = 0.065 ) ( table 7 , figure 7 ) . moreover , when ptc patients were subgrouped according to the clinicopathological variables , the kaplan - meier survival curves demonstrated that high expression of vcam-1 was remarkably associated with poor dfs in female patients and in the ptc patients postoperatively treated with ria therapy and/or rt .
it was observed that 12% ( 2/17 ) of the female ptc patients with high vcam-1 expression had significantly reduced dfs , whereas none of the female patients with lower vcam-1 expression had developed recurrence and/or distant metastasis during the follow - up period ( log rank = 3.881 , df = 1 , p = 0.049 ) ( figure 8) and 34% ( 5/19 ) of ptc patients treated with surgery followed by ria therapy and/or rt having high vcam-1 expression had reduced dfs in comparison to only 4% ( 1/27 ) of these patients with lower vcam-1 expression ( log rank = 4.760 , df = 1 , p = 0.029 ) ( figure 9 ) . on the other hand , the kaplan - meier survival analysis and the log rank test revealed that neither tnf- nor l - selectin expression emerged as significant prognosticators to predict dfs or os even when the ptc patients were grouped according to the clinicopathological features .
the current study revealed that serum tnf- was significantly higher in patients with benign thyroid diseases and in ptc patients , as compared to the healthy individuals .
roc curves also validated that tnf- was a potential marker for distinguishing patients with thyroid diseases from the healthy subjects . as compared to the patients with benign thyroid diseases , the circulating levels as well as the tumoural tissue expression of tnf-
the serum tnf- did not show significant correlation with any of the clinicopathological parameters , nor was it associated with dfs in these patients .
however , kaplan - meier survival analysis revealed that its elevated levels were significantly associated with shorter os in the ptc patients .
but in the multivariate survival analysis , as compared to the clinicopathological prognosticators , it lost its significance as an independent predictor of survival in ptc patients .
results of various other studies also show that , relative to normal healthy controls , a significant increase in circulating levels of tnf- was observed in hepatocellular carcinoma patients , cervical neoplasia [ 22 , 23 ] , epithelial ovarian cancer [ 24 , 25 ] , prostate cancer , and renal cell carcinoma . in concordance to our observation , high serum tnf- level has been associated with poor prognosis having reduced survival in prostate cancer and epithelial ovarian cancer .
also observed significantly higher plasma tnf- concentration in patients with chronic lymphocytic leukemia than in healthy control population and the elevated circulating tnf- correlated with the extent of disease and was suggested to be a novel prognostic factor for survival in patients with chronic lymphocytic leukemia .
observed that patients with thyroid diseases tended to have lower tnf- levels than the normal reference group , although the difference was not statistically significant and lumachi et al .
have reported that tnf- acts as a growth inhibitor of papillary thyroid cancer cell lines .
have reported that mean levels of tnf- were also significantly reduced in pretherapeutic serum samples of patients with bladder cancer as compared to the controls , whereas gendek - kubiak et al
. showed that the average tnf- levels measured in neoplastic patients were not significantly different from that observed in the control group .
differences in results of the reported studies may be related to the difference in the number of patients studied and in their distinct clinical and pathological characteristics .
further , in the existing study , expression of tnf- was localized in the cytoplasm and was found to be significantly higher in the primary tumours of ptc patients than that observed in patients with benign thyroid diseases . the tnf- expression was significantly positively correlated only with the presence of calcification in the tumours indicating that tnf- is likely to participate in the development of calcification in the tumours . moreover ,
tumoural protein expression of tnf- was not able to predict dfs or os in the ptc patients .
in accordance with the present study , cytoplasmic immunoexpression of tnf- has also been reported by de miguel et al . in epithelial cells of prostate cancer which were increased in comparison to normal prostate tissues .
higher expressions of tnf- have been reported in various tumour tissues [ 22 , 24 , 28 , 35 , 36 ] .
tnf- expression and its action have also been reported in esophageal cancer , ovarian cancer [ 38 , 39 ] , breast cancer , and follicular thyroid cancer as well .
the present study also found a significant inverse correlation between the circulating levels and tumoural protein expression of tnf-. this indicates that the circulating tnf- level was lower in patients having high tumoural expression of this protein and vice versa .
firstly , the absence of shedding - off of the protein overexpressed by tumour cells into the circulation and secondly the higher levels in circulation may be due to stimulation of its production by the immune cells in response to the tumourigenic condition .
thus , the current findings suggest that tnf- could be used as an indicator of thyroid cancer risk from benign conditions and may have a role in development of tumour and prognosis of thyroid cancer patients . the pretherapeutic circulating levels of both the adhesion molecules l - selectin and vcam-1 were significantly higher in patients with the benign disease as well as ptc patients than in the healthy individuals . these results
were further confirmed with roc curve analysis , where both l - selectin and vcam-1 could significantly discriminate patients with benign thyroid diseases and ptc from the healthy individuals .
however , circulating levels of only l - selectin , and not of vcam-1 , were significantly elevated in the ptc patients when compared with the benign thyroid disease patients .
similar to these , the results of roc curves also authenticated the efficacy of l - selectin to distinguish the ptc patients from those with benign thyroid diseases .
such results confirm essential roles of these adhesion molecules in the development of thyroid tumour . in accordance with our findings ,
studies by other authors have also reported significantly higher serum l - selectin levels in patients with ovarian cancer , small - cell and non - small - cell lung cancer , chronic myelogenic leukemia , and acute myeloid leukemia than those in healthy control groups . moreover , a study by chen et al
. also demonstrated markedly higher serum levels of l - selectin in patients with lung cancer or benign diseases than in the healthy controls .
they have also reported significant differences in levels of l - selectin between the lung cancer patients with advanced stage disease or with metastasis and those with early stage disease or with no metastasis .
the serum l - selectin levels were closely related to disease progression in ovarian and liver cancers . in a recent study ,
circulating l - selectin levels were also found to be higher in serum samples from patients with high grade metastatic versus high grade nonmetastatic muscle invasive bladder cancer . moreover , as concerned with the levels of serum vcam-1 , in a study by pasieka et al .
, significantly higher levels of vcam-1 were observed in the serum samples of only anaplastic thyroid carcinoma patients , while in ptc patients the circulating vcam-1 levels were comparable with those of healthy control group .
the increased levels of vcam-1 in the peripheral blood have also been demonstrated in progression of many forms of cancer : non - small - cell lung cancer , breast cancer [ 17 , 51 ] , rectal cancer , gastric cancer [ 53 , 54 ] , colorectal cancer [ 5559 ] , prostate cancer , bladder cancer , urological malignancies , head and neck cancer , pancreatic cancer , and ovarian cancer .
recently , martinez et al . have reported higher levels of vcam-1 in the bone marrow of advanced breast cancer patients than the healthy volunteers . however , tas et al .
also did not find significant difference in the levels of vcam-1 between epithelial ovarian cancer patients and control group . circulating vcam-1 levels
coskun et al . observed that serum vcam-1 level correlated with tumour stage in bladder cancer patients and was higher in patients with muscle invasive tumours than those with superficial tumours .
alexiou et al . have demonstrated that circulating vcam-1 was significantly associated with disease stage , gastric wall invasion , lymph node involvement , and distant metastasis in gastric cancer patients .
moreover , in colorectal cancer , it was to be associated with dukes ' d stage and distant metastasis .
however , in the present study , serum l - selectin and vcam-1 were predominantly positively correlated only with presence of haemorrhagic area and bilaterality of the tumours , respectively , while they were not significantly associated with the rest of the studied clinicopathological parameters in ptc patients .
this may be because , in thyroid carcinoma , the risk of disease relapse and occurrence of metastasis is relatively very low as compared to that in other malignancies arising from other organs .
further , only l - selectin and not vcam-1 was significantly overexpressed in the primary tumours of ptc patients when compared to that in patients with benign thyroid diseases .
moreover , l - selectin immunoreactivity predominantly correlated with larger tumour size and extrathyroidal extension of tumours .
similarly , protein expression of l - selectin has also been found to be significantly higher in the primary tumours of oral squamous cell carcinoma and salivary gland tumours .
further , in oral squamous cell carcinoma , it was associated with differentiation , tnm stages , and lymph node metastasis .
also in colorectal carcinoma , the l - selectin overexpression was observed , which was closely associated with the development of the disease and metastasis .
miao et al . also suggested that l - selectin expression increases in sentinel lymph node metastasis positive breast cancer and that it plays an important role in lymphatic chemotactic metastasis of breast cancer .
moreover , significant attenuation of metastasis was observed in the absence of l - selectin , indicating that this adhesion molecule actively contributes to leukocyte recruitment and formation of a metastatic niche [ 71 , 72 ] .
recently , higher expression of l - selectin in high grade muscle invasive bladder cancer ( mibc ) specimens versus low - grade bladder cancer ( lgbc ) specimens was observed by choudhary et al . .
further , they observed that l - selectin localization was seen in foci of metastatic tumour cells in lymph node specimens from patients with high grade mibc and known nodal involvement .
although not significant , the incidence of vcam-1 expression was higher in the thyroid cancer patients as compared to the patients with benign thyroid diseases . it has been suggested that proteolytic shedding of vcam-1 generates its soluble form which can be detected in circulation [ 73 , 74 ] . in thyroid carcinoma also
, there might be shedding of vcam-1 from the thyroid cancer cells into circulation which may be the factor accounting for the significantly elevated serum levels of vcam-1 .
further , similar to the present study , huang et al . and wang et al
. have observed cytoplasmic staining of vcam-1 in ovarian cancer and breast cancer patients , respectively .
however , vcam-1 expression has been found to be overexpressed in primary tumours of many human cancers : ovarian cancer , pancreatic cancer , gastric cancer , oral squamous cell carcinoma , and clear cell renal carcinoma as well as papillary renal cell carcinoma as compared to the corresponding noncancerous tissues .
further , in the present study , vcam-1 expression was significantly overexpressed in the ptc patients with larger tumour size , presence of lymph node metastasis and unifocal tumours , and presence of extrathyroidal extension of tumours as compared to their respective counterparts .
similar to this , vcam-1 expression was significantly higher in gastric cancer patients with lymph node metastasis than in those without lymph node metastasis .
they also demonstrated that vcam-1 overexpression was associated with clinicopathological stage and depth of infiltration .
moreover , shin et al . suggested that vcam-1 expression may contribute to the metastatic adhesion of tumour cells and thus facilitate malignant progression of human gastric tumours . in ovarian cancer ,
scalici et al . showed significant increase in vcam-1 expression with advancing tumour stage and its expression was found to reduce in patients who received neoadjuvant chemotherapy while , in a study by huang et al . ,
the overexpression of vcam-1 was associated with advanced age at diagnosis as well as with response to treatment with surgery and chemotherapy .
expression of vcam-1 was found to be highest in poorly differentiated cutaneous squamous cell carcinoma .
documented that , in oral squamous cell carcinoma , vcam-1 expression was closely correlated to depth of infiltration and lymph node metastasis . moreover , in this study , neither the circulating level nor the protein expression of l - selectin or vcam-1 was able to predict dfs or os in total ptc patients .
again such results might be due to better survival and overall very low mortality rates in ptc patients .
the results by other authors have suggested that serum l - selectin could be of prognostic value in ovarian cancer and liver cancer while li et al . suggested that the detection of protein expression of l - selectin conduces to judging the prognosis of colorectal cancer patients .
but in ptc patients , as the overall mortality is very low , these patients need to be followed up for a longer period of time .
moreover , acute myeloid leukemia patients with higher soluble l - selectin at diagnosis had high probability of relapse compared to those with normal levels and had shorter event - free survival than patients with lower levels .
although vcam-1 expression was not able to predict dfs or os in total ptc patients , kaplan - meier survival analysis demonstrated higher incidence of disease relapse in patients with high vcam-1 expression as compared to those with low vcam-1 expression . moreover , higher vcam-1 expression was significantly associated with the reduced dfs in female ptc patients and in those who were postoperatively treated with ria and/or rt .
thus , it can be suggested that the higher vcam-1 expression in the primary tumours may be helpful in predicting dfs in female ptc patients and also the extent of treatment that might be required in the ptc patients with vcam-1 overexpression .
however , although not independent of tumour stage , serum vcam-1 levels were significant prognostic factors for patient survival in gastric cancer and colorectal cancer . moreover , serum vcam-1 was the only prognostic factor for patients with stage 3 and stage 4 rectal cancer patients while , in prostate cancer , serum vcam-1 achieved the status of independent predictor after adjusting for the standard postoperative clinicopathological features .
ho et al . observed better dfs in hepatocellular carcinoma patients with low vcam-1 levels .
the serum vcam-1 levels were also found to have prognostic significance in breast cancer patients [ 51 , 85 ] .
further , expression of vcam-1 in high grade serous ovarian cancer was associated with poor prognosis .
there was a significant positive correlation between circulating l - selectin and tnf- in the studied ptc patients , while vcam-1 expression was predominantly and positively associated with tnf- immunoreactivity .
this is in relation to borsig , who suggested that selectins may be expressed and/or activated in presence of certain mediators like tnf- , interleukins , or other toxins .
radhakrishnan et al . showed that tnf- enhances the motility and invasiveness of human prostate cancer cells by stimulating the expression of selective glycosyl- and sulfotransferase genes involved in the synthesis of selectin ligands .
further , they suggested that interactions of selectins and their ligands play a crucial role in enhancing the potential of the cancer cells to target to the lymphoid organs and inflamed endothelium at distant sites .
moreover , there are also studies indicating that tnf- produced by stimulated kupffer cells appeared to promote expression of adhesion molecules early during the metastatic process [ 8890 ] . in breast cancer cell lines ,
ali et al . observed that vcam-1 expression was enhanced by treatment with tnf-. thus , collectively these results support the idea that host responses to cancer cells may cause expression of adhesion molecules via the production of inflammatory cytokine like tnf- , which can facilitate tumour promotion and progression and further substantiate the link between inflammation and cancer progression .
the results of the present study signify a probable role of tnf- and the adhesion molecules : l - selectin and vcam-1 in thyroid carcinogenesis . determining their circulating levels may serve as a promising noninvasive method to uncover diagnostically informative differences between benign and malignant thyroid conditions .
this may further aid in the results of indeterminate fine needle aspiration results and also in the preoperative prediction of malignancy in patients with nodular thyroid diseases . on the whole , the interaction between tnf- and the adhesion molecules may form a comprehensive network which may induce sustained activation of various signalling pathways in follicular cells .
thus , understanding this complexity may offer potential therapeutic targets for management of thyroid cancer which may help the clinicians to identify and perform more personalized approach to their patients with more aggressive tumours , sparing the vast majority of patients with indolent disease from unnecessary procedures .
developing preventive and therapeutic strategies targeted towards tnf- may help induce antitumour immunity , further reducing the rates of recurrence and mortality in thyroid cancer . | circulating levels of tnf- and the adhesion molecules l - selectin and vcam-1 as well as their expression in the primary tumors of patients with benign thyroid diseases and papillary thyroid carcinoma ( ptc ) have been determined in this study .
the serum levels of tnf- , l - selectin , and vcam-1 were significantly higher in patients with both benign thyroid diseases and ptc as compared to the healthy individuals .
however , the levels of only tnf- and l - selectin , and not vcam-1 , were significantly higher in patients with ptc in comparison to those observed in patients with benign thyroid diseases .
further the expression of tnf- and l - selectin was also significantly higher in the primary tumors of ptc patients , relative to the benign thyroid diseases .
the expression of l - selectin and vcam-1 significantly correlated with aggressive tumor behavior . in ptc patients , the circulating tnf- levels significantly positively correlated with the levels of l - selectin , while tnf- immunoreactivity was significantly associated with vcam-1 expression .
serum tnf- was found to be a significant prognosticator for os in ptc patients .
overall the results signify that the interaction between tnf- and the adhesion molecules may have a role in thyroid carcinogenesis and understanding this complexity may offer potential therapeutic targets for better management of thyroid cancer . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion | the major types of follicular cell derived thyroid cancer include papillary thyroid cancer ( ptc ) , follicular thyroid cancer ( ftc ) , and anaplastic thyroid cancer ( atc ) , while medullary thyroid cancer ( mtc ) is the parafollicular cell derived thyroid cancer . it implies that , on one hand , there is a need to avoid excessive treatment and psychological discomfort to the patient who has benign thyroid disease or is in the initial stage of differentiated thyroid cancer and , on the other hand , patients with aggressive disease need to be guaranteed effective management right at the initial stage of the disease when it is still curable . have stated that changes in the expression or function of the cell adhesion molecules have been implicated in all steps of tumour progression , including detachment of tumour cells from primary site , intravasation into the blood stream , extravasation into distant target organs , and formation of secondary lesions . hence , this study aimed to determine the circulating and tumoural protein expression of tnf- and the adhesion molecules : l - selectin and vcam-1 in primary tumours of ptc patients in relation to their expression in patients with benign thyroid diseases . total 150 untreated patients with pathologically confirmed benign thyroid diseases ( n = 67 ) and papillary thyroid cancer ( n = 83 ) , enrolled between 2008 and 2012 at our institute , were included in this study . the patients with benign thyroid diseases comprised those diagnosed with having nodular goitre , multinodular goitre , colloid goitre , adenomatoid goitre , follicular adenoma , hrthle cell adenoma , and adenohyperplasia . pretherapeutic blood samples were collected from all patients as well as from 67 healthy individuals to detect the circulating levels of tnf- , l - selectin , and vcam-1 . the circulating levels of the tnf- , vcam-1 , and l - selectin were estimated from the serum samples using specific commercially available elisa kits ( tnf- : krishgen biosystems , l - selectin : abcam , and vcam-1 : invitrogen ) . immunohistochemical staining was performed for detection of tumoural expression tnf- , l - selectin , and vcam-1 in primary tumours of ptc patients and in patients with benign thyroid diseases . independent samples t - test was used to compare the means of circulating levels of analytes between two groups of subjects and also to assess the association of the analytes with the clinicopathological parameters of thyroid cancer patients . the circulating levels ( mean standard error , m se ) of tnf- , l - selectin , and vcam-1 in healthy individuals and patients with benign thyroid diseases and ptc are depicted in table 2 . it was observed that the circulating levels of tnf- , l - selectin , and vcam-1 were significantly higher in patients with benign thyroid diseases , as compared to that in healthy individuals ( p < 0.001 for each ) . further , the levels of tnf- and l - selectin , but not vcam-1 , were found to be significantly higher in ptc patients as compared to those in patients with benign thyroid diseases ( tnf- : p = 0.009 , l - selectin : p < 0.001 , and vcam-1 : p = 0.912 ) . further , the roc curves were generated to reveal the efficacy of these significantly elevated serum cytokine levels in order to differentiate the healthy individuals and patients with different thyroid diseases . the results demonstrated that tnf- , l - selectin , and vcam-1 exhibited a good discriminatory efficacy between healthy individuals and patients with benign thyroid diseases ( figure 1 ) as well as between healthy individuals and ptc patients ( figure 2 ) . moreover , tnf- ( auc = 0.598 , p = 0.040 ) and l - selectin ( auc = 0.692 , p
< 0.001 ) but not vcam-1 ( auc = 0.513 , p = 0.788 ) showed good power to discriminate between patients with benign thyroid diseases and ptc ( figure 3 ) . the correlation of circulating levels of tnf- , l - selectin , and vcam-1 with the clinicopathological parameters of ptc patients has been depicted in table 3 . the circulating levels of l - selectin were significantly higher in ptc patients whose tumours showed haemorrhagic area as compared to those in whom haemorrhagic area was absent ( p = 0.010 ) and vcam-1 levels were significantly higher in patients having bilateral tumours than those with unilateral tumours ( p = 0.037 ) . the expression of tnf- and vcam-1 was observed in the cytoplasm of the thyroid follicles , while cytoplasmic and membranous staining was observed for l - selectin . tnf- expression was observed in 67% ( 30/45 ) of patients with benign thyroid diseases with median irs-3 and in 89% ( 74/83 ) of ptc patients and irs-3 emerged as the median score . l - selectin immunoreactivity was observed in 82% ( 37/45 ) of benign thyroid disease patients , and the median score was irs-6 . further , 51% ( 23/45 ) of patients with benign thyroid diseases showed expression for vcam-1 having irs-1 as the median score . on the other hand , 99% ( 82/83 ) of the ptc patients
exhibited l - selectin expression and the irs-9 was observed as the median score and vcam-1 expression was positive in 84% ( 70/83 ) of the tumours with median score as irs-2 . the immunoreactivity of tnf- and l - selectin was found to be significantly high in ptc patients as compared to the benign thyroid disease patients ( tnf- : = 7.657 , r = + 0.245 , p = 0.005 and l - selectin : = 5.276 , r = + 0.203 , p = 0.022 ) ( table 4 , figure 5 ) . on the other hand ,
the incidence of expression of vcam-1 in ptc patients was not significantly different from that in the benign thyroid disease patients ( = 1.831 , r = + 0.120 , p = 0.179 ) ( table 4 ) . moreover , when correlated with the clinicopathological parameters of ptc patients , the tnf- expression was significantly positively correlated only with presence of calcification ( = 5.706 , r = + 0.262 , p = 0.017 ) . further , significant positive correlation of l - selectin expression in ptc patients with larger tumour size ( t3 + t4 ) ( = 7.955 , r = + 0.310 , p = 0.004 ) and presence of extrathyroidal extension of tumours ( = 12.120 , r = + 0.382 ,
vcam-1 expression showed a significantly positive correlation with larger tumour size ( t3 + t4 ) ( = 6.219 , r = + 0.274 , p = 0.012 ) , presence of lymph node metastasis ( = 3.971 , r = + 0.219 , p = 0.047 ) , and extrathyroidal extension of tumours ( = 11.350 , r = + 0.370 , p = 0.001 ) as compared to their respective counterparts . a significant inverse correlation was observed in the circulating tnf- levels and tnf- protein expression in the primary tumour tissues of ptc patients ( r = 0.254 , p = 0.021 ) . however
, statistical data analysis did not express any significant correlation between the circulating adhesion molecules and their respective protein expressions in ptc patients . further , the circulating levels of tnf- showed a significant positive correlation with the circulating levels of l - selectin ( r = + 0.303 , p = 0.005 ) , while the protein expression of tnf- ( r = + 0.347 , p = 0.001 ) showed a significant positive correlation with vcam-1 expression in the primary tumours of ptc patients ( table 6 ) . in univariate survival analysis ,
the circulating levels of none of the studied parameters could predict dfs in ptc patients ( table 7 ) . further , log rank test indicated that besides age , gender , metastasis , stage , and multifocality , serum tnf- ( log rank = 5.12 , df = 1 , p = 0.024 ) was a significant prognosticator for os in ptc patients ( table 8) . the kaplan - meier survival curve indicated significantly reduced os in ptc patients having higher levels of serum tnf- than those having lower circulating tnf- levels . the event of death was significantly higher in ptc patients with high serum tnf- levels ( 18% , 7/38 ) than in patients with low serum tnf- levels ( 3% , 1/38 ) ( log rank = 5.129 , df = 1 , p = 0.024 ) ( figure 6 ) . moreover , the circulating levels of l - selectin and vcam-1 were not able to predict os in the ptc patients . the kaplan - meier survival analysis demonstrated higher incidence of disease relapse in patients with high vcam-1 expression ( 18% , 5/27 ) as compared to those with low vcam-1 expression ( 5% , 2/42 ) . moreover , when ptc patients were subgrouped according to the clinicopathological variables , the kaplan - meier survival curves demonstrated that high expression of vcam-1 was remarkably associated with poor dfs in female patients and in the ptc patients postoperatively treated with ria therapy and/or rt . it was observed that 12% ( 2/17 ) of the female ptc patients with high vcam-1 expression had significantly reduced dfs , whereas none of the female patients with lower vcam-1 expression had developed recurrence and/or distant metastasis during the follow - up period ( log rank = 3.881 , df = 1 , p = 0.049 ) ( figure 8) and 34% ( 5/19 ) of ptc patients treated with surgery followed by ria therapy and/or rt having high vcam-1 expression had reduced dfs in comparison to only 4% ( 1/27 ) of these patients with lower vcam-1 expression ( log rank = 4.760 , df = 1 , p = 0.029 ) ( figure 9 ) . on the other hand , the kaplan - meier survival analysis and the log rank test revealed that neither tnf- nor l - selectin expression emerged as significant prognosticators to predict dfs or os even when the ptc patients were grouped according to the clinicopathological features . the current study revealed that serum tnf- was significantly higher in patients with benign thyroid diseases and in ptc patients , as compared to the healthy individuals . as compared to the patients with benign thyroid diseases , the circulating levels as well as the tumoural tissue expression of tnf-
the serum tnf- did not show significant correlation with any of the clinicopathological parameters , nor was it associated with dfs in these patients . however , kaplan - meier survival analysis revealed that its elevated levels were significantly associated with shorter os in the ptc patients . but in the multivariate survival analysis , as compared to the clinicopathological prognosticators , it lost its significance as an independent predictor of survival in ptc patients . results of various other studies also show that , relative to normal healthy controls , a significant increase in circulating levels of tnf- was observed in hepatocellular carcinoma patients , cervical neoplasia [ 22 , 23 ] , epithelial ovarian cancer [ 24 , 25 ] , prostate cancer , and renal cell carcinoma . also observed significantly higher plasma tnf- concentration in patients with chronic lymphocytic leukemia than in healthy control population and the elevated circulating tnf- correlated with the extent of disease and was suggested to be a novel prognostic factor for survival in patients with chronic lymphocytic leukemia . have reported that mean levels of tnf- were also significantly reduced in pretherapeutic serum samples of patients with bladder cancer as compared to the controls , whereas gendek - kubiak et al
. further , in the existing study , expression of tnf- was localized in the cytoplasm and was found to be significantly higher in the primary tumours of ptc patients than that observed in patients with benign thyroid diseases . moreover ,
tumoural protein expression of tnf- was not able to predict dfs or os in the ptc patients . the present study also found a significant inverse correlation between the circulating levels and tumoural protein expression of tnf-. this indicates that the circulating tnf- level was lower in patients having high tumoural expression of this protein and vice versa . thus , the current findings suggest that tnf- could be used as an indicator of thyroid cancer risk from benign conditions and may have a role in development of tumour and prognosis of thyroid cancer patients . the pretherapeutic circulating levels of both the adhesion molecules l - selectin and vcam-1 were significantly higher in patients with the benign disease as well as ptc patients than in the healthy individuals . these results
were further confirmed with roc curve analysis , where both l - selectin and vcam-1 could significantly discriminate patients with benign thyroid diseases and ptc from the healthy individuals . however , circulating levels of only l - selectin , and not of vcam-1 , were significantly elevated in the ptc patients when compared with the benign thyroid disease patients . similar to these , the results of roc curves also authenticated the efficacy of l - selectin to distinguish the ptc patients from those with benign thyroid diseases . in accordance with our findings ,
studies by other authors have also reported significantly higher serum l - selectin levels in patients with ovarian cancer , small - cell and non - small - cell lung cancer , chronic myelogenic leukemia , and acute myeloid leukemia than those in healthy control groups . also demonstrated markedly higher serum levels of l - selectin in patients with lung cancer or benign diseases than in the healthy controls . they have also reported significant differences in levels of l - selectin between the lung cancer patients with advanced stage disease or with metastasis and those with early stage disease or with no metastasis . in a recent study ,
circulating l - selectin levels were also found to be higher in serum samples from patients with high grade metastatic versus high grade nonmetastatic muscle invasive bladder cancer . , significantly higher levels of vcam-1 were observed in the serum samples of only anaplastic thyroid carcinoma patients , while in ptc patients the circulating vcam-1 levels were comparable with those of healthy control group . however , in the present study , serum l - selectin and vcam-1 were predominantly positively correlated only with presence of haemorrhagic area and bilaterality of the tumours , respectively , while they were not significantly associated with the rest of the studied clinicopathological parameters in ptc patients . this may be because , in thyroid carcinoma , the risk of disease relapse and occurrence of metastasis is relatively very low as compared to that in other malignancies arising from other organs . further , only l - selectin and not vcam-1 was significantly overexpressed in the primary tumours of ptc patients when compared to that in patients with benign thyroid diseases . similarly , protein expression of l - selectin has also been found to be significantly higher in the primary tumours of oral squamous cell carcinoma and salivary gland tumours . also in colorectal carcinoma , the l - selectin overexpression was observed , which was closely associated with the development of the disease and metastasis . moreover , significant attenuation of metastasis was observed in the absence of l - selectin , indicating that this adhesion molecule actively contributes to leukocyte recruitment and formation of a metastatic niche [ 71 , 72 ] . although not significant , the incidence of vcam-1 expression was higher in the thyroid cancer patients as compared to the patients with benign thyroid diseases . in thyroid carcinoma also
, there might be shedding of vcam-1 from the thyroid cancer cells into circulation which may be the factor accounting for the significantly elevated serum levels of vcam-1 . however , vcam-1 expression has been found to be overexpressed in primary tumours of many human cancers : ovarian cancer , pancreatic cancer , gastric cancer , oral squamous cell carcinoma , and clear cell renal carcinoma as well as papillary renal cell carcinoma as compared to the corresponding noncancerous tissues . further , in the present study , vcam-1 expression was significantly overexpressed in the ptc patients with larger tumour size , presence of lymph node metastasis and unifocal tumours , and presence of extrathyroidal extension of tumours as compared to their respective counterparts . moreover , in this study , neither the circulating level nor the protein expression of l - selectin or vcam-1 was able to predict dfs or os in total ptc patients . suggested that the detection of protein expression of l - selectin conduces to judging the prognosis of colorectal cancer patients . moreover , acute myeloid leukemia patients with higher soluble l - selectin at diagnosis had high probability of relapse compared to those with normal levels and had shorter event - free survival than patients with lower levels . although vcam-1 expression was not able to predict dfs or os in total ptc patients , kaplan - meier survival analysis demonstrated higher incidence of disease relapse in patients with high vcam-1 expression as compared to those with low vcam-1 expression . moreover , higher vcam-1 expression was significantly associated with the reduced dfs in female ptc patients and in those who were postoperatively treated with ria and/or rt . thus , it can be suggested that the higher vcam-1 expression in the primary tumours may be helpful in predicting dfs in female ptc patients and also the extent of treatment that might be required in the ptc patients with vcam-1 overexpression . there was a significant positive correlation between circulating l - selectin and tnf- in the studied ptc patients , while vcam-1 expression was predominantly and positively associated with tnf- immunoreactivity . the results of the present study signify a probable role of tnf- and the adhesion molecules : l - selectin and vcam-1 in thyroid carcinogenesis . this may further aid in the results of indeterminate fine needle aspiration results and also in the preoperative prediction of malignancy in patients with nodular thyroid diseases . on the whole , the interaction between tnf- and the adhesion molecules may form a comprehensive network which may induce sustained activation of various signalling pathways in follicular cells . thus , understanding this complexity may offer potential therapeutic targets for management of thyroid cancer which may help the clinicians to identify and perform more personalized approach to their patients with more aggressive tumours , sparing the vast majority of patients with indolent disease from unnecessary procedures . | [
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we had better courageously face the fact that prejudices are themselves elements of the interpretation of the social world and even one of the mainsprings that make it tick .
it makes little sense to tell the negrophobe in the south that in terms of biological science there is no such thing as a negro race.1 we had better courageously face the fact that prejudices are themselves elements of the interpretation of the social world and even one of the mainsprings that make it tick .
it makes little sense to tell the negrophobe in the south that in terms of biological science there is no such thing as a negro race.1 what makes schutz s insight , which he formulates in his 1955 essay equality and the social meaning structure , philosophically remarkable is that it not only considers the negativity of a specific type of social action with unusual acuity , but also recognizes such negativity as the social action s very mainspring . with this in mind , in this paper , i seek to analyze racism as a phenomenon of negative sociality and , thereby , to view it from a socio - phenomenological perspective .
first , it is necessary to reflect on what i understand by negative sociality .
in the light of these reflections , it should become clear from the outset why i view racism as an exemplary phenomenon of negative sociality .
third , i will address , in brief , why i consider phenomenology suitable for an analysis of racism as being such a phenomenon .
following these preliminary considerations , in the main section of my paper , i will turn to the analysis of the phenomenon itself . in doing so ,
i am not concerned , however , with tracing the concrete historical manifestations of racism .
rather , my analysis will center on , to my mind , the invariant methods , which characterize racism as a specific form of social practice ( or social technology ) that exploits our dependency upon bodily identifications .
following hund , these methods could be aptly described as negative socialization,2 which are of identity - relevance not only for those subjected to them , but also for those that
drawing on concrete phenomenological descriptions , i aim to exhibit these methods by way of examples , two of which i shall focus on . in the concluding section ,
i seek to clarify , at least in outline , whether and to what extent racism can be understood as an irreducible phenomenon of negative sociality and how such an understanding of racism bears on the way in which it is dealt with socially .
indeed , it seems to speak directly to the suspicion that [ ] negative experiences [ such as mistrust , discrimination , contempt , or violence ] can not be cancelled out by a community or a society that is characterized by responsibility , justice , and recognition .
if this were the case , then we would be faced with the necessity fundamentally to revise these concepts , and eventually even the concept of the social itself . for according to the thesis deduced thereof , the social , under these conditions ,
can not be consistently brought under the rule of law or be pacified by the rules of coexistence.3from this follows that the focus must rest on negativity
, concretely lived negative experiencesin terms of its irreducibility or indissolubility in social life itself and on the coherence and intelligibility related to it .
thus the term negativity does not refer to a phenomenon of contingent deficiency that could be caught up with discursively , mediated dialectically , or overcome procedurally to name but a few approaches to coping with contingency
. negativity does not merely amount to a contingent deficiency of justice , trust , responsibility , communalization , solidarity , communication , and so on , as tends to be assumed by the mainstream of social philosophy , where it is at all sensitive to this issue .
whether a philosophical perspective assumes that such negative experiences can be countered by a unity that is to be realized through harmony , universality , concordia , homonoia , the reciprocity of perspectives , the non - violent discourse , or the ideal communication society , whilst a political perspective opposes them to the ideals of a fully integrated society or an organic community , under the unifying force of which such negativity should simply dissolve : in all these cases , negativity is presented as relative , as pure deficiency or as an exception in social functioning,4 i.e. , as socially anomalous , as unusual , an individual aberration or institutional hangover.5 in contrast , i suggest that it is indeed necessary that negativity be understood as a
factum in the sense of the kantian factum of reason , which can not be sublated into a more comprehensive legal or political order .
. to conceive of negativity in its extraordinariness , however , also entails a reflection on its order - constitutive function . for orders ( re)generate through exclusion and selection through the production of an extra - ordinary .
, there is the violent character of order itself , to the extent to which it always functions on a selective and exclusive basis , i.e. , to the extent to which itexcludes the extraordinary by including it.6 thus , within order there inheres a constitutive moment of negativity .
this moment , however , is not contingent because it renders possible the order s accomplishment of order ( and therefore provides identity - relevant motivations to act ) .
therefore , it is imperative to recognize that in its extraordinariness and indissolubility , negativity plays a constitutive role for the accomplishment of social order and thus concretely for socialization .
this implies that the aforementioned phenomena of negative sociality are , on the one hand , social phenomena through and through , while , on the other , they negate the facticity of the social in - between , the horizon within which we tend to think
. phenomena of negative sociality bear de - socializing effects and remind us of the limits of socialization and communality,7 whilst at the same time remaining socially overdetermined and tied to concrete social action in their effectiveness . the expression negative sociality appears , at first , eminently problematic .
indeed , it seems to speak directly to the suspicion that [ ] negative experiences [ such as mistrust , discrimination , contempt , or violence ] can not be cancelled out by a community or a society that is characterized by responsibility , justice , and recognition .
if this were the case , then we would be faced with the necessity fundamentally to revise these concepts , and eventually even the concept of the social itself . for according to the thesis deduced thereof ,
the social , under these conditions , can not be consistently brought under the rule of law or be pacified by the rules of coexistence.3 from this follows that the focus must rest on negativity
, concretely lived negative experiencesin terms of its irreducibility or indissolubility in social life itself and on the coherence and intelligibility related to it .
thus the term negativity does not refer to a phenomenon of contingent deficiency that could be caught up with discursively , mediated dialectically , or overcome procedurally to name but a few approaches to coping with contingency
. negativity does not merely amount to a contingent deficiency of justice , trust , responsibility , communalization , solidarity , communication , and so on , as tends to be assumed by the mainstream of social philosophy , where it is at all sensitive to this issue . whether a philosophical perspective assumes that such negative experiences can be countered by a unity that is to be realized through harmony , universality , concordia , homonoia , the reciprocity of perspectives , the non - violent discourse , or the ideal communication society , whilst a political perspective opposes them to the ideals of a fully integrated society or an organic community , under the unifying force of which such negativity should simply dissolve : in all these cases , negativity is presented as relative , as pure deficiency or as an exception in social functioning,4 i.e. , as socially anomalous , as unusual , an individual aberration or institutional hangover.5 in contrast , i suggest that it is indeed necessary that negativity be understood as a
factum in the sense of the kantian factum of reason , which can not be sublated into a more comprehensive legal or political order .
. to conceive of negativity in its extraordinariness , however , also entails a reflection on its order - constitutive function . for orders ( re)generate through exclusion and selection through the production of an extra - ordinary .
in particular , there is the violent character of order itself , to the extent to which it always functions on a selective and exclusive basis , i.e. , to the extent to which itexcludes the extraordinary by including it.6 thus , within order there inheres a constitutive moment of negativity .
this moment , however , is not contingent because it renders possible the order s accomplishment of order ( and therefore provides identity - relevant motivations to act ) .
therefore , it is imperative to recognize that in its extraordinariness and indissolubility , negativity plays a constitutive role for the accomplishment of social order and thus concretely for socialization .
this implies that the aforementioned phenomena of negative sociality are , on the one hand , social phenomena through and through , while , on the other , they negate the facticity of the social in - between , the horizon within which we tend to think .
phenomena of negative sociality bear de - socializing effects and remind us of the limits of socialization and communality,7 whilst at the same time remaining socially overdetermined and tied to concrete social action in their effectiveness .
( b)for a considerable time , research on racism has attempted to demonstrate that it would be more felicitous to speak not of racism , but of racisms.8 as shown by several analyses , the reference to the concept of race is by no means constitutive for the historical manifestations of racism .
historical contexts ; as a consequence , race has to be viewed as a social construct.9 at the same time , the opposite view has been put forth arguing for a racism without races,10 i.e. , a racism that succeeds culturalistically without any longer requiring the concept of race to this end . as a result of these insights , a distinction has consistently been drawn between racisms of
race , and those within class , gender , and nationality,11 which on their part rely on ideological , religious , or culturalistic discourses of legitimization . at stake in all these cases
, there is and at least until here , the minimum consensus seems to reacha process of the social construction of purportedly natural inequality , which is an attempt to find a natural explanation for social differences.12 as a hegemonic scheme of explanation , racism therefore always also mirrors the power relations that shape the integrity of societies internally and externally .
in contrast to social discrimination , which structures individuals within the social context according to hierarchical contrasts , racism taken in the broadest sense posits these individuals as others outside of the social context , in robbing them of their social particularity , or denying their ability for it.13 in this vein , racism functions as a motive or a means of threat for social integration or exclusion on the one hand , and assimilation or segregation on the other .
this dual direction of the impact of racist processes of socialization , which as a result works on the basis of a dual logic of including exclusion and excluding inclusion , is to my mind
constitutive for the phenomenon.in accordance with what has been stated so far , i understand racism as a social process of hetero - normative meaning - constitution,14 which holds out the prospect of an illusory sense of community through bodily desocializing others and thus charts identity - relevant motivations for actions for those who seek to belong to this imagined community . what vary in the history of racism are the legitimization patterns of these processes and the practices of exclusion that substantiate them
ideal typical constructions fail to grasp the diversity and heterogeneity of racist practices .
they do seem comparable , however , insofar as they construct the otherswithin society as well as outside of itas deficient beings and locate them beyond all respective social dualisms.15 from this insight follows moreover that[t]he achievement of identity at the expense and to the detriment of others does not only generate different degrees of being human , but also destabilizes the very context that it founds . at the same time as the border to inferiority
is maintained outwardly , it is drawn into the very inner of society in form of a flexible predetermined breakage point.16thus it follows that as a form of negative socialization , racism must be viewed as an exemplary phenomenon of negative sociality because it is not only a negating social practice , but also makes us aware of the negativity of the social itself , i.e. , the social violence that is an integral part of its constitution . for a considerable time , research on racism has attempted to demonstrate that it would be more felicitous to speak not of racism , but of racisms.8 as shown by several analyses , the reference to the concept of race is by no means constitutive for the historical manifestations of racism .
historical contexts ; as a consequence , race has to be viewed as a social construct.9 at the same time , the opposite view has been put forth arguing for a racism without races,10
i.e. , a racism that succeeds culturalistically without any longer requiring the concept of race to this end . as a result of these insights , a distinction has consistently been drawn between racisms of
race , and those within class , gender , and nationality,11 which on their part rely on ideological , religious , or culturalistic discourses of legitimization . at stake in all these cases
, there is and at least until here , the minimum consensus seems to reacha process of the social construction of purportedly natural inequality , which is an attempt to find a natural explanation for social differences.12 as a hegemonic scheme of explanation , racism therefore always also mirrors the power relations that shape the integrity of societies internally and externally .
in contrast to social discrimination , which structures individuals within the social context according to hierarchical contrasts , racism taken in the broadest sense posits these individuals as others outside of the social context , in robbing them of their social particularity , or denying their ability for it.13 in this vein , racism functions as a motive or a means of threat for social integration or exclusion on the one hand , and assimilation or segregation on the other .
this dual direction of the impact of racist processes of socialization , which as a result works on the basis of a dual logic of including exclusion and excluding inclusion , is to my mind
constitutive for the phenomenon . in accordance with what has been stated so far , i understand racism as a social process of hetero - normative meaning - constitution,14 which holds out the prospect of an illusory sense of community through bodily desocializing others and thus charts identity - relevant motivations for actions for those who seek to belong to this imagined community .
what vary in the history of racism are the legitimization patterns of these processes and the practices of exclusion that substantiate them .
ideal typical constructions fail to grasp the diversity and heterogeneity of racist practices .
they do seem comparable , however , insofar as they construct the otherswithin society as well as outside of itas deficient beings and locate them beyond all respective social dualisms.15 from this insight follows moreover that[t]he achievement of identity at the expense and to the detriment of others does not only generate different degrees of being human , but also destabilizes the very context that it founds . at the same time as the border to inferiority is maintained outwardly , it is drawn into the very inner of society in form of a flexible predetermined breakage point.16 [ t]he achievement of identity at the expense and to the detriment of others does not only generate different degrees of being human , but also destabilizes the very context that it founds . at the same time as the border to inferiority is maintained outwardly , it is drawn into the very inner of society in form of a flexible predetermined breakage point.16 thus it follows that as a form of negative socialization , racism must be viewed as an exemplary phenomenon of negative sociality because it is not only a negating social practice , but also makes us aware of the negativity of the social itself , i.e. , the social violence that is an integral part of its constitution .
( c)can phenomenology , then , make a contribution to an analysis of racism as a process of negative socializationan undoubtedly historical process , into which flow concrete material foundations , structural forms of social organization , and ideological justifications ? on the assumption that racism , or to speak with r. miles , racializations are in many cases processes of meaning - constitution17 that are transmitted on a pre - reflexive level and that carry with them identity - relevant choices of identification or experiences of exclusion , a phenomenological or , more precisely , a socio - phenomenological approach to analysis seems fitting .
the first concerns the specificity of the subject matter violence or a relation of violence , the second relates to the specific methodology of phenomenological analysis.the practice of phenomenology allows for what , following husserl , we could designate as an ethical epoch.18 i understand the task of such an epoch to be the bracketing of all those discourses on the reasons and the justifications of violence , in the normatively overdetermined ( and oftentimes moralizing ) light of which the analysis of direct violence and indirect relations of violence both implied by racism has to a large extent taken place hitherto .
thus , phenomenology must seek to thematize the conditions of possibility of violence and relations of violence ; it must focus on what is responsible for rendering such in this case racist violence possible in the first case , and must ask how what is identified can then subsequently become the subject of discourses of justification.19 in this , it emerges as necessary that a phenomenology of racist violence and respectively , a phenomenology of violence in general
get to the bottom of those processes of desensibilization that precede all direct violence . focusing their structure , pre - reflective functioning , and genesis
, a phenomenology of violence intends to show how subjects grow indifferent to the suffering of others , and that they can hence no longer perceive the singularity of their
ethical demand.20here , the second reason for my choice of the phenomenological approach becomes perspicuous : it is the only approach that is in a position to steer clear of the nominalistic and essentialist misinterpretations of the phenomenon , in order to trace , conversely , the effective reality of racializations in theeverydayness of lived experience , personal habitualities , and the institutions of our life - world.21 this implies , more concisely , that so - called meaning - constitutions can be comprehensively reconstructed neither in reference to the motives of a self - reflective subject capable of acting instrumentally nor in taking recourse to structures by which actions are functionally governed .
rather , i would suggest , the focus must lie on those pre - reflective dimensions of lived experience that predetermine the habitus of our experiencing , and interactions in a bodily manner i.e .
racialize it and , hence , make overt racist violence possible.thus in terms of methodology , i consider the phenomenological approach suitable insofar as it admits for the illumination of the interplay between different registers of experience in their identity - relevance . without elaborating this thought any further here , i propose to understand the meaning - constitution and therefore subject - constitution as the interplay between subjective sense - bestowal , anonymous processes of sense - formation , and symbolic institution .
the open and indefinite unity of subjectivity thus emerges , to follow merleau - ponty , in correlation with the open unity of the world , the autochthonous significance22 of which essentially determines its genesis . in viewing meaning - constitution and self - constitution as such a relational and basically inter - corporeal occurrence
ultimately , it is only in the latter s light that phenomenology is able to zoom in adequately on the efficiency previously mentioned , i.e. , on the performative force and apparent magic of culturally overdetermined nexuses of sense , and respectively , of hegemonic regimes of sense - formation ( such as the concept of race or the pseudo - scientific discourse on race).23 can phenomenology , then , make a contribution to an analysis of racism as a process of negative socializationan undoubtedly historical process , into which flow concrete material foundations , structural forms of social organization , and ideological justifications ? on the assumption that racism , or to speak with r. miles , racializations are in many cases processes of meaning - constitution17 that are transmitted on a pre - reflexive level and that carry with them identity - relevant choices of identification or experiences of exclusion , a phenomenological or , more precisely , a socio - phenomenological approach to analysis seems fitting .
the first concerns the specificity of the subject matter violence or a relation of violence , the second relates to the specific methodology of phenomenological analysis.the practice of phenomenology allows for what , following husserl , we could designate as an ethical epoch.18 i understand the task of such an epoch to be the bracketing of all those discourses on the reasons and the justifications of violence , in the normatively overdetermined ( and oftentimes moralizing ) light of which the analysis of direct violence and indirect relations of violence both implied by racism has to a large extent taken place hitherto . thus , phenomenology must seek to thematize the conditions of possibility of violence and relations of violence ; it must focus on what is responsible for rendering such in this case racist violence possible in the first case , and must ask how what is identified can then subsequently become the subject of discourses of justification.19 in this , it emerges as necessary that a phenomenology of racist violence and respectively , a phenomenology of violence in general get to the bottom of those processes of desensibilization that precede all direct violence .
focusing their structure , pre - reflective functioning , and genesis , a phenomenology of violence intends to show how subjects grow indifferent to the suffering of others , and that they can hence no longer perceive the singularity of their
ethical demand.20here , the second reason for my choice of the phenomenological approach becomes perspicuous : it is the only approach that is in a position to steer clear of the nominalistic and essentialist misinterpretations of the phenomenon , in order to trace , conversely , the effective reality of racializations in theeverydayness of lived experience , personal habitualities , and the institutions of our life - world.21 this implies , more concisely , that so - called meaning - constitutions can be comprehensively reconstructed neither in reference to the motives of a self - reflective subject capable of acting instrumentally nor in taking recourse to structures by which actions are functionally governed .
rather , i would suggest , the focus must lie on those pre - reflective dimensions of lived experience that predetermine the habitus of our experiencing , and interactions in a bodily manner i.e .
the practice of phenomenology allows for what , following husserl , we could designate as an ethical epoch.18 i understand the task of such an epoch to be the bracketing of all those discourses on the reasons and the justifications of violence , in the normatively overdetermined ( and oftentimes moralizing ) light of which the analysis of direct violence and indirect relations of violence both implied by racism has to a large extent taken place hitherto .
thus , phenomenology must seek to thematize the conditions of possibility of violence and relations of violence ; it must focus on what is responsible for rendering such in this case racist violence possible in the first case , and must ask how what is identified can then subsequently become the subject of discourses of justification.19 in this , it emerges as necessary that a phenomenology of racist violence and respectively , a phenomenology of violence in general get to the bottom of those processes of desensibilization that precede all direct violence .
focusing their structure , pre - reflective functioning , and genesis , a phenomenology of violence intends to show how subjects grow indifferent to the suffering of others , and that they can hence no longer perceive the singularity of their
ethical demand.20 here , the second reason for my choice of the phenomenological approach becomes perspicuous : it is the only approach that is in a position to steer clear of the nominalistic and essentialist misinterpretations of the phenomenon , in order to trace , conversely , the effective reality of racializations in theeverydayness of lived experience , personal habitualities , and the institutions of our life - world.21 this implies , more concisely , that so - called meaning - constitutions can be comprehensively reconstructed neither in reference to the motives of a self - reflective subject capable of acting instrumentally nor in taking recourse to structures by which actions are functionally governed .
rather , i would suggest , the focus must lie on those pre - reflective dimensions of lived experience that predetermine the habitus of our experiencing , and interactions in a bodily manner i.e .
thus in terms of methodology , i consider the phenomenological approach suitable insofar as it admits for the illumination of the interplay between different registers of experience in their identity - relevance . without elaborating
this thought any further here , i propose to understand the meaning - constitution and therefore subject - constitution as the interplay between subjective sense - bestowal , anonymous processes of sense - formation , and symbolic institution .
the open and indefinite unity of subjectivity thus emerges , to follow merleau - ponty , in correlation with the open unity of the world , the autochthonous significance22 of which essentially determines its genesis . in viewing meaning - constitution and self - constitution as such a relational and basically inter - corporeal occurrence
ultimately , it is only in the latter s light that phenomenology is able to zoom in adequately on the efficiency previously mentioned , i.e. , on the performative force and apparent magic of culturally overdetermined nexuses of sense , and respectively , of hegemonic regimes of sense - formation ( such as the concept of race or the pseudo - scientific discourse on race).23
thus , the direction of the impact of a phenomenological recourse to lived experience is clearly determined . yet , does it not , in the same gesture , confront us with a dire problem ?
does it not delineate a phenomenology that can no longer rely on the undeniable meaningfulness of the social world , but rather encounters the incompossible , the resistance of which undermines the sheltering canopies of our symbolic universes24 ? do not at least two mutually irreducible starting points of a reflection on the sense of violence offer themselves to such a pursuit ?
is it not either a phenomenology of the oppressor25 as sartre proposed , or a phenomenology of suffered violence , hence a phenomenology of the experience of the victim of racist discrimination or violence ? and matters seem to get even more complicated : from a phenomenological standpoint , are we not required also to consider the perspectives of a third party , be it actual ( such as witnesses , followers , bystanders , uninvolved observers ) or virtual ( for instance , one s conscience as the innermost form of otherness , or the apparent universality of the law ) . for after all , the third party s presence or absence and intervention or passivity is , without doubt , of decisive importance for the genesis of the respective contexts of experience and their identity - relevant implications . to sum up my argument : is the category of sense the category of phenomenology and hermeneutics at all suitable to cope with the structural difference , which patently seems to separate violence as an intention and violence as an experience that befalls us ( to refrain here from a discussion of violence as a
second - order construct)?26 despite the irreducibility , on which it insists , this problem appears to me to be only a pseudo - problem . for
the experience of the otherto use a husserlian concept is relational through and through ; social phenomena are phenomena of the in - between , to use the language of bernhard waldenfels . the experience of the other is endowed with a sense , which occurs between me and the other , between what is my own and what is foreign to me .
more precisely , it possesses a sense , which can neither be fully traced back to the initiative and the capacity of single individuals or groups nor to a mediating authority of order nor to codified regulations.27 to put it phenomenologically , it possesses a sense , which can neither be entirely ascribed to the ego s sense - bestowals nor explained as the result of processes of symbolic institution .
neither is it a sense that emerges completely behind the back of the active i. rather , for its identity - relevant articulation , this sense requires the transition through the historic - cultural interworld of human symbolisms as well as the creative re - ordering of fact and hazard.28 in other words , the experience of the other thus bears a sense that is only articulated in the concrete intersubjective encounter , even though in this case and this point is crucial it does not indicate a reciprocity of perspectives , which would secure for us at least a minimal access to a shared world . on the contrary , it is the concrete modality of racist practices , the mode in which it addresses others , which precludes this possibility , i.e. , de - subjectivizes , de - humanizes the other . with great force ,
frantz fanon gives voice to this experience of de - subjectivized subjectivization in the chapter the fact of blackness of his book black skin , white masks . to my mind
, this is , in fact , one of the first times that the problem of racism is described phenomenologically .
although it rests essentially on sartre s theory of a bodily being - for - others,29 fanon , at the same time , also seeks to do justice to the basic existential factum that is masked by sartre : for the black man , the white man is not simply only the other but also the master , whether real or imaginary.30 not only does the concrete failure of the dialectic of recognition that is constitutive for sartre s approach become manifest thereby ; with fanon , further , we are in the position to demonstrate that in the form of symbolically instituted power - relations , this factum informs the habitus of bodily self - experience of the
racialized objects31 and is thus performatively reproduced.the black man among his own in the twentieth century does not know at what moment his inferiority comes into being through the other ( ) and then the occasion arose when i had to meet the white man s eyes .
in the white world the man of color encounters difficulties in the development of his bodily schema .
i know that if i want to smoke , i shall have to reach out my right arm and take the pack of cigarettes lying at the other end of the table .
( ) and all these movements are made not out of habit but out of implicit knowledge .
a slow composition of my self as a body in the middle of a spatial and temporal world such seems to be the schema .
it does not impose itself on me ; it is , rather , a definitive structuring of the self and the world definitive because it creates a real dialectic between my body and the world.32 the black man among his own in the twentieth century does not know at what moment his inferiority comes into being through the other ( ) and then the occasion arose when i had to meet the white man s eyes .
in the white world the man of color encounters difficulties in the development of his bodily schema .
i know that if i want to smoke , i shall have to reach out my right arm and take the pack of cigarettes lying at the other end of the table .
( ) and all these movements are made not out of habit but out of implicit knowledge . a slow composition of my self as a body in the middle of a spatial and temporal world such seems to be the schema
. it does not impose itself on me ; it is , rather , a definitive structuring of the self and the world definitive because it creates a real dialectic between my body and the world.32 following fanon , the body schema that is referred to here , which merleau - ponty thematized as the linchpin of the being - for - the - world mentioned earlier , describes a merely superficial mode of experiencing lived embodiment .
what fanon designates as the historico - racial schema underlies and structures it : for several years certain laboratories have been trying to produce a serum for denegrification ; with all the earnestness of the world ,
laboratories have sterilized their test tubes , checked their scales , and embarked on researches that might make it possible for the miserable negro to whiten himself and thus to throw off the burden of that corporeal malediction . below the corporeal schema
the elements that i used had been provided for me not by residual sensations and perceptions primarily of a tactile , vestibular , kinesthetic , and visual character , but by the other , the white man , who had woven me out of a thousand details , anecdotes , stories .
i thought that what i had in hand was to construct a physiological self , to balance space , to localize sensations , and here i was called on for more.33 for several years certain laboratories have been trying to produce a serum for denegrification ; with all the earnestness of the world ,
laboratories have sterilized their test tubes , checked their scales , and embarked on researches that might make it possible for the miserable negro to whiten himself and thus to throw off the burden of that corporeal malediction . below the corporeal schema
the elements that i used had been provided for me not by residual sensations and perceptions primarily of a tactile , vestibular , kinesthetic , and visual character , but by the other , the white man , who had woven me out of a thousand details , anecdotes , stories .
i thought that what i had in hand was to construct a physiological self , to balance space , to localize sensations , and here i was called on for more.33 the body schema as thematized by phenomenology seems , in this regard , to be the schema of a body that is in the world with - itself , a body that lives in a pre - reflexive balance with its surroundings , a body whose habitual performance knowledge is lived unproblematically .
the white gaze ( which is , of course , never merely a gaze nor simply white ) racializes this schema .
more precisely , it makes the one who is gazed at aware that the schema was always already racialized . as a consequence ,
his orientation in the world becomes questionable , the accessibility of certain objects seems suddenly problematic , and more generally the body schema breaks down to give room to a
racial epidermal schema.34 this schema forces itself upon the perception of the social space and its institutions and organizes action in the light of a retrospective reoccupation of a space that i already inhabited.35 the lived space as such becomes white , in the sense of that absent centre against which others appear only as deviants , or points of deviation.36 the habitual incorporation of lived space re - oriented in this vein thus creates habit worlds and leads to the institutionalization of a certain likeness, which makes non - white bodies feel uncomfortable , exposed , visible , different , when they take up this place.37 the
i.e. , a violence that functions on basis of the pre - reflective embodiment of oppressive social power relations .
precisely because of its pre - reflexivity , it is , however , not conceived as violence , but rather succeeds in appearing as
naturally given.39i could no longer laugh , because i already knew that there were legends , stories , history , and above all historicity , which i had learned about from jaspers . then , assailed at various points , the corporeal schema crumbled , its place taken by a racial epidermal schema . [
] i was responsible at the same time for my body , for my race , for my ancestors .
i subjected myself to an objective examination , i discovered my blackness , my ethnic characteristics ; and i was battered down by tom - toms , cannibalism , intellectual deficiency , fetichism , racial defects , slave - ships , and above all else , above all :
[ y a bon banania]40 i could no longer laugh , because i already knew that there were legends , stories , history , and above all historicity , which i had learned about from jaspers .
then , assailed at various points , the corporeal schema crumbled , its place taken by a racial epidermal schema .
[ ] i was responsible at the same time for my body , for my race , for my ancestors .
i subjected myself to an objective examination , i discovered my blackness , my ethnic characteristics ; and i was battered down by tom - toms , cannibalism , intellectual deficiency , fetichism , racial defects , slave - ships , and above all else , above all :
, on racism as a symbolically transmitted and reproducedrelation of domination that generates patterns of interaction prone of violence
therefore brings something else to the fore , something on which anti - racist discussions would only later begin to focus : the insight that in correlation to the symbolic violence of racism mentioned already , i.e. , correlative to the symbolically embodied pre - formation of subjective action , the room to maneuver for the interaction of the racialized subjects undergoes a pre - formation too.41 sartre became aware of this correlation subsequent to his first visit to the usa in the year 1945 , when he began to reflect upon the invisibility of the black gaze,42 a gaze that was unable to enter into the struggle for recognition at all .
sartre realized that this imposed and habitually lived invisibility correlatively required an effort on the part of those that were completely absorbed by whiteness as norm,43 whilst constantly living in fear of not meeting this norm44a fear which , as a consequence , became projected onto the other.45look , a negro ! it was an external stimulus that flicked over me as i passed by .
it amused me.look , a negro ! the circle was drawing a bit tighter .
i made up my mind to laugh myself to tears , but laughter had become impossible.46
i made up my mind to laugh myself to tears , but laughter had become impossible.46 by focusing on the lived experience of blackness ,
fanon s descriptions also provide decisive starting points for an analytics of the racist subject .
it does so in proffering evidence that the negro , because of his body , impedes the closing of the postural schema of the white man47which characterizes him as the principle of evil48 ( of bad , impure , biological , sexual , etc . ) .
analogously , and in addition to the central modalities of the experience of racism , his descriptions also enable us to trace the central methods of racism , methods that have also been unearthed by comparative historical studies.49 first , we come upon the experience of inferiorization through the attribution of cultural difference .
such attribution practices employ a logic of mutual exclusion that is expressed in pairs of opposites such as culture / barbarism , civilization / savageness , purity / impurity , chosenness / fallenness or significance / inferiority.50 this process of an inferiorizing positing of difference represents a heteronormative form of socialization , which is ultimately based not on an always discriminating inclusion , but in principle on segregation and exclusion , i.e. , it proceeds merely negatively .
secondly and closely linked to this is the resulting experience of self - alienation , which can be traced back to desocialization in principle as the objective inherent to inferiorization.51 the alienating splitting of consciousness , i.e. , the doubling of personality according to sartre52 or the
double consciousness in the sense of du bois,53 results from the self - denigrating adoption of racist norms the fulfillment of which is impossible by definition .
the corresponding practices of desocialization are widespread and range from the denial of social particularization to a fundamental questioning of cultural singularity.54 finally and most crucially , fanon makes us aware of stigmatizing forms of embodiment that render possible and transmit desocialization and inferiorization , and which thus must be retained as the third central method of racism .
what has been said so far can , however , by no means only be demonstrated in the context of the infernal machine of colonialism,55 which is , of course , of decisive importance from a historical perspective , for the genesis of a racial racism and from a genealogical perspective , for subsequent national racism.56 rather to take a big leap we could say that it also becomes manifest in the affective micro - practices of everyday racist interactions . to illustrate this point , i will refer to audre lordes practical - phenomenological description of such an affectively overdetermined situation of interbodily encounter in her sister outsider : the aa subway train to harlem .
i clutch my mother s sleeve , her arms full of shopping bags , christmas - heavy .
on one side of me a man reading a paper . on the other , a woman in a fur hat staring at me .
her mouth twitches as she stares and then her gaze drops down , pulling mine with it .
her leather - gloved hand plucks at the line where my new blue snowpants and her sleek fur coat meet .
i do not see whatever terrible thing she is seeing on the seat between us probably a roach .
it must be something very bad from the way she s looking , so i pull my snowsuit closer to me away from it , too .
when i look up the woman is still staring at me , her nose holes and eyes huge .
and suddenly i realise there is nothing crawling up the seat between us ; it is me she does nt want her coat to touch .
the fur brushes my face as she stands with a shudder and holds on to a strap in the speeding train .
born and bred a new york city child , i quickly slide over to make room for my mother to sit down .
i m afraid to say anything to my mother because i do nt know what i have done .
something s going on here i do not understand , but i will never forget it . her eyes .
i clutch my mother s sleeve , her arms full of shopping bags , christmas - heavy . the wet smell of winter clothes , the train s lurching .
on one side of me a man reading a paper . on the other , a woman in a fur hat staring at me .
her mouth twitches as she stares and then her gaze drops down , pulling mine with it .
her leather - gloved hand plucks at the line where my new blue snowpants and her sleek fur coat meet .
i do not see whatever terrible thing she is seeing on the seat between us probably a roach .
it must be something very bad from the way she s looking , so i pull my snowsuit closer to me away from it , too .
when i look up the woman is still staring at me , her nose holes and eyes huge .
and suddenly i realise there is nothing crawling up the seat between us ; it is me she does nt want her coat to touch .
the fur brushes my face as she stands with a shudder and holds on to a strap in the speeding train .
born and bred a new york city child , i quickly slide over to make room for my mother to sit down .
i m afraid to say anything to my mother because i do nt know what i have done .
something s going on here i do not understand , but i will never forget it . her eyes .
the hate.57 in this description of an intercorporeal encounter , the above mentioned methods of racism can be found again .
moreover , it exhibits with particular clarity how interpersonal encounters unfold in a medium of
interkinesthetic affectivity,58 a dimension of lateral sense - formation that is rarely accounted for in mainstream social theory as well as in philosophy .
this becomes clear , first , with regard to the experience of inferiorization through the attribution of difference , which , in this case , occurs through the misperception of a phobic object.59 it is audre herself who becomes this object she experiences being the disgusting cause of the hatred that she meets in the other s gaze .
second , this leads to the experience of self - alienation , an experience , in this case , of an i can not , which influences the lived organization of social and bodily space .
since in this experience , the social space itself undergoes a change and is even affectively reconstituted , practices of desocialization come to the fore , which take shape in the expulsion of blackness from white social space and correlatively in a
re - forming [ of ] the apartness of the white body.60 thereby , a stigmatizing mode of embodiment gains ground .
for the affective over - determination of the encounter , which is described as unforgettable , does not merely call into question the ways audre inhabits the lived space and coincides with the ease of movement of her body . rather , it interferes with the body schema , by creating disoriented bodies the movement of which becomes a defensive posture in the social space , which is geared at avoiding social stressand precisely thereby reproduces that same affective racist economy , which signalizes its exclusion from this space . summing up
, i want to emphasize clearly once again that the racialization of identities through the said methods functions on two planes . not only does it concern the discursive status of the subject in public , where it is rendered quasi invisible as a social agent,61 in order to secure thereby the normalizing invisibility of the dominant identity - formation .
further , it bears especially on the manner of bodily self - experience of the subject . as a result of the subject s stigmatizing embodiment
, its self - experience is haunted by difficulties in the development of [ the ] bodily schema,62 whilst our i can is usually , to put it with sartre , the neglected , passed by in silence.63 the historical - racist schema , that comes to overlay the body schema,64 thus produces a habitus that seems to carry the stigma of difference , which is mediated discursively , always already inscribed into it : regardless of whether this difference is taken up affirmatively and thus performatively reproduced , or whether it is discarded as objectively non - existing , to be evoked as an even more dangerous , for invisible danger as difference , it is powerfully reflected in the amputated body schema , as fanon describes it , or in the inner overdetermination of the actions of the racially dominated , to which sartre refers in his anti - semite and jew.65 in a nutshell , in light of the examples mentioned , racism can be grasped as the bodily inferiorization of the other which practices la limite the other s desocialization .
its social sense consists , besides , in imagining community and creating individual identity - offers , which pretend to transcend the contingency of factual forms of subjectivity , whilst effectively securing given power relations .
current research has often underlined the structural character of racism , which , as it seems , can be abolished neither through a social critique nor through the intensified combat against ideological forms of prejudices and discriminations . in this context ,
common - sense [ ] feature of everyday life and global social structure.66 from a structural functional perspective , hund emphasizes its integrating function in the process of class - specific socialization.67 goldberg makes the simple and disillusioned statement : race is irrelevant , but all is race . even if all racist ideologies could be abolished , racism , it follows , would by no means be disposed of once and for all .
it appears to be an irreducible phenomenon of negative sociality as it settles into pre - reflectively lived schemata of perceptions , permeates the subjective system of relevance , and enters into the micro - practices of interaction ( such as the posture or the gaze ) , in the daily reactivation of which it is tacitly reproduced .
yet , the question remains to which level these practices effectively reach . in this context
, i have in mind not only the institutionalized or systemic forms , in shape of which a racism against one s will68 characterizes also those societies in which but very few would explicitly understand or define themselves as racist and where , in contrast , the irrelevance of racializing identifications seems to prevail .
rather , i am thinking of the humanistic universalism , which demands the abolishment of such particularizing identifications and which , in doing so , fixes the selective normativity of its position , i.e. , fixes the way the other is to appear in a world in which i do not need to thematize my identity precisely because it is an invisible norm.69 and finally , i have in mind in particular the institutional desire for good practice , which almost teleologically glorifies the social promise of diversity , with the result that the desire for signs of resistance changes all too rapidly into a resistance to understanding racism as an ongoing and unfinished history.70 to conclude , the performative magic of racism is not only embedded into the meaningful constitution of the social world , i.e. , into the normal societal functioning.71 it finally returns in a dialectics of equality and alterity that haunts all our necessary attempts of a comparison of the incomparable.72 insofar as the desire for identity and thus the identification of the other is constitutive for this constitution,73 racism structurally inhabits it . as levinas
it is [ indeed ] obvious that it is in the knowledge of the other as a simple individual the individual of a genus , a class , or a race that peace with the other changes into hatred ; it is the approach to the other as a
kind of this or that.74 this reflection is important , but fails to provide us with an adequate demonstration of the reasons of such hate . from a phenomenological point of view
, such hatred can be shown to be ignited by our anxiety to lose our grip on the other ( in order to enable her to appear in her likeness ) as well as the unsettling experience that we ourselves might not fit the norm of this us . to show this | this paper addresses racism from a phenomenological viewpoint . its main task is , ultimately , to show that racism as a process of negative socialization does not amount to a contingent deficiency that simply disappears under the conditions of a fully integrated society .
in other words , i suspect that racism does not only indicate a lack of integration , solidarity , responsibility , recognition , etc . ;
rather , that it is , in its extraordinary negativity , a socially constitutive phenomenon per se . after suggesting phenomenology s potential to tackle the question of racism , i will focus on the experiential oppressiveness of racism , i.e. , the ways in which it affects its victims lived experiences , in transforming their habitual ways of life and , finally , their subjectivities .
my major thesis is that racism works via both inter - kinaesthetically as well as symbolically inflicted distortions of the victim s body schema .
as such a process of negative socialization , racism , however , influences the embodied self - conception of the oppressor , who finds himself compelled to adhere to some kind of invisible norm such as , e.g. , whiteness . | Introduction
Three preliminary considerations
On the phenomenology of racist methods
Conclusion | with this in mind , in this paper , i seek to analyze racism as a phenomenon of negative sociality and , thereby , to view it from a socio - phenomenological perspective . third , i will address , in brief , why i consider phenomenology suitable for an analysis of racism as being such a phenomenon . rather , my analysis will center on , to my mind , the invariant methods , which characterize racism as a specific form of social practice ( or social technology ) that exploits our dependency upon bodily identifications . in the concluding section ,
i seek to clarify , at least in outline , whether and to what extent racism can be understood as an irreducible phenomenon of negative sociality and how such an understanding of racism bears on the way in which it is dealt with socially . negativity does not merely amount to a contingent deficiency of justice , trust , responsibility , communalization , solidarity , communication , and so on , as tends to be assumed by the mainstream of social philosophy , where it is at all sensitive to this issue . whether a philosophical perspective assumes that such negative experiences can be countered by a unity that is to be realized through harmony , universality , concordia , homonoia , the reciprocity of perspectives , the non - violent discourse , or the ideal communication society , whilst a political perspective opposes them to the ideals of a fully integrated society or an organic community , under the unifying force of which such negativity should simply dissolve : in all these cases , negativity is presented as relative , as pure deficiency or as an exception in social functioning,4 i.e. negativity does not merely amount to a contingent deficiency of justice , trust , responsibility , communalization , solidarity , communication , and so on , as tends to be assumed by the mainstream of social philosophy , where it is at all sensitive to this issue . whether a philosophical perspective assumes that such negative experiences can be countered by a unity that is to be realized through harmony , universality , concordia , homonoia , the reciprocity of perspectives , the non - violent discourse , or the ideal communication society , whilst a political perspective opposes them to the ideals of a fully integrated society or an organic community , under the unifying force of which such negativity should simply dissolve : in all these cases , negativity is presented as relative , as pure deficiency or as an exception in social functioning,4 i.e. at stake in all these cases
, there is and at least until here , the minimum consensus seems to reacha process of the social construction of purportedly natural inequality , which is an attempt to find a natural explanation for social differences.12 as a hegemonic scheme of explanation , racism therefore always also mirrors the power relations that shape the integrity of societies internally and externally . this dual direction of the impact of racist processes of socialization , which as a result works on the basis of a dual logic of including exclusion and excluding inclusion , is to my mind
constitutive for the phenomenon.in accordance with what has been stated so far , i understand racism as a social process of hetero - normative meaning - constitution,14 which holds out the prospect of an illusory sense of community through bodily desocializing others and thus charts identity - relevant motivations for actions for those who seek to belong to this imagined community . they do seem comparable , however , insofar as they construct the otherswithin society as well as outside of itas deficient beings and locate them beyond all respective social dualisms.15 from this insight follows moreover that[t]he achievement of identity at the expense and to the detriment of others does not only generate different degrees of being human , but also destabilizes the very context that it founds . at the same time as the border to inferiority
is maintained outwardly , it is drawn into the very inner of society in form of a flexible predetermined breakage point.16thus it follows that as a form of negative socialization , racism must be viewed as an exemplary phenomenon of negative sociality because it is not only a negating social practice , but also makes us aware of the negativity of the social itself , i.e. at stake in all these cases
, there is and at least until here , the minimum consensus seems to reacha process of the social construction of purportedly natural inequality , which is an attempt to find a natural explanation for social differences.12 as a hegemonic scheme of explanation , racism therefore always also mirrors the power relations that shape the integrity of societies internally and externally . this dual direction of the impact of racist processes of socialization , which as a result works on the basis of a dual logic of including exclusion and excluding inclusion , is to my mind
constitutive for the phenomenon . they do seem comparable , however , insofar as they construct the otherswithin society as well as outside of itas deficient beings and locate them beyond all respective social dualisms.15 from this insight follows moreover that[t]he achievement of identity at the expense and to the detriment of others does not only generate different degrees of being human , but also destabilizes the very context that it founds . at the same time as the border to inferiority is maintained outwardly , it is drawn into the very inner of society in form of a flexible predetermined breakage point.16 [ t]he achievement of identity at the expense and to the detriment of others does not only generate different degrees of being human , but also destabilizes the very context that it founds . at the same time as the border to inferiority is maintained outwardly , it is drawn into the very inner of society in form of a flexible predetermined breakage point.16 thus it follows that as a form of negative socialization , racism must be viewed as an exemplary phenomenon of negative sociality because it is not only a negating social practice , but also makes us aware of the negativity of the social itself , i.e. ( c)can phenomenology , then , make a contribution to an analysis of racism as a process of negative socializationan undoubtedly historical process , into which flow concrete material foundations , structural forms of social organization , and ideological justifications ? on the assumption that racism , or to speak with r. miles , racializations are in many cases processes of meaning - constitution17 that are transmitted on a pre - reflexive level and that carry with them identity - relevant choices of identification or experiences of exclusion , a phenomenological or , more precisely , a socio - phenomenological approach to analysis seems fitting . the first concerns the specificity of the subject matter violence or a relation of violence , the second relates to the specific methodology of phenomenological analysis.the practice of phenomenology allows for what , following husserl , we could designate as an ethical epoch.18 i understand the task of such an epoch to be the bracketing of all those discourses on the reasons and the justifications of violence , in the normatively overdetermined ( and oftentimes moralizing ) light of which the analysis of direct violence and indirect relations of violence both implied by racism has to a large extent taken place hitherto . focusing their structure , pre - reflective functioning , and genesis
, a phenomenology of violence intends to show how subjects grow indifferent to the suffering of others , and that they can hence no longer perceive the singularity of their
ethical demand.20here , the second reason for my choice of the phenomenological approach becomes perspicuous : it is the only approach that is in a position to steer clear of the nominalistic and essentialist misinterpretations of the phenomenon , in order to trace , conversely , the effective reality of racializations in theeverydayness of lived experience , personal habitualities , and the institutions of our life - world.21 this implies , more concisely , that so - called meaning - constitutions can be comprehensively reconstructed neither in reference to the motives of a self - reflective subject capable of acting instrumentally nor in taking recourse to structures by which actions are functionally governed . in viewing meaning - constitution and self - constitution as such a relational and basically inter - corporeal occurrence
ultimately , it is only in the latter s light that phenomenology is able to zoom in adequately on the efficiency previously mentioned , i.e. , on the performative force and apparent magic of culturally overdetermined nexuses of sense , and respectively , of hegemonic regimes of sense - formation ( such as the concept of race or the pseudo - scientific discourse on race).23 can phenomenology , then , make a contribution to an analysis of racism as a process of negative socializationan undoubtedly historical process , into which flow concrete material foundations , structural forms of social organization , and ideological justifications ? the first concerns the specificity of the subject matter violence or a relation of violence , the second relates to the specific methodology of phenomenological analysis.the practice of phenomenology allows for what , following husserl , we could designate as an ethical epoch.18 i understand the task of such an epoch to be the bracketing of all those discourses on the reasons and the justifications of violence , in the normatively overdetermined ( and oftentimes moralizing ) light of which the analysis of direct violence and indirect relations of violence both implied by racism has to a large extent taken place hitherto . focusing their structure , pre - reflective functioning , and genesis , a phenomenology of violence intends to show how subjects grow indifferent to the suffering of others , and that they can hence no longer perceive the singularity of their
ethical demand.20here , the second reason for my choice of the phenomenological approach becomes perspicuous : it is the only approach that is in a position to steer clear of the nominalistic and essentialist misinterpretations of the phenomenon , in order to trace , conversely , the effective reality of racializations in theeverydayness of lived experience , personal habitualities , and the institutions of our life - world.21 this implies , more concisely , that so - called meaning - constitutions can be comprehensively reconstructed neither in reference to the motives of a self - reflective subject capable of acting instrumentally nor in taking recourse to structures by which actions are functionally governed . focusing their structure , pre - reflective functioning , and genesis , a phenomenology of violence intends to show how subjects grow indifferent to the suffering of others , and that they can hence no longer perceive the singularity of their
ethical demand.20 here , the second reason for my choice of the phenomenological approach becomes perspicuous : it is the only approach that is in a position to steer clear of the nominalistic and essentialist misinterpretations of the phenomenon , in order to trace , conversely , the effective reality of racializations in theeverydayness of lived experience , personal habitualities , and the institutions of our life - world.21 this implies , more concisely , that so - called meaning - constitutions can be comprehensively reconstructed neither in reference to the motives of a self - reflective subject capable of acting instrumentally nor in taking recourse to structures by which actions are functionally governed . in viewing meaning - constitution and self - constitution as such a relational and basically inter - corporeal occurrence
ultimately , it is only in the latter s light that phenomenology is able to zoom in adequately on the efficiency previously mentioned , i.e. , on the performative force and apparent magic of culturally overdetermined nexuses of sense , and respectively , of hegemonic regimes of sense - formation ( such as the concept of race or the pseudo - scientific discourse on race).23
thus , the direction of the impact of a phenomenological recourse to lived experience is clearly determined . neither is it a sense that emerges completely behind the back of the active i. rather , for its identity - relevant articulation , this sense requires the transition through the historic - cultural interworld of human symbolisms as well as the creative re - ordering of fact and hazard.28 in other words , the experience of the other thus bears a sense that is only articulated in the concrete intersubjective encounter , even though in this case and this point is crucial it does not indicate a reciprocity of perspectives , which would secure for us at least a minimal access to a shared world . on the contrary , it is the concrete modality of racist practices , the mode in which it addresses others , which precludes this possibility , i.e. although it rests essentially on sartre s theory of a bodily being - for - others,29 fanon , at the same time , also seeks to do justice to the basic existential factum that is masked by sartre : for the black man , the white man is not simply only the other but also the master , whether real or imaginary.30 not only does the concrete failure of the dialectic of recognition that is constitutive for sartre s approach become manifest thereby ; with fanon , further , we are in the position to demonstrate that in the form of symbolically instituted power - relations , this factum informs the habitus of bodily self - experience of the
racialized objects31 and is thus performatively reproduced.the black man among his own in the twentieth century does not know at what moment his inferiority comes into being through the other ( ) and then the occasion arose when i had to meet the white man s eyes . it does not impose itself on me ; it is , rather , a definitive structuring of the self and the world definitive because it creates a real dialectic between my body and the world.32 the black man among his own in the twentieth century does not know at what moment his inferiority comes into being through the other ( ) and then the occasion arose when i had to meet the white man s eyes . it does not impose itself on me ; it is , rather , a definitive structuring of the self and the world definitive because it creates a real dialectic between my body and the world.32 following fanon , the body schema that is referred to here , which merleau - ponty thematized as the linchpin of the being - for - the - world mentioned earlier , describes a merely superficial mode of experiencing lived embodiment . i thought that what i had in hand was to construct a physiological self , to balance space , to localize sensations , and here i was called on for more.33 the body schema as thematized by phenomenology seems , in this regard , to be the schema of a body that is in the world with - itself , a body that lives in a pre - reflexive balance with its surroundings , a body whose habitual performance knowledge is lived unproblematically . as a consequence ,
his orientation in the world becomes questionable , the accessibility of certain objects seems suddenly problematic , and more generally the body schema breaks down to give room to a
racial epidermal schema.34 this schema forces itself upon the perception of the social space and its institutions and organizes action in the light of a retrospective reoccupation of a space that i already inhabited.35 the lived space as such becomes white , in the sense of that absent centre against which others appear only as deviants , or points of deviation.36 the habitual incorporation of lived space re - oriented in this vein thus creates habit worlds and leads to the institutionalization of a certain likeness, which makes non - white bodies feel uncomfortable , exposed , visible , different , when they take up this place.37 the
i.e. i subjected myself to an objective examination , i discovered my blackness , my ethnic characteristics ; and i was battered down by tom - toms , cannibalism , intellectual deficiency , fetichism , racial defects , slave - ships , and above all else , above all :
, on racism as a symbolically transmitted and reproducedrelation of domination that generates patterns of interaction prone of violence
therefore brings something else to the fore , something on which anti - racist discussions would only later begin to focus : the insight that in correlation to the symbolic violence of racism mentioned already , i.e. such attribution practices employ a logic of mutual exclusion that is expressed in pairs of opposites such as culture / barbarism , civilization / savageness , purity / impurity , chosenness / fallenness or significance / inferiority.50 this process of an inferiorizing positing of difference represents a heteronormative form of socialization , which is ultimately based not on an always discriminating inclusion , but in principle on segregation and exclusion , i.e. what has been said so far can , however , by no means only be demonstrated in the context of the infernal machine of colonialism,55 which is , of course , of decisive importance from a historical perspective , for the genesis of a racial racism and from a genealogical perspective , for subsequent national racism.56 rather to take a big leap we could say that it also becomes manifest in the affective micro - practices of everyday racist interactions . as a result of the subject s stigmatizing embodiment
, its self - experience is haunted by difficulties in the development of [ the ] bodily schema,62 whilst our i can is usually , to put it with sartre , the neglected , passed by in silence.63 the historical - racist schema , that comes to overlay the body schema,64 thus produces a habitus that seems to carry the stigma of difference , which is mediated discursively , always already inscribed into it : regardless of whether this difference is taken up affirmatively and thus performatively reproduced , or whether it is discarded as objectively non - existing , to be evoked as an even more dangerous , for invisible danger as difference , it is powerfully reflected in the amputated body schema , as fanon describes it , or in the inner overdetermination of the actions of the racially dominated , to which sartre refers in his anti - semite and jew.65 in a nutshell , in light of the examples mentioned , racism can be grasped as the bodily inferiorization of the other which practices la limite the other s desocialization . it appears to be an irreducible phenomenon of negative sociality as it settles into pre - reflectively lived schemata of perceptions , permeates the subjective system of relevance , and enters into the micro - practices of interaction ( such as the posture or the gaze ) , in the daily reactivation of which it is tacitly reproduced . in this context
, i have in mind not only the institutionalized or systemic forms , in shape of which a racism against one s will68 characterizes also those societies in which but very few would explicitly understand or define themselves as racist and where , in contrast , the irrelevance of racializing identifications seems to prevail . , fixes the way the other is to appear in a world in which i do not need to thematize my identity precisely because it is an invisible norm.69 and finally , i have in mind in particular the institutional desire for good practice , which almost teleologically glorifies the social promise of diversity , with the result that the desire for signs of resistance changes all too rapidly into a resistance to understanding racism as an ongoing and unfinished history.70 to conclude , the performative magic of racism is not only embedded into the meaningful constitution of the social world , i.e. as levinas
it is [ indeed ] obvious that it is in the knowledge of the other as a simple individual the individual of a genus , a class , or a race that peace with the other changes into hatred ; it is the approach to the other as a
kind of this or that.74 this reflection is important , but fails to provide us with an adequate demonstration of the reasons of such hate . from a phenomenological point of view
, such hatred can be shown to be ignited by our anxiety to lose our grip on the other ( in order to enable her to appear in her likeness ) as well as the unsettling experience that we ourselves might not fit the norm of this us . | [
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] |
les infections fongiques invasives sassocient la morbidit et la mortalit en cas de neutropnie secondaire un cancer hmatologique . les antifongiques empiriques sont utiliss pour en rduire les consquences .
la micafongine lui a t compare pour traiter des patients atteints dun cancer hmatologique et dune neutropnie prolonge .
les chercheurs ont men une tude de cohorte rtrospective auprs de patients atteints dun cancer hmatologique et dune neutropnie profonde pendant au moins dix jours et qui avaient reu de la micafongine ou de la caspofongine empirique pendant au moins cinq jours entre avril 2005 et novembre 2009 .
les rsultats positifs se fondaient sur un paramtre ultime composite : survie au moins sept jours aprs larrt de lantifongique , rsolution de linfection fongique de dpart , absence deffets indsirables et de perce de linfection fongique .
les infections fongiques taient dfinies conformment aux critres des dfinitions rvises de maladie fongique invasive de lorganisation europenne de recherche sur le traitement du cancer et linvasive fungal infections cooperative group et du national institute of allergy and infectious diseases mycoses study group ( eortc - msg ) , avec la modification aux critres dimagerie diagnostique .
la micafongine fonctionnait de manire similaire la caspofongine ( 60,4 % [ 29 sur 48 ] plutt que 57,3 % [ 47 sur 82 ] , respectivement ; p=0,729 ) .
la survie tait plus leve dans le groupe sous micafongine que sous caspofongine ( 100 % [ 48 sur 48 ] plutt que 89 % [ 73 sur 82 ] ; p=0,02 ) .
aucune infection fongique invasive na t constate au dpart dans le groupe prenant de la micafongine , mais trois infections dmontres traites la caspofongine ( 3,7 % ; p=0,18 ) lont t .
trois perces dinfections dmontres t observes dans le groupe prenant de la micafongine ( trois sur 48 [ 27,3 % ] ) , mais aucune dans celui prenant de la caspofongine ( zro 82 ; p=0,02 ) .
la micafongine a une efficacit similaire celle de la caspofongine comme thrapie antifongique empirique chez les patients neutropniques fbriles atteints dun cancer hmatologique .
a retrospective cohort analysis involving patients with acute leukemia admitted to the princess margaret cancer centre ( university health network , toronto , ontario ) between april 2005 and november 2009 was conducted .
all patients reviewed had received empirical antifungal therapy with either caspofungin or micafungin for persistent fever during a period of profound neutropenia .
caspofungin was the sole echinocandin used for persistent fever until june 2008 , when micafungin was introduced as therapy for persistent fever in profoundly neutropenic patients .
patients were identified through the department of pharmacy dispensing records at the hospital and were enrolled in a 1:2 manner for micafungin versus caspofungin .
approval for data collection was obtained from the research ethics board of the university health network ( toronto , ontario ) .
patients diagnosed with hematological malignancies housed in specific patient wards at the princess margaret cancer centre and who received caspofungin or micafungin were initially identified from pharmacy records .
thereafter , patients were only identified by their medical record number and data were retrieved from the electronic patient record . to be included in the present study , patients had to meet stipulated criteria : have a hematological malignancy ; undergo induction , reinduction for failed remission or relapse , or consolidation chemotherapy , or have myelodysplastic syndrome ( mds ) treated with azacytadine ; be profoundly neutropenic , defined by < 0.510 neutrophils / l for a minimum of 10 days ; and be treated with caspofungin or micafungin for a minimum of five days .
the following information was collected : age ; sex ; length of hospital stay ; duration of neutropenia ( the first day the neutrophil count fell to < 0.510/l cells was considered to be day 1 of neutropenia , and the first day of neutrophil recovery 0.510/l cells was considered to be the end of the neutropenic period ) ; primary diagnosis ( acute myeloid leukemia , acute lymphoblastic leukemia [ all ] , mds , chronic myeloid leukemia ) ; antifungal prophylaxis ( drug and dose ) ; type of chemotherapy ( induction , reinduction or consolidation ) ; chemotherapeutic agents ; duration of neutropenia ; start and end date of systemic antifungal therapy ; computed tomography ( ct ) scan studies ; treatment of baseline fungal infection ; adverse events related to caspofungin and micafungin necessitating discontinuation of therapy ; survival ; and breakthrough fungal infections .
data regarding the systemic empirical antifungal treatments were accrued as follows : the day on which either antifungal treatment was started was considered to be day 0 ; and assessments were performed again for each patient at days three to five of therapy , two weeks and four weeks .
a composite end point was evaluated for each antifungal treatment course of micafungin and caspofungin , respectively .
antifungal treatment success reflected survival at seven days of antifungal treatment , successful treatment of baseline fungal infections , absence of adverse events due to the antifungal medication and the absence of breakthrough fungal infections .
fever , which is usually included as one of the variables of the composite end point , was not assessed due to the lack of consistent temperature records in the electronic patient record .
baseline fungal infection was defined as a fungal infection that was already identified on inclusion in the study or within 48 h of starting micafungin or caspofungin .
breakthrough fungal infection was defined as a fungal infection diagnosed > 48 h after starting antifungal treatment .
however , a possible ifi was determined on the basis of historical commentary of persistent fever unresponsive to appropriate antibacterial therapy with ct imaging findings consistent with pulmonary infection that did not meet the prespecified ct scan findings outlined in the eortc - msg criteria ( 7 ) .
abnormal chest ct findings similar to those previously documented by others were considered to be consistent with ifi ( 8) . the diagnosis of presumed fungal infection was made based on the presence of infectious symptoms of presumed fungal origin , regardless of whether antifungal prophylaxis was being administered , because appropriate antibacterial therapy was being used and there was no obvious focus of infection documented .
demographic information for the treatment groups was assessed and compared using tests for categorical variables , student s t test for normally distributed continuous variables and rank - sum test for non - normally distributed continuous variables .
overall success rates between the treatment groups were evaluated on the basis of the four parameters comprising the composite end point as stated above .
differences between the groups in overall success and each of the four end points were evaluated using testing .
a forest plot was generated using r version 3.1.0 and the 95% cis for the absolute difference between the treatment groups were calculated using a cochran - mantel haenszel method ( 9 ) .
a retrospective cohort analysis involving patients with acute leukemia admitted to the princess margaret cancer centre ( university health network , toronto , ontario ) between april 2005 and november 2009 was conducted .
all patients reviewed had received empirical antifungal therapy with either caspofungin or micafungin for persistent fever during a period of profound neutropenia .
caspofungin was the sole echinocandin used for persistent fever until june 2008 , when micafungin was introduced as therapy for persistent fever in profoundly neutropenic patients .
patients were identified through the department of pharmacy dispensing records at the hospital and were enrolled in a 1:2 manner for micafungin versus caspofungin .
approval for data collection was obtained from the research ethics board of the university health network ( toronto , ontario ) .
patients diagnosed with hematological malignancies housed in specific patient wards at the princess margaret cancer centre and who received caspofungin or micafungin were initially identified from pharmacy records .
thereafter , patients were only identified by their medical record number and data were retrieved from the electronic patient record . to be included in the present study , patients had to meet stipulated criteria : have a hematological malignancy ; undergo induction , reinduction for failed remission or relapse , or consolidation chemotherapy , or have myelodysplastic syndrome ( mds ) treated with azacytadine ; be profoundly neutropenic , defined by < 0.510 neutrophils / l for a minimum of 10 days ; and be treated with caspofungin or micafungin for a minimum of five days .
the following information was collected : age ; sex ; length of hospital stay ; duration of neutropenia ( the first day the neutrophil count fell to < 0.510/l cells was considered to be day 1 of neutropenia , and the first day of neutrophil recovery 0.510/l cells was considered to be the end of the neutropenic period ) ; primary diagnosis ( acute myeloid leukemia , acute lymphoblastic leukemia [ all ] , mds , chronic myeloid leukemia ) ; antifungal prophylaxis ( drug and dose ) ; type of chemotherapy ( induction , reinduction or consolidation ) ; chemotherapeutic agents ; duration of neutropenia ; start and end date of systemic antifungal therapy ; computed tomography ( ct ) scan studies ; treatment of baseline fungal infection ; adverse events related to caspofungin and micafungin necessitating discontinuation of therapy ; survival ; and breakthrough fungal infections .
data regarding the systemic empirical antifungal treatments were accrued as follows : the day on which either antifungal treatment was started was considered to be day 0 ; and assessments were performed again for each patient at days three to five of therapy , two weeks and four weeks .
a composite end point was evaluated for each antifungal treatment course of micafungin and caspofungin , respectively .
antifungal treatment success reflected survival at seven days of antifungal treatment , successful treatment of baseline fungal infections , absence of adverse events due to the antifungal medication and the absence of breakthrough fungal infections .
fever , which is usually included as one of the variables of the composite end point , was not assessed due to the lack of consistent temperature records in the electronic patient record .
baseline fungal infection was defined as a fungal infection that was already identified on inclusion in the study or within 48 h of starting micafungin or caspofungin .
breakthrough fungal infection was defined as a fungal infection diagnosed > 48 h after starting antifungal treatment .
however , a possible ifi was determined on the basis of historical commentary of persistent fever unresponsive to appropriate antibacterial therapy with ct imaging findings consistent with pulmonary infection that did not meet the prespecified ct scan findings outlined in the eortc - msg criteria ( 7 ) .
abnormal chest ct findings similar to those previously documented by others were considered to be consistent with ifi ( 8) . the diagnosis of presumed fungal infection was made based on the presence of infectious symptoms of presumed fungal origin , regardless of whether antifungal prophylaxis was being administered , because appropriate antibacterial therapy was being used and there was no obvious focus of infection documented .
demographic information for the treatment groups was assessed and compared using tests for categorical variables , student s t test for normally distributed continuous variables and rank - sum test for non - normally distributed continuous variables .
overall success rates between the treatment groups were evaluated on the basis of the four parameters comprising the composite end point as stated above .
differences between the groups in overall success and each of the four end points were evaluated using testing .
a forest plot was generated using r version 3.1.0 and the 95% cis for the absolute difference between the treatment groups were calculated using a cochran - mantel haenszel method ( 9 ) .
the investigators initially identified 340 patients through retrospective chart review , who received echinocandin therapy with caspofungin or micafungin for fever and neutropenia during the study period .
a total of 92 and 118 patients were excluded from the caspofungin and micafungin groups , respectively .
reasons for exclusions were : duplicate entries ; neutropenia for < 10 days ; receipt of the study drug for < 5 days ; or receipt of the study drug concomitantly with another agent .
patients who received interrupted courses of the study drug or received it while non - neutropenic , and those who were stem cell transplant recipients were also excluded .
in addition , 58 patients were excluded from the micafungin group because they received prophylactic micafungin at a dose of 50 mg per day .
the demographic characteristics of the patients analyzed in the study are summarized in table 1 .
the median ages of the patients were 47 and 54 years of age in the micafungin and caspofungin groups , respectively ( p=0.02 ) .
there was no difference in the sex distribution between the groups , with 56% ( 27 of 48 ) and 61% ( 50 of 82 ) being male in the micafungin and the caspofungin groups , respectively ( p=0.6 ) .
the median length of admission was similar in both groups ( 36 days in the micafungin and 39 days in the caspofungin patients ; p=0.8 ) .
the most common underlying diagnosis was acute myeloid leukemia ( 79% [ 38 of 48 ] in the micafungin group and 83% [ 68 of 82 ] in the caspofungin group ) .
the remaining diagnoses were all , relapsed all and mds . there were no significant differences in chemotherapeutic regimens used for the patients .
forty percent ( 19 of 48 ) of the micafungin group compared with 54% ( 44 of 82 ) of the caspofungin patients received induction regimens , while 42% ( 20 of 48 ) versus 30% ( 25 of 82 ) received reinduction chemotherapy .
the median duration of neutropenia was similar in in the micafungin and caspofungin groups ( 25 days and 29 days , respectively ; p=0.7 ) .
seventy - three percent ( 35 of 48 ) of the patients in the micafungin group received antifungal prophylaxis , in contrast to 57% ( 47 of 82 ) of the patients in the caspofungin group ( p=0.08 ) .
of the 48 patients who received antifungal prophylaxis in the micafungin group , 31 received fluconazole , while three received micafungin 50 mg and one received voriconazole ( table 2 ) . in the caspofungin group ,
38 of 82 received fluconazole , one received caspofungin , five received voriconazole , one received itraconazole and two received amphotericin b. caspofungin prophylaxis was provided to one patient in the caspofungin group for seven days before the development of neutropenia ; the patient was then transitioned to empirical antifungal therapy .
no baseline fungal infection was noted ; however , the patient subsequently developed pulmonary ct findings indicative of a possible breakthrough infection and was considered to represent a failure .
all patients received antibacterial agents for their febrile neutropenic episode , but no significant differences were noted between the two groups .
in addition , there was a minority of patients in each group ( nine in the micafungin group and 19 in the caspofungin group ) who required previous antifungal therapy due to presumed fungal infection but were judged to have failed this therapy due to ongoing infectious symptoms and/or abnormal radiological findings and were , thus , placed on one of the study medications .
the median duration of therapy for the micafungin and caspofungin groups was 16 days and 14.5 days , respectively ( p=0.62 ) ( table 1 ) .
as mentioned above , a successful treatment outcome was adjudicated based on four parameters : survival 7 days after the discontinuation of antifungal therapy ; treatment of baseline fungal infection ; absence of breakthrough fungal infections ; and absence of discontinuation of antifungal therapy due to adverse events .
all patients in the micafungin group ( 100% [ 48 of 48 ] ) survived for a minimum of seven days after discontinuation of micafungin compared with 89% ( 73 of 82 ) in the caspofungin group ( p=0.02 ) .
as mentioned previously , all fungal infections were categorized according to the eortc - msg criteria as proven , probable and possible infections ( 7 ) .
the modified ct findings of the chest indicating a fungal infection , as described by others ( 8) , were also adopted .
baseline fungal infections were defined as those diagnosed before or within 72 h after starting the study antifungal .
however , there were 19 patients ( 40% ) with possible ifis at baseline and the remaining patients ( n=29 ) received micafungin as empirical treatment ( 60% ) for various symptoms ( predominantly fever ) .
in the caspofungin group , there were three ( 4% ) proven baseline infections ( p=0.18 ) .
no patients in the caspofungin group had a probable fungal infection , while 56% ( 46 of 82 ) had a possible fungal infection ( p=0.07 compared with micafungin ) and the remaining 40% ( 33 of 82 ) ( p=0.03 ) received caspofungin as empirical antifungal treatment .
overall , baseline fungal infections ( proven , probable and possible ) were successfully treated in eight ( 42% ) patients in the micafungin treatment group compared with 26 ( 53% ) in the caspofungin group ( p=0.42 ) .
breakthrough ifi was once again classified as per the eortc - msg criteria as proven , probable and possible infection ( 7 ) .
there were three proven breakthrough ifis ( three of 48 [ 6% ] ) among the micafungin therapy patients ( two candidemias cause by candida parapsilosis and one biopsy - proven mucormycosis of the skin ) .
however , eight ( 17% ) of 48 patients were classified as having possible ifis with no identifiable pathogen noted .
thirty - seven ( 77% ) individuals had no breakthrough fungal infections . in contrast , none of the caspofungin - treated patients developed a proven ( p=0.02 ) or probable ifi .
a possible breakthrough ifi occurred in 11 ( 13% ) ( p=0.61 ) ; while 71 of the 82 patients ( 86.6% ) had no breakthrough ifi ; this was not statistically significant compared with the micafungin group ( p=0.16 ) ( table 3 ) .
no adverse events led to discontinuation of micafungin , whereas caspofungin therapy had to be stopped in one of the 82 patients ( 1.2% ) due to an adverse event manifested by elevated liver enzymes ( aspartate aminotransferase > 5 times the baseline value ) ( p=0.44 ) ( table 3 ) .
failure in any of the four parameters used to assess successful therapy resulted in determining failure of the course of empirical therapy for either antifungal agent .
thus , the overall success rate for micafungin was 60.4% ( 29 of 48 ) , whereas for caspofungin it was 57.3% ( 47 of 82 ) ( p=0.729 ) .
the absolute differences between the micafungin and caspofungin patient groups ( 95% cis ) with regard to each end point as well as overall success are presented in figure 2 .
the median duration of therapy for the micafungin and caspofungin groups was 16 days and 14.5 days , respectively ( p=0.62 ) ( table 1 ) .
as mentioned above , a successful treatment outcome was adjudicated based on four parameters : survival 7 days after the discontinuation of antifungal therapy ; treatment of baseline fungal infection ; absence of breakthrough fungal infections ; and absence of discontinuation of antifungal therapy due to adverse events .
the treatment outcomes are summarized in table 3 . both drugs performed similarly in all parameters except for survival .
all patients in the micafungin group ( 100% [ 48 of 48 ] ) survived for a minimum of seven days after discontinuation of micafungin compared with 89% ( 73 of 82 ) in the caspofungin group ( p=0.02 ) .
as mentioned previously , all fungal infections were categorized according to the eortc - msg criteria as proven , probable and possible infections ( 7 ) .
the modified ct findings of the chest indicating a fungal infection , as described by others ( 8) , were also adopted .
baseline fungal infections were defined as those diagnosed before or within 72 h after starting the study antifungal . there were no proven or probable baseline fungal infections among the micafungin patients .
however , there were 19 patients ( 40% ) with possible ifis at baseline and the remaining patients ( n=29 ) received micafungin as empirical treatment ( 60% ) for various symptoms ( predominantly fever ) . in the caspofungin group , there were three ( 4% ) proven baseline infections ( p=0.18 ) .
no patients in the caspofungin group had a probable fungal infection , while 56% ( 46 of 82 ) had a possible fungal infection ( p=0.07 compared with micafungin ) and the remaining 40% ( 33 of 82 ) ( p=0.03 ) received caspofungin as empirical antifungal treatment .
overall , baseline fungal infections ( proven , probable and possible ) were successfully treated in eight ( 42% ) patients in the micafungin treatment group compared with 26 ( 53% ) in the caspofungin group ( p=0.42 ) .
breakthrough ifi was once again classified as per the eortc - msg criteria as proven , probable and possible infection ( 7 ) .
there were three proven breakthrough ifis ( three of 48 [ 6% ] ) among the micafungin therapy patients ( two candidemias cause by candida parapsilosis and one biopsy - proven mucormycosis of the skin ) .
however , eight ( 17% ) of 48 patients were classified as having possible ifis with no identifiable pathogen noted .
in contrast , none of the caspofungin - treated patients developed a proven ( p=0.02 ) or probable ifi .
a possible breakthrough ifi occurred in 11 ( 13% ) ( p=0.61 ) ; while 71 of the 82 patients ( 86.6% ) had no breakthrough ifi ; this was not statistically significant compared with the micafungin group ( p=0.16 ) ( table 3 ) .
no adverse events led to discontinuation of micafungin , whereas caspofungin therapy had to be stopped in one of the 82 patients ( 1.2% ) due to an adverse event manifested by elevated liver enzymes ( aspartate aminotransferase > 5 times the baseline value ) ( p=0.44 ) ( table 3 ) .
failure in any of the four parameters used to assess successful therapy resulted in determining failure of the course of empirical therapy for either antifungal agent .
thus , the overall success rate for micafungin was 60.4% ( 29 of 48 ) , whereas for caspofungin it was 57.3% ( 47 of 82 ) ( p=0.729 ) .
the absolute differences between the micafungin and caspofungin patient groups ( 95% cis ) with regard to each end point as well as overall success are presented in figure 2 .
prompted by the high rate of ifis in patients with acute leukemia and the subsequent morbidity and mortality of these infections in persistently febrile neutropenic patients ( 10 ) , empirical antifungal therapy , aimed at targeting the most commonly encountered fungal pathogens ( ie , candida and aspergillus species ) , is a commonly used strategy for managing persistently febrile neutropenic patients ( 1 ) .
the echinocandins , caspofungin ( 3 ) , micafungin ( 11 ) and anidulafungin ( 12 ) , have demonstrated efficacy against candida .
moreover , caspofungin , anidulafungin and micafungin have demonstrated efficacy in treating invasive aspergillosis alone or in combination with other antifungal agents ( 1315 ) .
although micafungin was shown to be superior to fluconazole when used for primary antifungal prophylaxis in stem cell transplant recipients , with reduction in the rate of breakthrough invasive aspergillus infections in the micafungin arm ( 16 ) , the optimal micafungin dose to be used in treating invasive aspergillosis is still unknown ( 15 ) .
due to its lower wholesale noncontractual drug acquisition cost compared with caspofungin , micafungin offers a potential advantage .
however , while caspofungin has been approved as an empirical antifungal treatment for persistently febrile neutropenic cancer patients , the efficacy of micafungin for that indication is still unproven .
this issue prompted kubiak et al ( 6 ) to explore the use of micafungin as an empirical antifungal therapy in febrile neutropenic patients in a retrospective study .
that study , however , had a number of limitations : specifically , a heterogeneous patient population with varying degrees and durations of neutropenia , as previously mentioned .
we mandated a 10-day minimum duration of neutropenia for inclusion , which is the duration associated with the development of fungal infections ( 1 ) .
moreover , a minimum duration of five days of treatment in each arm was required for inclusion to ensure proper assessment for efficacy and side effects . in the present retrospective study
, we demonstrated that micafungin had a similar overall efficacy compared with caspofungin when used for treating persistently neutropenic patients with acute leukemia ( overall efficacy of 60.4% for micafungin versus 57.3% for caspofungin ) .
efficacy was substantiated in a robust fashion by using four of five efficacy end points based on previously used criteria to evaluate and compare efficacy of antifungals for this purpose ( 2,17,18 ) . with regard to the evaluation parameters and a comparison of the treatment groups , proven baseline fungal infections
were all candidemias , and occurred only in the caspofungin group , all of which were successfully treated .
we also included possible baseline ifis to enhance our results , demonstrating response rates of 42% in the micafungin group and 53% with caspofungin ( p=0.42 ) .
breakthrough infections occurred only in the micafungin group ; two were c parapsilosis candidemias , an expected breakthrough infection considering the well - documented inherent increased minimum inhibitory concentrations of c parapsilosis species to echinocandins ( 19 ) .
the third was due to biopsy - proven mucormycosis of the skin once more , a weakness in the antifungal coverage of the echinocandins ( 19 ) .
although our data demonstrate similar efficacy of micafungin compared with caspofungin when used to treat persistently neutropenic patients , one may question the robustness of this conclusion given the exclusion of fever as an end point .
nonetheless , the utility of including fever as a primary composite end point has been questioned by investigators , primarily due to the lack of specificity of fever as a sign of occult fungal infection , particularly in the studied population ( 20,21 ) and its lack of accuracy as a harbinger for ifi ( 22,23 ) .
the retrospective nature limited the ability to control for all the potential confounding factors , although the cohort design lessened this issue somewhat .
there were also few proven baseline fungal infections and all occurred in the caspofungin group .
in addition , there were no proven mold infections among the included patients , which makes drawing firm conclusions regarding the efficacy of micafungin to treat mold infections impossible .
furthermore , surrogate markers for invasive mold infection , such as galactomannan assays , were not available at the institution at the time of patients hospitalizations , which may partially explain the high number of possible ifis and the absence of probable ifis .
the lack of adherence to the standardized eortc - msg radiological criteria in our study may be viewed as another limitation . however , strict adherence to these radiological criteria may result in unnecessary exclusion of patients from trials that assess efficacy of antifungal therapies in treating ifis in neutropenic populations , as previously suggested ( 8) . to substantiate false identification of patients with bacterial respiratory tract infections as possible invasive pulmonary fungal infections , all patients with possible or probable infections based on abnormal radiological findings had negative sputum or bronchoalveolar lavage bacterial cultures at the time of inclusion .
micafungin demonstrated similar efficacy to caspofungin when used to empirically treat febrile neutropenic patients with hematological malignancies , as shown by the composite end point .
our study also substantiates the safety profile of micafungin at a dose of 100 mg .
a prospective , randomized clinical trial comparing caspofungin and micafungin for treatment of persistently febrile neutropenic patients with focus on outcomes with mold infections is warranted . | patients with neutropenia resulting from chemotherapy for hematological malignancies are at risk for considerable morbidity and mortality due to invasive fungal infections and should , thus , be treated with antifungal agents .
caspofungin has been one of the most common antifungal agents used for this purpose ; its analogue micafungin may also be appropriate , but has not been tested as extensively .
accordingly , the authors of this article conducted a retrospective study to compare these two agents ; the results contribute to the literature regarding the use of micafungin for the treatment of invasive fungal infections . | HISTORIQUE :
MTHODOLOGIE :
RSULTATS :
CONCLUSION :
METHODS
Study design
Patients
Treatment outcomes
Statistical analysis
RESULTS
Treatment outcomes
DISCUSSION
CONCLUSION | a retrospective cohort analysis involving patients with acute leukemia admitted to the princess margaret cancer centre ( university health network , toronto , ontario ) between april 2005 and november 2009 was conducted . all patients reviewed had received empirical antifungal therapy with either caspofungin or micafungin for persistent fever during a period of profound neutropenia . approval for data collection was obtained from the research ethics board of the university health network ( toronto , ontario ) . patients diagnosed with hematological malignancies housed in specific patient wards at the princess margaret cancer centre and who received caspofungin or micafungin were initially identified from pharmacy records . to be included in the present study , patients had to meet stipulated criteria : have a hematological malignancy ; undergo induction , reinduction for failed remission or relapse , or consolidation chemotherapy , or have myelodysplastic syndrome ( mds ) treated with azacytadine ; be profoundly neutropenic , defined by < 0.510 neutrophils / l for a minimum of 10 days ; and be treated with caspofungin or micafungin for a minimum of five days . the following information was collected : age ; sex ; length of hospital stay ; duration of neutropenia ( the first day the neutrophil count fell to < 0.510/l cells was considered to be day 1 of neutropenia , and the first day of neutrophil recovery 0.510/l cells was considered to be the end of the neutropenic period ) ; primary diagnosis ( acute myeloid leukemia , acute lymphoblastic leukemia [ all ] , mds , chronic myeloid leukemia ) ; antifungal prophylaxis ( drug and dose ) ; type of chemotherapy ( induction , reinduction or consolidation ) ; chemotherapeutic agents ; duration of neutropenia ; start and end date of systemic antifungal therapy ; computed tomography ( ct ) scan studies ; treatment of baseline fungal infection ; adverse events related to caspofungin and micafungin necessitating discontinuation of therapy ; survival ; and breakthrough fungal infections . data regarding the systemic empirical antifungal treatments were accrued as follows : the day on which either antifungal treatment was started was considered to be day 0 ; and assessments were performed again for each patient at days three to five of therapy , two weeks and four weeks . antifungal treatment success reflected survival at seven days of antifungal treatment , successful treatment of baseline fungal infections , absence of adverse events due to the antifungal medication and the absence of breakthrough fungal infections . fever , which is usually included as one of the variables of the composite end point , was not assessed due to the lack of consistent temperature records in the electronic patient record . demographic information for the treatment groups was assessed and compared using tests for categorical variables , student s t test for normally distributed continuous variables and rank - sum test for non - normally distributed continuous variables . overall success rates between the treatment groups were evaluated on the basis of the four parameters comprising the composite end point as stated above . differences between the groups in overall success and each of the four end points were evaluated using testing . a forest plot was generated using r version 3.1.0 and the 95% cis for the absolute difference between the treatment groups were calculated using a cochran - mantel haenszel method ( 9 ) . a retrospective cohort analysis involving patients with acute leukemia admitted to the princess margaret cancer centre ( university health network , toronto , ontario ) between april 2005 and november 2009 was conducted . all patients reviewed had received empirical antifungal therapy with either caspofungin or micafungin for persistent fever during a period of profound neutropenia . caspofungin was the sole echinocandin used for persistent fever until june 2008 , when micafungin was introduced as therapy for persistent fever in profoundly neutropenic patients . patients diagnosed with hematological malignancies housed in specific patient wards at the princess margaret cancer centre and who received caspofungin or micafungin were initially identified from pharmacy records . to be included in the present study , patients had to meet stipulated criteria : have a hematological malignancy ; undergo induction , reinduction for failed remission or relapse , or consolidation chemotherapy , or have myelodysplastic syndrome ( mds ) treated with azacytadine ; be profoundly neutropenic , defined by < 0.510 neutrophils / l for a minimum of 10 days ; and be treated with caspofungin or micafungin for a minimum of five days . the following information was collected : age ; sex ; length of hospital stay ; duration of neutropenia ( the first day the neutrophil count fell to < 0.510/l cells was considered to be day 1 of neutropenia , and the first day of neutrophil recovery 0.510/l cells was considered to be the end of the neutropenic period ) ; primary diagnosis ( acute myeloid leukemia , acute lymphoblastic leukemia [ all ] , mds , chronic myeloid leukemia ) ; antifungal prophylaxis ( drug and dose ) ; type of chemotherapy ( induction , reinduction or consolidation ) ; chemotherapeutic agents ; duration of neutropenia ; start and end date of systemic antifungal therapy ; computed tomography ( ct ) scan studies ; treatment of baseline fungal infection ; adverse events related to caspofungin and micafungin necessitating discontinuation of therapy ; survival ; and breakthrough fungal infections . data regarding the systemic empirical antifungal treatments were accrued as follows : the day on which either antifungal treatment was started was considered to be day 0 ; and assessments were performed again for each patient at days three to five of therapy , two weeks and four weeks . a composite end point was evaluated for each antifungal treatment course of micafungin and caspofungin , respectively . antifungal treatment success reflected survival at seven days of antifungal treatment , successful treatment of baseline fungal infections , absence of adverse events due to the antifungal medication and the absence of breakthrough fungal infections . fever , which is usually included as one of the variables of the composite end point , was not assessed due to the lack of consistent temperature records in the electronic patient record . demographic information for the treatment groups was assessed and compared using tests for categorical variables , student s t test for normally distributed continuous variables and rank - sum test for non - normally distributed continuous variables . overall success rates between the treatment groups were evaluated on the basis of the four parameters comprising the composite end point as stated above . differences between the groups in overall success and each of the four end points were evaluated using testing . a forest plot was generated using r version 3.1.0 and the 95% cis for the absolute difference between the treatment groups were calculated using a cochran - mantel haenszel method ( 9 ) . the investigators initially identified 340 patients through retrospective chart review , who received echinocandin therapy with caspofungin or micafungin for fever and neutropenia during the study period . reasons for exclusions were : duplicate entries ; neutropenia for < 10 days ; receipt of the study drug for < 5 days ; or receipt of the study drug concomitantly with another agent . the demographic characteristics of the patients analyzed in the study are summarized in table 1 . the median ages of the patients were 47 and 54 years of age in the micafungin and caspofungin groups , respectively ( p=0.02 ) . the most common underlying diagnosis was acute myeloid leukemia ( 79% [ 38 of 48 ] in the micafungin group and 83% [ 68 of 82 ] in the caspofungin group ) . there were no significant differences in chemotherapeutic regimens used for the patients . forty percent ( 19 of 48 ) of the micafungin group compared with 54% ( 44 of 82 ) of the caspofungin patients received induction regimens , while 42% ( 20 of 48 ) versus 30% ( 25 of 82 ) received reinduction chemotherapy . seventy - three percent ( 35 of 48 ) of the patients in the micafungin group received antifungal prophylaxis , in contrast to 57% ( 47 of 82 ) of the patients in the caspofungin group ( p=0.08 ) . of the 48 patients who received antifungal prophylaxis in the micafungin group , 31 received fluconazole , while three received micafungin 50 mg and one received voriconazole ( table 2 ) . no baseline fungal infection was noted ; however , the patient subsequently developed pulmonary ct findings indicative of a possible breakthrough infection and was considered to represent a failure . all patients received antibacterial agents for their febrile neutropenic episode , but no significant differences were noted between the two groups . in addition , there was a minority of patients in each group ( nine in the micafungin group and 19 in the caspofungin group ) who required previous antifungal therapy due to presumed fungal infection but were judged to have failed this therapy due to ongoing infectious symptoms and/or abnormal radiological findings and were , thus , placed on one of the study medications . the median duration of therapy for the micafungin and caspofungin groups was 16 days and 14.5 days , respectively ( p=0.62 ) ( table 1 ) . as mentioned above , a successful treatment outcome was adjudicated based on four parameters : survival 7 days after the discontinuation of antifungal therapy ; treatment of baseline fungal infection ; absence of breakthrough fungal infections ; and absence of discontinuation of antifungal therapy due to adverse events . all patients in the micafungin group ( 100% [ 48 of 48 ] ) survived for a minimum of seven days after discontinuation of micafungin compared with 89% ( 73 of 82 ) in the caspofungin group ( p=0.02 ) . as mentioned previously , all fungal infections were categorized according to the eortc - msg criteria as proven , probable and possible infections ( 7 ) . the modified ct findings of the chest indicating a fungal infection , as described by others ( 8) , were also adopted . baseline fungal infections were defined as those diagnosed before or within 72 h after starting the study antifungal . overall , baseline fungal infections ( proven , probable and possible ) were successfully treated in eight ( 42% ) patients in the micafungin treatment group compared with 26 ( 53% ) in the caspofungin group ( p=0.42 ) . there were three proven breakthrough ifis ( three of 48 [ 6% ] ) among the micafungin therapy patients ( two candidemias cause by candida parapsilosis and one biopsy - proven mucormycosis of the skin ) . thirty - seven ( 77% ) individuals had no breakthrough fungal infections . in contrast , none of the caspofungin - treated patients developed a proven ( p=0.02 ) or probable ifi . a possible breakthrough ifi occurred in 11 ( 13% ) ( p=0.61 ) ; while 71 of the 82 patients ( 86.6% ) had no breakthrough ifi ; this was not statistically significant compared with the micafungin group ( p=0.16 ) ( table 3 ) . no adverse events led to discontinuation of micafungin , whereas caspofungin therapy had to be stopped in one of the 82 patients ( 1.2% ) due to an adverse event manifested by elevated liver enzymes ( aspartate aminotransferase > 5 times the baseline value ) ( p=0.44 ) ( table 3 ) . failure in any of the four parameters used to assess successful therapy resulted in determining failure of the course of empirical therapy for either antifungal agent . thus , the overall success rate for micafungin was 60.4% ( 29 of 48 ) , whereas for caspofungin it was 57.3% ( 47 of 82 ) ( p=0.729 ) . the median duration of therapy for the micafungin and caspofungin groups was 16 days and 14.5 days , respectively ( p=0.62 ) ( table 1 ) . as mentioned above , a successful treatment outcome was adjudicated based on four parameters : survival 7 days after the discontinuation of antifungal therapy ; treatment of baseline fungal infection ; absence of breakthrough fungal infections ; and absence of discontinuation of antifungal therapy due to adverse events . the treatment outcomes are summarized in table 3 . all patients in the micafungin group ( 100% [ 48 of 48 ] ) survived for a minimum of seven days after discontinuation of micafungin compared with 89% ( 73 of 82 ) in the caspofungin group ( p=0.02 ) . as mentioned previously , all fungal infections were categorized according to the eortc - msg criteria as proven , probable and possible infections ( 7 ) . the modified ct findings of the chest indicating a fungal infection , as described by others ( 8) , were also adopted . there were no proven or probable baseline fungal infections among the micafungin patients . overall , baseline fungal infections ( proven , probable and possible ) were successfully treated in eight ( 42% ) patients in the micafungin treatment group compared with 26 ( 53% ) in the caspofungin group ( p=0.42 ) . there were three proven breakthrough ifis ( three of 48 [ 6% ] ) among the micafungin therapy patients ( two candidemias cause by candida parapsilosis and one biopsy - proven mucormycosis of the skin ) . in contrast , none of the caspofungin - treated patients developed a proven ( p=0.02 ) or probable ifi . a possible breakthrough ifi occurred in 11 ( 13% ) ( p=0.61 ) ; while 71 of the 82 patients ( 86.6% ) had no breakthrough ifi ; this was not statistically significant compared with the micafungin group ( p=0.16 ) ( table 3 ) . no adverse events led to discontinuation of micafungin , whereas caspofungin therapy had to be stopped in one of the 82 patients ( 1.2% ) due to an adverse event manifested by elevated liver enzymes ( aspartate aminotransferase > 5 times the baseline value ) ( p=0.44 ) ( table 3 ) . failure in any of the four parameters used to assess successful therapy resulted in determining failure of the course of empirical therapy for either antifungal agent . thus , the overall success rate for micafungin was 60.4% ( 29 of 48 ) , whereas for caspofungin it was 57.3% ( 47 of 82 ) ( p=0.729 ) . prompted by the high rate of ifis in patients with acute leukemia and the subsequent morbidity and mortality of these infections in persistently febrile neutropenic patients ( 10 ) , empirical antifungal therapy , aimed at targeting the most commonly encountered fungal pathogens ( ie , candida and aspergillus species ) , is a commonly used strategy for managing persistently febrile neutropenic patients ( 1 ) . moreover , caspofungin , anidulafungin and micafungin have demonstrated efficacy in treating invasive aspergillosis alone or in combination with other antifungal agents ( 1315 ) . although micafungin was shown to be superior to fluconazole when used for primary antifungal prophylaxis in stem cell transplant recipients , with reduction in the rate of breakthrough invasive aspergillus infections in the micafungin arm ( 16 ) , the optimal micafungin dose to be used in treating invasive aspergillosis is still unknown ( 15 ) . due to its lower wholesale noncontractual drug acquisition cost compared with caspofungin , micafungin offers a potential advantage . however , while caspofungin has been approved as an empirical antifungal treatment for persistently febrile neutropenic cancer patients , the efficacy of micafungin for that indication is still unproven . this issue prompted kubiak et al ( 6 ) to explore the use of micafungin as an empirical antifungal therapy in febrile neutropenic patients in a retrospective study . we mandated a 10-day minimum duration of neutropenia for inclusion , which is the duration associated with the development of fungal infections ( 1 ) . in the present retrospective study
, we demonstrated that micafungin had a similar overall efficacy compared with caspofungin when used for treating persistently neutropenic patients with acute leukemia ( overall efficacy of 60.4% for micafungin versus 57.3% for caspofungin ) . efficacy was substantiated in a robust fashion by using four of five efficacy end points based on previously used criteria to evaluate and compare efficacy of antifungals for this purpose ( 2,17,18 ) . with regard to the evaluation parameters and a comparison of the treatment groups , proven baseline fungal infections
were all candidemias , and occurred only in the caspofungin group , all of which were successfully treated . the third was due to biopsy - proven mucormycosis of the skin once more , a weakness in the antifungal coverage of the echinocandins ( 19 ) . although our data demonstrate similar efficacy of micafungin compared with caspofungin when used to treat persistently neutropenic patients , one may question the robustness of this conclusion given the exclusion of fever as an end point . nonetheless , the utility of including fever as a primary composite end point has been questioned by investigators , primarily due to the lack of specificity of fever as a sign of occult fungal infection , particularly in the studied population ( 20,21 ) and its lack of accuracy as a harbinger for ifi ( 22,23 ) . there were also few proven baseline fungal infections and all occurred in the caspofungin group . in addition , there were no proven mold infections among the included patients , which makes drawing firm conclusions regarding the efficacy of micafungin to treat mold infections impossible . the lack of adherence to the standardized eortc - msg radiological criteria in our study may be viewed as another limitation . to substantiate false identification of patients with bacterial respiratory tract infections as possible invasive pulmonary fungal infections , all patients with possible or probable infections based on abnormal radiological findings had negative sputum or bronchoalveolar lavage bacterial cultures at the time of inclusion . micafungin demonstrated similar efficacy to caspofungin when used to empirically treat febrile neutropenic patients with hematological malignancies , as shown by the composite end point . a prospective , randomized clinical trial comparing caspofungin and micafungin for treatment of persistently febrile neutropenic patients with focus on outcomes with mold infections is warranted . | [
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liver ischemia and reperfusion ( i / r)-induced injury is a major complication associated with hemorrhagic or endotoxin shock and thermal injury as well as liver transplantation and resectional surgery . the vast majority of studies performed in experimental animals have used in situ models of liver i / r suggesting that data obtained from these investigations are most relevant to pathophysiological situations or surgical manipulations involving warm
( 37c ) i / r such as hemorrhagic or endotoxin shock or resectional surgery .
indeed , the pathophysiological mechanisms responsible for warm i / r - induced liver injury may be significantly different than those that occur with the cold ischemia ( 4c ) associated with liver storage prior to transplantation . nevertheless , a great deal of mechanistic information has been derived from in situ i / r studies .
these studies demonstrate that reperfusion of ischemic tissue initiates a cascade of molecular and cellular events that culminate in the superoxide anion radical ( o2 ) dependent , nuclear transcription factor-b ( nf-b)-mediated expression of both injurious and protective mediators in favor of the former ( fig . 1 ) .
post - ischemic liver injury is biphasic in nature consisting of an acute or early phase and a subacute or late phase .
the early phase of injury occurs in the absence of leukocyte infiltration and is thought to be initiated by a rapid alteration in the redox state of the tissue in favor of a more oxidative environment .
the late phase of injury is dependent upon the production of several different cytokines and chemokines that promote the infiltration of large numbers polymorphonuclear neutrophils ( pmns ) and lymphocytes into the liver interstitium via the up - regulation of endothelial cell adhesion molecules and formation of chemotactic gradients .
interstitial pmns become fully activated and release copius amounts of reactive oxygen species ( ros ) together with extracellular matrix degrading enzymes such as collagenase and matrix metalloproteases .
the net result of this inflammatory infiltrate is an amplification of the acute injurious response resulting in extensive inflammatory tissue injury . over the past 10 years
, an emerging body of experimental data suggest that endothelial cell nitric oxide synthase ( enos)-derived no may limit ros- and pmn - mediated tissue injury thereby regulating the subsequent inflammatory response in vivo .
indeed , it is becoming clear that reperfusion of ischemic tissue induces a rapid reduction in the bio - availability of no which is thought to represent an important initiating event in the pathophysiology of post - ischemic injury in a variety of different tissues including the liver , heart , kidney and gut .
this chapter presents evidence supporting the hypothesis that : a ) i / r enhances the generation of the o2 and b ) the i / r - induced decrease in the bioavailability of no is mediated directly or indireclty by the o2-dependent decomposition of endogenous no .
in addition to reducing the steady state levels of this protective nitrogen oxide , post - ischemic overproduction of o2 as well as other ros would also alter the redox potential of the liver creating a more oxidative environment within the major cell types of liver including the hepatocytes , kupffer cells ( kcs ) and sinusoidal endothelial cells ( secs ; fig . 1 ) . increasing
the redox potential within these cells would activate nf-b thereby promoting the expression of pro - inflammatory cytokines ( e.g. , tnf- , il-12 , il-1 ) that may injure the tissue directly in the acute phase as well as induce chemokine and endothelial cell adhesion molecule expression in the later subacute phase .
activation of this transcription factor would also induce the expression of certain protective and anti - apopototic genes that could limit tissue injury following i / r .
the evidence implicating endogenous enos - dependent and - independent generation of no as important regulatory pathways that limit the early and late phases of i / r - induced liver injury are also presented in this review .
the early phase of hepatocellular i / r occurs within 16 h following reperfusion and is associated with kupffer cell ( kc ) and possibly lymphocyte activation .
data obtained from several different laboratories demonstrate that this acute , pmn - independent injury may be initiated by alterations in the redox state of the post - ischemic liver such that the redox potential becomes more positive thereby creating a more oxidative environment .
exactly how this happens has been the subject of active debate for more than a decade .
we do know that one of the earliest events associated with i / r is a remarkable dysfunction of the sec characterized by profound decreases in the steady state production of enos - derived no .
this decrease in steady state levels of no occurs very quickly ( within 5 min of reperfusion ) and appears to be due to decreased synthesis of no , enhanced inactivation of no by certain ros or both .
co - incident with the decrease in no production is the enhanced production of ros such as o2 and hydrogen peroxide ( h2o2 ) during the first few minutes of reperfusion .
the cellular source(s ) of these ros are not known with certainty but several candidate enzymes / pathways have been suggested including hepatocyte xanthine oxidase and/or mitochondrial electron transport as well as kc- and sec - associated nadph oxidase .
interest in xanthine oxidase has waned over the years in favor of mitochondrial sources of ros and/or kc - associated nadph oxidase .
it is well known that certain oxygen - or hemoprotein - derived free radicals such as o2 and ferryl ( fe ) hemoproteins may interact with and decompose no .
in addition , h2o2 can indirectly decrease steady state levels of no via the peroxidase - catalyzed consumption of no in different tissues .
therefore , i / r - induced over - production of ros not only reduces ( or eliminates ) the important vasoactive mediator and cytoprotective no free radical , but it also induces the rapid oxidation of cellular gsh creating a more positive redox potential within the liver . indeed , a number of different studies , using pharmacologic interventions or genetic approaches have demonstrated that the protective effects of certain nonenzymatic or enzymatic antioxidants administered prior to inducing liver ischemia correlates with restoration of gsh levels within the tissue .
there is a large body of work suggesting that the major ros responsible for post - ischemic hepatocellular injury is o2 .
a variety of different studies have demonstrated that adenoviral transfection of the liver with manganese superoxide dismutase ( mnsod ) as well as exogenous administration of long - lived sods attenuate i / r - induced injury suggesting that o2 per se rather than secondary oxidants is directly or indirectly responsible for i / r - induced tissue damage ( fig . 2 ) .
a number of cellular sources of o2 have been proposed to account for post - ischemic tissue injury in the absence of an inflammatory infiltrate .
the most likely candidate is kc - associated nadph oxidase although sec nadph oxidase may play an as yet - to - be discovered role .
this multimeric o2-producing enzymatic complex has been well - characterized in phagocytic leukocytes and kupffer cells .
several studies have determined that kc - derived nadph oxidase is critically involved in promoting post ischemic liver injury .
exactly how cell - derived o2 promotes liver injury is not known but emerging data suggest that multiple mechanisms are likely involved .
several studies had originally suggested that o2 dependent lipid peroxidation represented an important pathway for i / r - induced damage ; however other investigations cautioned that oxidative degradation of membrane lipids may , in reality , be a consequence rather than a cause of i / r - induced liver injury .
more recent studies suggest that o2 may directly or indirectly mediate cell and tissue injury by rapidly interacting with and damaging mitochondrial membrane proteins leading to the loss of inner membrane potential and atp generating capacity .
another mechanism that has been proposed to account for o2 dependent injury to the postischemic liver is through the generation of even more potent oxidants via its interaction with no .
this interaction is known to produce the potent cytotoxic oxidizing and nitrating specie peroxynitrite ( onoo ) and its conjugate acid peroxynitrous acid ( onooh ) .
although an attractive hypothesis , the data do not support a role for this pathway .
indeed , we have found that i / r - induced liver injury in enos - deficient ( enos ) mice or wild type mice pretreated with the nos inhibitor n(g)-nitro - l - arginine methyl ester ( l - name ) exacerbates liver injury suggesting that no is protective in nature ( fig .
in addition to its direct biochemical effects , o2 may mediate hepatocellular damage indirectly via the up - regulation ( or down - regulation ) of certain redox - sensitive genes known to be important in cell proliferation , apoptosis and the inflammatory response .
in addition to the above mentioned mechanisms , there is good evidence to suggest that i / r - induced o2 production may mediate hepatocellular injury and inflammation by creating a more oxidative environment within the different cells of the liver thereby activating redox - sensitive transcription factors such as nf-b .
studies from several different laboratories have shown that i / r - induces the rapid up regulation of several different cytokines ( tnf- , il-1 , il-2 , il-6 , il-8 , il-12 ) , endothelial cell adhesion molecules ( icam-1 , vcam-1 , e - selectin and mucosal addressin cell adhesion molecule-1 ) , cxc chemokines ( macrophage inflammatory protein-2 , keritinocyte - derived chemokine , and epithelial neutrophil - activating peptide ) as well as nitric oxide synthase-2 ( inos ) and cyclooxygenase-2 ( cox2 ) .
the expression of the majority of these genes is regulated by the transcription factor nf-b and thus there is great interest in understanding how the molecular events associated with i / r - mediated activation of nf-b and the subsequent induction and roles of the different cytokines , chemokines , adhesion molecules and enzymes ( fig .
indeed , it is now well - appreciated that the expression of tnf- , il-1 and il-12 are important for mediating post - ischemic liver injury .
a large body of experimental data suggest o2 and/or h2o2 may act as downstream signaling and/or costimulatory molecules required for the activation of nf-b .
although an attractive hypothesis that continues to receive substantial attention in the literature , there is in fact little data directly demonstrating that ros mediate nf-b activation in vivo . it may be that ros function to activate nf-b indirectly by promoting the expression of certain cytokines and/or mediators ( e.g. , tnf- and il-1 ) which can in turn activate this transcription factor .
indeed , it has been demonstrated that a major mediator of post - ischemic liver injury is tnf-. the mechanisms by which tnf- promotes hepatocellular injury are not entirely clear but are most likely multifactorial .
previous studies have suggested that much of the post - ischemic liver injury may be a result of tnf--induced endothelial cell and hepatocyte apoptosis .
it is known that tnf- will induce hepatocyte injury via the ros - dependent activation of numerous cell signaling pathways resulting in apoptosis .
however , jaeschke et al . found , using well - defined histopathological criteria , that virtually all liver injury occurred by
nevertheless , it should be remembered that cells that begin to die by apoptosis may also undergo necrosis if intracellular atp is low or absent as would be the case during ischemia .
it is clear that i / r - induced activation of nf-b within kcs and secs induces an injurious / pro - inflammatory response via the upregulation of numerous cytokines , enzymes and chemokines ( fig . 1 ) .
however , the function of nf-b in hepatocytes appears to be more complicated ( fig . 1 ) .
emerging evidence suggests that nf-b activation may be to protect a variety of cells from the injurious effects of certain pro - inflammatory cytokines such as tnf-. it is well known that pre - treatment of cultured cells with non - lethal concentrations of tnf- protects these cells from a subsequent challenge by lethal amounts of this same cytokine and that this protective response is dependent upon nf-b activation . in addition , ikk has been shown to be required for embryonic development of skin and limbs whereas ikk appears essential for normal liver development with massive liver apoptosis , injury and embryonic lethality occurring in ikk-deficient mice .
similar effects have been observed in mice that genetically lack the p65 ( rela ) component of nf-b . taken together
, these studies suggest that nf-b activation is essential for normal liver development and that inhibition of nf-b nuclear translocation promotes liver injury via massive apoptosis .
studies by brenner and coworkers have confirmed that over - expression of liver ib- in mice inhibited nf-b activation resulting in massive hepatocyte injury and apoptosis following partial hepatectomy in these animals .
it is known that nf-b activation induces several anti - apoptotic genes including mnsod , a zinc finger protein termed a20 , cellular inhibitors of apoptosis-2 ( c - iap-2 ) , and bcl-2 family members such as a1 .
most of these genes ( i.e. mnsod , a20 and a1 ) are also known to inhibit nf-b activation and thus may control the expression of certain pro - inflammatory genes ( fig . 1 ) .
despite the recent body of experimental data demonstrating that nf-b may limit hepatocyte injury , there is also an equally convincing body of data demonstrating that i / r - induced , tnf--mediated liver injury is mediated by nf-b activation .
for example , it has been shown that much of the post - ischemic liver injury is abrogated in tnf--deficient mice .
these are important studies as they demonstrate that i / r - induced liver injury is attenuated in the complete absence of nf-b activation ie in the absence of both the injurious / inflammatory and the protective responses .
it may be that the balance between injurious vs protective genes upregulated during i / r as well as the cellular location of nf-b activation dictates whether nf-b activation will promote injury or protection .
a more direct approach to the role of hepatocyte nf-b in the pathophysiology of post - ischemic tissue injury has been provided by investigators who showed that i / r - induced injury was attenuated in mice with hepatocyte - specific ablation of ikk. they found that the protective effect was associated with decreased expression of hepatocyte - derived tnf-. in apparent contradiction to these results , these same investigators reported that i / r - induced liver injury in mice with a hepatocyte - specific deletion of ikk ( nemo ) was dramatically increased compared to wild type mice suggesting a protective effect of hepatocyte nf-b . in an attempt to reconcile these seemingly contradictory results
, the authors suggested that hepatocyte - specific deletion of ikk may possess some residual nf-b activity whereas ikk ( nemo)-deficient hepatocytes have no detectable nf-b activity and thus no protective response to limit liver damage . taken together , these data suggest that further work is needed to define the role of hepatocyte nf-b .
there is a large body of work suggesting that the major ros responsible for post - ischemic hepatocellular injury is o2 .
a variety of different studies have demonstrated that adenoviral transfection of the liver with manganese superoxide dismutase ( mnsod ) as well as exogenous administration of long - lived sods attenuate i / r - induced injury suggesting that o2 per se rather than secondary oxidants is directly or indirectly responsible for i / r - induced tissue damage ( fig . 2 ) .
a number of cellular sources of o2 have been proposed to account for post - ischemic tissue injury in the absence of an inflammatory infiltrate .
the most likely candidate is kc - associated nadph oxidase although sec nadph oxidase may play an as yet - to - be discovered role .
this multimeric o2-producing enzymatic complex has been well - characterized in phagocytic leukocytes and kupffer cells .
several studies have determined that kc - derived nadph oxidase is critically involved in promoting post ischemic liver injury .
exactly how cell - derived o2 promotes liver injury is not known but emerging data suggest that multiple mechanisms are likely involved .
several studies had originally suggested that o2 dependent lipid peroxidation represented an important pathway for i / r - induced damage ; however other investigations cautioned that oxidative degradation of membrane lipids may , in reality , be a consequence rather than a cause of i / r - induced liver injury .
more recent studies suggest that o2 may directly or indirectly mediate cell and tissue injury by rapidly interacting with and damaging mitochondrial membrane proteins leading to the loss of inner membrane potential and atp generating capacity .
another mechanism that has been proposed to account for o2 dependent injury to the postischemic liver is through the generation of even more potent oxidants via its interaction with no .
this interaction is known to produce the potent cytotoxic oxidizing and nitrating specie peroxynitrite ( onoo ) and its conjugate acid peroxynitrous acid ( onooh ) .
although an attractive hypothesis , the data do not support a role for this pathway .
indeed , we have found that i / r - induced liver injury in enos - deficient ( enos ) mice or wild type mice pretreated with the nos inhibitor n(g)-nitro - l - arginine methyl ester ( l - name ) exacerbates liver injury suggesting that no is protective in nature ( fig .
in addition to its direct biochemical effects , o2 may mediate hepatocellular damage indirectly via the up - regulation ( or down - regulation ) of certain redox - sensitive genes known to be important in cell proliferation , apoptosis and the inflammatory response .
in addition to the above mentioned mechanisms , there is good evidence to suggest that i / r - induced o2 production may mediate hepatocellular injury and inflammation by creating a more oxidative environment within the different cells of the liver thereby activating redox - sensitive transcription factors such as nf-b .
studies from several different laboratories have shown that i / r - induces the rapid up regulation of several different cytokines ( tnf- , il-1 , il-2 , il-6 , il-8 , il-12 ) , endothelial cell adhesion molecules ( icam-1 , vcam-1 , e - selectin and mucosal addressin cell adhesion molecule-1 ) , cxc chemokines ( macrophage inflammatory protein-2 , keritinocyte - derived chemokine , and epithelial neutrophil - activating peptide ) as well as nitric oxide synthase-2 ( inos ) and cyclooxygenase-2 ( cox2 ) .
the expression of the majority of these genes is regulated by the transcription factor nf-b and thus there is great interest in understanding how the molecular events associated with i / r - mediated activation of nf-b and the subsequent induction and roles of the different cytokines , chemokines , adhesion molecules and enzymes ( fig .
indeed , it is now well - appreciated that the expression of tnf- , il-1 and il-12 are important for mediating post - ischemic liver injury .
a large body of experimental data suggest o2 and/or h2o2 may act as downstream signaling and/or costimulatory molecules required for the activation of nf-b . although an attractive hypothesis that continues to receive substantial attention in the literature , there is in fact little data directly demonstrating that ros mediate nf-b activation in vivo
. it may be that ros function to activate nf-b indirectly by promoting the expression of certain cytokines and/or mediators ( e.g. , tnf- and il-1 ) which can in turn activate this transcription factor .
indeed , it has been demonstrated that a major mediator of post - ischemic liver injury is tnf-. the mechanisms by which tnf- promotes hepatocellular injury are not entirely clear but are most likely multifactorial .
previous studies have suggested that much of the post - ischemic liver injury may be a result of tnf--induced endothelial cell and hepatocyte apoptosis .
it is known that tnf- will induce hepatocyte injury via the ros - dependent activation of numerous cell signaling pathways resulting in apoptosis .
however , jaeschke et al . found , using well - defined histopathological criteria , that virtually all liver injury occurred by
nevertheless , it should be remembered that cells that begin to die by apoptosis may also undergo necrosis if intracellular atp is low or absent as would be the case during ischemia .
it is clear that i / r - induced activation of nf-b within kcs and secs induces an injurious / pro - inflammatory response via the upregulation of numerous cytokines , enzymes and chemokines ( fig . 1 ) .
however , the function of nf-b in hepatocytes appears to be more complicated ( fig . 1 ) .
emerging evidence suggests that nf-b activation may be to protect a variety of cells from the injurious effects of certain pro - inflammatory cytokines such as tnf-. it is well known that pre - treatment of cultured cells with non - lethal concentrations of tnf- protects these cells from a subsequent challenge by lethal amounts of this same cytokine and that this protective response is dependent upon nf-b activation . in addition , ikk has been shown to be required for embryonic development of skin and limbs whereas ikk appears essential for normal liver development with massive liver apoptosis , injury and embryonic lethality occurring in ikk-deficient mice .
similar effects have been observed in mice that genetically lack the p65 ( rela ) component of nf-b . taken together
, these studies suggest that nf-b activation is essential for normal liver development and that inhibition of nf-b nuclear translocation promotes liver injury via massive apoptosis .
studies by brenner and coworkers have confirmed that over - expression of liver ib- in mice inhibited nf-b activation resulting in massive hepatocyte injury and apoptosis following partial hepatectomy in these animals .
it is known that nf-b activation induces several anti - apoptotic genes including mnsod , a zinc finger protein termed a20 , cellular inhibitors of apoptosis-2 ( c - iap-2 ) , and bcl-2 family members such as a1 .
most of these genes ( i.e. mnsod , a20 and a1 ) are also known to inhibit nf-b activation and thus may control the expression of certain pro - inflammatory genes ( fig . 1 ) .
despite the recent body of experimental data demonstrating that nf-b may limit hepatocyte injury , there is also an equally convincing body of data demonstrating that i / r - induced , tnf--mediated liver injury is mediated by nf-b activation .
for example , it has been shown that much of the post - ischemic liver injury is abrogated in tnf--deficient mice .
these are important studies as they demonstrate that i / r - induced liver injury is attenuated in the complete absence of nf-b activation ie in the absence of both the injurious / inflammatory and the protective responses .
it may be that the balance between injurious vs protective genes upregulated during i / r as well as the cellular location of nf-b activation dictates whether nf-b activation will promote injury or protection .
a more direct approach to the role of hepatocyte nf-b in the pathophysiology of post - ischemic tissue injury has been provided by investigators who showed that i / r - induced injury was attenuated in mice with hepatocyte - specific ablation of ikk. they found that the protective effect was associated with decreased expression of hepatocyte - derived tnf-. in apparent contradiction to these results , these same investigators reported that i / r - induced liver injury in mice with a hepatocyte - specific deletion of ikk ( nemo ) was dramatically increased compared to wild type mice suggesting a protective effect of hepatocyte nf-b . in an attempt to reconcile these seemingly contradictory results
, the authors suggested that hepatocyte - specific deletion of ikk may possess some residual nf-b activity whereas ikk ( nemo)-deficient hepatocytes have no detectable nf-b activity and thus no protective response to limit liver damage . taken together , these data suggest that further work is needed to define the role of hepatocyte nf-b .
the early phase of i / r injury is a pmn - independent process in which kcs are activated , ros are generated by these resident phagocytes and by dysfunctional mitochondria in kcs , secs and hepatocytes and proinflammatory cytokines ( tnf- , il-12 , il-1 ) are synthesized .
this initial response induces the upregulation of additional inflammatory mediators including activated complement factors ( e.g. , c5a ) , platelet activating factor and different cxc chemokines , such as interleukin-8 ( il-8 ) , macrophage inflammatory protein-2 ( mip-2 ) , keratinocyte - derived chemokine , and cytokine - induced neutrophil chemoattractant ( cinc-1 ) .
these inflammatory mediators promote the enhanced expression of cd11b / cd18 which prime pmns and promote their extravasation from the sinusoids and post - sinusoidal venules into the tissue thereby initiating the late or subacute phase of post - ischemic tissue injury .
this inflammatory response is characterized by the activation of pmns resulting in the production of large amounts of ros and release of a variety of different proteases ( gelatinase , collagenase ) .
the net result of this acute inflammatory response is oxidant - mediated mitochondrial dysfunction as well as degradation of the interstitial matrix and damage to the parenchymal cells .
in addition , tlr4-mediated production of cytokines and inflammatory mediators may represent an additional important pathway for the amplification of the inflammatory response .
although it is well - recognized that pmns play an important role in the pathophysiology of iiver i / r injury , other leukocytes may also be involved in this innate immune response .
for example , it has been reported that cd4 + t - cells play an important role in recruiting pmns into the post - ischemic liver and that cd4 + t - cell - deficiency remarkably attenuated both pmn infiltration and tissue injury following i / r .
however , more recent studies have found that cd4 + t - cell - deficient mice responded to liver i / r with an exacerbation in liver injury despite the fact that these mice displayed fewer pmns infiltrating the post - ischemic liver .
the mechanisms responsible for these seemingly contradictory results are not clear at the present time but may relate to the suppressive or regulatory nature of the infiltrating cd4 + t - cells in the later study .
it is becoming increasingly appreciated that i / r results in rapid endothelial dysfunction in a number of different tissues that is characterized by a marked decrease in steady - state production of endothelial cell - derived no .
the decrease in no bioavailability occurs within the first few minutes following reperfusion and appears to be due to decreased synthesis of no , enhanced inactivation of no by o2 ( or other ros ) or both . because endogenous no is known to interact with and
rapidly decompose certain ros such as o2 , ho , and ferryl hemoproteins , decreased steady state production of no would in all likelihood increase the redox potential of hepatocytes , kcs and/or secs in favor of a more oxidative environment .
indeed , we have demonstrated that many of the pathophysiological characteristics induced by i / r ( i.e. oxidative stress , leukocyte adhesion , enhanced vascular permeability ) may be recapitulated in normal tissue by administration of certain nos inhibitors suggesting that enos - derived no may act as an endogenous anti - inflammatory mediator in the microcirculation . the use of certain nos inhibitors to evaluate the role of no in vivo has proven problematic .
it is known for example that l - name and l - nmma are nonspecific inhibitors of both enos and inos as well as potent vasoconstrictors in vivo .
thus , the non - selective nature of these nos inhibitors coupled to the fact that their exacerbatory effects may be due to their vasoconstrictor effects in a model of hypoperfusion makes their usefulness less than ideal to probe the role for no in liver i / r .
the potentially protective role of endogenous no in liver i / r injury is supported by studies demonstrating enhanced hepatocellular injury in post - ischemic animals rendered deficient in enos ( fig . 3 ) .
in addition , investigators have demonstrated that no donors or over - expression of liver enos protects mice from liver i / r - induced injury .
although enos has been shown by several groups of investigators to play a critical protective role in liver
studies from our laboratory suggest that i / r - induced liver injury is enhanced in inos - deficient mice however interpretation is complicated by the fact that we could not demonstrate upregulation of inos message in post - ischemic livers of wild type mice suggesting that the source of inos is not in the liver ( e.g. , in the intestine ) or that a small population of cells such as kcs or secs upregulate inos which we are unable to detect in whole liver preparations . the mechanisms by which endogenous no protects the liver against the injurious effects of i / r are now beginning to be defined ( fig . 5 )
no is known to interact with and decompose o2 or other reactive radicals or oxidants thereby limiting the formation of o2-derived oxidants and preventing the downstream oxidant - induced signaling pathways .
if this antioxidant / free radical scavenging activity of no is a major protective mechanism , then one would predict that administration of a long lived sod such as the fusion protein sod2/3 to enos - deficient mice would attenuate i / r - induced injury to the liver .
in fact , we found that although sod2/3 markedly attenuates post - ischemic injury in wild type mice , it does not attenuate liver injury in enos - deficient animals .
these data suggested that the protective effect of sod2/3 in wild type mice is due to its ability to increase the bioavailability of no by preventing the o2 mediated inactivation of no .
we then return to the question : how does no limit i / r - induced tissue damage ?
for example , decaterina and coworkers provide evidence that no enhances the denovo synthesis and/or stabilization of the natural inhibitor ib-. in addition , no - mediated s - nitrosation of a specific cysteine on the p50 and/or p65 subunits of nf-b results in inhibition of binding of this heterodimer to it consensus sequences upstream of the different pro - inflammatory genes .
another possible mechanism may be that no - dependent activation of soluble guanylyl cyclase ( sgc ) with the subsequent production of the vasorelaxant cgmp may protect against reperfusion injury by enhancing blood flow thereby limiting the degree of ischemia to the liver .
it has also been proposed that no - mediated activation of protein kinase g via the sgc / cgmp pathway opens mitochondrial katp channels thereby reducing calcium accumulation within the mitochondria and preventing the loss of cytochrome c from the mitochondrial intermembranal space .
alternatively or in addition to , no may reversibly inhibit mitochondrial respiration via interaction with complex i and/or cytochrome c oxidase .
this would inhibit apoptosis , maintain small but significant amounts of oxygen during ischemia and allow for a more controlled resumption of respiration following reperfusion .
similar observations have been made with no - dependent s - nitrosation of caspase-3 resulting in inactivation of this enzyme and inhibition of apoposis .
this would minimize free radical - mediated damage to the mitochondrial membrane and preserve cellular function .
no may attenuate the later stages of post - ischemic tissue damage by inhibiting platelet / leukocyte - endothelial cell interactions .
there is ample evidence demonstrating that enos - derived no as well as exogenous no - releasing compounds attenuate i / r - induced liver injury .
more recent studies demonstrate that animals and humans are capable of producing substantial quantities of no via enos - independent pathways that utilize nitrogen oxides that are produced from the decomposition of no or are ingested in the diet . like any important signaling molecule , the generation and lifetime of no is very short - lived in vivo .
a physiologically - relevant set of pathways for the inactivation of no are mediated by metal - catalyzed oxidation reactions .
the copper - containing protein ceruloplasmin has been shown to rapidly oxidize no to no2 under physiological conditions .
in addition to ceruloplasmin , ferrous dioxygenated hemoglobin ( hb - feo2 ; oxyhemoglobin ) or myoblobin rapidly decomposes no to yield nitrate .
historically , it has been assumed that blood and tissue nitrate and nitrite simply reflect inert endproducts of no metabolism and food contaminants .
however , it is now recognized that these oxidized metabolites of nitrogen can be metabolized in vivo to regenerate no ( fig .
because the rate of no generation from nitrite is linearly dependent on reductions in tissue po2 and ph , nitrite may be metabolized to no in ischemic tissue ( via deoxyhb or mb ) and exert no - dependent protective effects site - specifically .
indeed , the tissue - specific reduction of nitrite to generate cytoprotective no has important clinical significance in that treatment of different ischemic disorders with nitrite would reduce the untoward cardiovascular effects of systemic delivery of inhaled no or intravenous administration of s - nitrosothiols .
a growing number of studies demonstrate that nitrite is cytoprotective in different models of tissue i / r including the liver , heart , kidney and brain ( reviewed in ref .
the clinical usefulness of nitrite is currently being investigated as a potential therapeutic agent to treat ischemic disorders .
there is now overwhelming evidence supporting the concept that i / r - induced tissue damage is associated with the o2-dependent decrease in steady - state levels of no .
furthermore , accumulating evidence suggests that enos - dependent as well as enos - independent generation of no acts to limit post - ischemic tissue injury .
the mechanisms by which this nitrogen oxide protects the post - ischemic liver remain to be defined and are the subject of active investigation .
therapeutic uses of no or nitrite are currently underway in several different laboratories to treat a variety of ischemic disorders . | liver ischemia and reperfusion - induced injury is a major clinical complication associated with hemorrhagic or endotoxin shock and thermal injury as well as liver transplantation and resectional surgery .
data obtained from several different studies suggest that an important initiating event in the pathophysiology of ischemia and reperfusion - induced tissue injury is enhanced production of superoxide concomitant with a decrease in the bioavailability of endothelial cell - derived nitric oxide .
this review will summarize the evidence supporting the hypothesis that the redox imbalance induced by alterations in superoxide and nitric oxide generation creates a more oxidative environment within the different cells of the liver that enhances the nuclear transcription factor-b - dependent expression of a variety of different cytokines and mediators that may promote as well as limit ischemia and reperfusion - induced hepatocellular injury .
in addition , the evidence implicating endothelial cell nitric oxide synthase - dependent and -independent generation of nitric oxide as important regulatory pathways that act to limit ischemia and reperfusion - induced liver injury and inflammation is also presented . | Introduction
Ischemia and Reperfusion Increases Reactive Oxygen Production, Decreases Nitric Oxide Bioavailability and Alters the Redox State of the Liver:
Superoxide is the major ROS involved in post-ischemic liver injury
Role of Leukocytes in I/R Injury
Suppression of I/R-Induced Liver Damage by Endogenous or Exogenous Nitric Oxide
Concluding Remarks | liver ischemia and reperfusion ( i / r)-induced injury is a major complication associated with hemorrhagic or endotoxin shock and thermal injury as well as liver transplantation and resectional surgery . the vast majority of studies performed in experimental animals have used in situ models of liver i / r suggesting that data obtained from these investigations are most relevant to pathophysiological situations or surgical manipulations involving warm
( 37c ) i / r such as hemorrhagic or endotoxin shock or resectional surgery . indeed , the pathophysiological mechanisms responsible for warm i / r - induced liver injury may be significantly different than those that occur with the cold ischemia ( 4c ) associated with liver storage prior to transplantation . these studies demonstrate that reperfusion of ischemic tissue initiates a cascade of molecular and cellular events that culminate in the superoxide anion radical ( o2 ) dependent , nuclear transcription factor-b ( nf-b)-mediated expression of both injurious and protective mediators in favor of the former ( fig . the early phase of injury occurs in the absence of leukocyte infiltration and is thought to be initiated by a rapid alteration in the redox state of the tissue in favor of a more oxidative environment . the late phase of injury is dependent upon the production of several different cytokines and chemokines that promote the infiltration of large numbers polymorphonuclear neutrophils ( pmns ) and lymphocytes into the liver interstitium via the up - regulation of endothelial cell adhesion molecules and formation of chemotactic gradients . over the past 10 years
, an emerging body of experimental data suggest that endothelial cell nitric oxide synthase ( enos)-derived no may limit ros- and pmn - mediated tissue injury thereby regulating the subsequent inflammatory response in vivo . indeed , it is becoming clear that reperfusion of ischemic tissue induces a rapid reduction in the bio - availability of no which is thought to represent an important initiating event in the pathophysiology of post - ischemic injury in a variety of different tissues including the liver , heart , kidney and gut . this chapter presents evidence supporting the hypothesis that : a ) i / r enhances the generation of the o2 and b ) the i / r - induced decrease in the bioavailability of no is mediated directly or indireclty by the o2-dependent decomposition of endogenous no . in addition to reducing the steady state levels of this protective nitrogen oxide , post - ischemic overproduction of o2 as well as other ros would also alter the redox potential of the liver creating a more oxidative environment within the major cell types of liver including the hepatocytes , kupffer cells ( kcs ) and sinusoidal endothelial cells ( secs ; fig . , tnf- , il-12 , il-1 ) that may injure the tissue directly in the acute phase as well as induce chemokine and endothelial cell adhesion molecule expression in the later subacute phase . activation of this transcription factor would also induce the expression of certain protective and anti - apopototic genes that could limit tissue injury following i / r . the evidence implicating endogenous enos - dependent and - independent generation of no as important regulatory pathways that limit the early and late phases of i / r - induced liver injury are also presented in this review . data obtained from several different laboratories demonstrate that this acute , pmn - independent injury may be initiated by alterations in the redox state of the post - ischemic liver such that the redox potential becomes more positive thereby creating a more oxidative environment . we do know that one of the earliest events associated with i / r is a remarkable dysfunction of the sec characterized by profound decreases in the steady state production of enos - derived no . co - incident with the decrease in no production is the enhanced production of ros such as o2 and hydrogen peroxide ( h2o2 ) during the first few minutes of reperfusion . therefore , i / r - induced over - production of ros not only reduces ( or eliminates ) the important vasoactive mediator and cytoprotective no free radical , but it also induces the rapid oxidation of cellular gsh creating a more positive redox potential within the liver . indeed , a number of different studies , using pharmacologic interventions or genetic approaches have demonstrated that the protective effects of certain nonenzymatic or enzymatic antioxidants administered prior to inducing liver ischemia correlates with restoration of gsh levels within the tissue . there is a large body of work suggesting that the major ros responsible for post - ischemic hepatocellular injury is o2 . a variety of different studies have demonstrated that adenoviral transfection of the liver with manganese superoxide dismutase ( mnsod ) as well as exogenous administration of long - lived sods attenuate i / r - induced injury suggesting that o2 per se rather than secondary oxidants is directly or indirectly responsible for i / r - induced tissue damage ( fig . a number of cellular sources of o2 have been proposed to account for post - ischemic tissue injury in the absence of an inflammatory infiltrate . exactly how cell - derived o2 promotes liver injury is not known but emerging data suggest that multiple mechanisms are likely involved . several studies had originally suggested that o2 dependent lipid peroxidation represented an important pathway for i / r - induced damage ; however other investigations cautioned that oxidative degradation of membrane lipids may , in reality , be a consequence rather than a cause of i / r - induced liver injury . more recent studies suggest that o2 may directly or indirectly mediate cell and tissue injury by rapidly interacting with and damaging mitochondrial membrane proteins leading to the loss of inner membrane potential and atp generating capacity . indeed , we have found that i / r - induced liver injury in enos - deficient ( enos ) mice or wild type mice pretreated with the nos inhibitor n(g)-nitro - l - arginine methyl ester ( l - name ) exacerbates liver injury suggesting that no is protective in nature ( fig . in addition to the above mentioned mechanisms , there is good evidence to suggest that i / r - induced o2 production may mediate hepatocellular injury and inflammation by creating a more oxidative environment within the different cells of the liver thereby activating redox - sensitive transcription factors such as nf-b . studies from several different laboratories have shown that i / r - induces the rapid up regulation of several different cytokines ( tnf- , il-1 , il-2 , il-6 , il-8 , il-12 ) , endothelial cell adhesion molecules ( icam-1 , vcam-1 , e - selectin and mucosal addressin cell adhesion molecule-1 ) , cxc chemokines ( macrophage inflammatory protein-2 , keritinocyte - derived chemokine , and epithelial neutrophil - activating peptide ) as well as nitric oxide synthase-2 ( inos ) and cyclooxygenase-2 ( cox2 ) . the expression of the majority of these genes is regulated by the transcription factor nf-b and thus there is great interest in understanding how the molecular events associated with i / r - mediated activation of nf-b and the subsequent induction and roles of the different cytokines , chemokines , adhesion molecules and enzymes ( fig . indeed , it is now well - appreciated that the expression of tnf- , il-1 and il-12 are important for mediating post - ischemic liver injury . although an attractive hypothesis that continues to receive substantial attention in the literature , there is in fact little data directly demonstrating that ros mediate nf-b activation in vivo . indeed , it has been demonstrated that a major mediator of post - ischemic liver injury is tnf-. previous studies have suggested that much of the post - ischemic liver injury may be a result of tnf--induced endothelial cell and hepatocyte apoptosis . in addition , ikk has been shown to be required for embryonic development of skin and limbs whereas ikk appears essential for normal liver development with massive liver apoptosis , injury and embryonic lethality occurring in ikk-deficient mice . taken together
, these studies suggest that nf-b activation is essential for normal liver development and that inhibition of nf-b nuclear translocation promotes liver injury via massive apoptosis . despite the recent body of experimental data demonstrating that nf-b may limit hepatocyte injury , there is also an equally convincing body of data demonstrating that i / r - induced , tnf--mediated liver injury is mediated by nf-b activation . for example , it has been shown that much of the post - ischemic liver injury is abrogated in tnf--deficient mice . these are important studies as they demonstrate that i / r - induced liver injury is attenuated in the complete absence of nf-b activation ie in the absence of both the injurious / inflammatory and the protective responses . it may be that the balance between injurious vs protective genes upregulated during i / r as well as the cellular location of nf-b activation dictates whether nf-b activation will promote injury or protection . a more direct approach to the role of hepatocyte nf-b in the pathophysiology of post - ischemic tissue injury has been provided by investigators who showed that i / r - induced injury was attenuated in mice with hepatocyte - specific ablation of ikk. they found that the protective effect was associated with decreased expression of hepatocyte - derived tnf-. in apparent contradiction to these results , these same investigators reported that i / r - induced liver injury in mice with a hepatocyte - specific deletion of ikk ( nemo ) was dramatically increased compared to wild type mice suggesting a protective effect of hepatocyte nf-b . there is a large body of work suggesting that the major ros responsible for post - ischemic hepatocellular injury is o2 . a variety of different studies have demonstrated that adenoviral transfection of the liver with manganese superoxide dismutase ( mnsod ) as well as exogenous administration of long - lived sods attenuate i / r - induced injury suggesting that o2 per se rather than secondary oxidants is directly or indirectly responsible for i / r - induced tissue damage ( fig . a number of cellular sources of o2 have been proposed to account for post - ischemic tissue injury in the absence of an inflammatory infiltrate . exactly how cell - derived o2 promotes liver injury is not known but emerging data suggest that multiple mechanisms are likely involved . several studies had originally suggested that o2 dependent lipid peroxidation represented an important pathway for i / r - induced damage ; however other investigations cautioned that oxidative degradation of membrane lipids may , in reality , be a consequence rather than a cause of i / r - induced liver injury . more recent studies suggest that o2 may directly or indirectly mediate cell and tissue injury by rapidly interacting with and damaging mitochondrial membrane proteins leading to the loss of inner membrane potential and atp generating capacity . indeed , we have found that i / r - induced liver injury in enos - deficient ( enos ) mice or wild type mice pretreated with the nos inhibitor n(g)-nitro - l - arginine methyl ester ( l - name ) exacerbates liver injury suggesting that no is protective in nature ( fig . in addition to the above mentioned mechanisms , there is good evidence to suggest that i / r - induced o2 production may mediate hepatocellular injury and inflammation by creating a more oxidative environment within the different cells of the liver thereby activating redox - sensitive transcription factors such as nf-b . studies from several different laboratories have shown that i / r - induces the rapid up regulation of several different cytokines ( tnf- , il-1 , il-2 , il-6 , il-8 , il-12 ) , endothelial cell adhesion molecules ( icam-1 , vcam-1 , e - selectin and mucosal addressin cell adhesion molecule-1 ) , cxc chemokines ( macrophage inflammatory protein-2 , keritinocyte - derived chemokine , and epithelial neutrophil - activating peptide ) as well as nitric oxide synthase-2 ( inos ) and cyclooxygenase-2 ( cox2 ) . the expression of the majority of these genes is regulated by the transcription factor nf-b and thus there is great interest in understanding how the molecular events associated with i / r - mediated activation of nf-b and the subsequent induction and roles of the different cytokines , chemokines , adhesion molecules and enzymes ( fig . indeed , it is now well - appreciated that the expression of tnf- , il-1 and il-12 are important for mediating post - ischemic liver injury . indeed , it has been demonstrated that a major mediator of post - ischemic liver injury is tnf-. previous studies have suggested that much of the post - ischemic liver injury may be a result of tnf--induced endothelial cell and hepatocyte apoptosis . in addition , ikk has been shown to be required for embryonic development of skin and limbs whereas ikk appears essential for normal liver development with massive liver apoptosis , injury and embryonic lethality occurring in ikk-deficient mice . taken together
, these studies suggest that nf-b activation is essential for normal liver development and that inhibition of nf-b nuclear translocation promotes liver injury via massive apoptosis . studies by brenner and coworkers have confirmed that over - expression of liver ib- in mice inhibited nf-b activation resulting in massive hepatocyte injury and apoptosis following partial hepatectomy in these animals . despite the recent body of experimental data demonstrating that nf-b may limit hepatocyte injury , there is also an equally convincing body of data demonstrating that i / r - induced , tnf--mediated liver injury is mediated by nf-b activation . for example , it has been shown that much of the post - ischemic liver injury is abrogated in tnf--deficient mice . these are important studies as they demonstrate that i / r - induced liver injury is attenuated in the complete absence of nf-b activation ie in the absence of both the injurious / inflammatory and the protective responses . it may be that the balance between injurious vs protective genes upregulated during i / r as well as the cellular location of nf-b activation dictates whether nf-b activation will promote injury or protection . a more direct approach to the role of hepatocyte nf-b in the pathophysiology of post - ischemic tissue injury has been provided by investigators who showed that i / r - induced injury was attenuated in mice with hepatocyte - specific ablation of ikk. they found that the protective effect was associated with decreased expression of hepatocyte - derived tnf-. in apparent contradiction to these results , these same investigators reported that i / r - induced liver injury in mice with a hepatocyte - specific deletion of ikk ( nemo ) was dramatically increased compared to wild type mice suggesting a protective effect of hepatocyte nf-b . these inflammatory mediators promote the enhanced expression of cd11b / cd18 which prime pmns and promote their extravasation from the sinusoids and post - sinusoidal venules into the tissue thereby initiating the late or subacute phase of post - ischemic tissue injury . this inflammatory response is characterized by the activation of pmns resulting in the production of large amounts of ros and release of a variety of different proteases ( gelatinase , collagenase ) . the net result of this acute inflammatory response is oxidant - mediated mitochondrial dysfunction as well as degradation of the interstitial matrix and damage to the parenchymal cells . in addition , tlr4-mediated production of cytokines and inflammatory mediators may represent an additional important pathway for the amplification of the inflammatory response . although it is well - recognized that pmns play an important role in the pathophysiology of iiver i / r injury , other leukocytes may also be involved in this innate immune response . for example , it has been reported that cd4 + t - cells play an important role in recruiting pmns into the post - ischemic liver and that cd4 + t - cell - deficiency remarkably attenuated both pmn infiltration and tissue injury following i / r . the mechanisms responsible for these seemingly contradictory results are not clear at the present time but may relate to the suppressive or regulatory nature of the infiltrating cd4 + t - cells in the later study . it is becoming increasingly appreciated that i / r results in rapid endothelial dysfunction in a number of different tissues that is characterized by a marked decrease in steady - state production of endothelial cell - derived no . because endogenous no is known to interact with and
rapidly decompose certain ros such as o2 , ho , and ferryl hemoproteins , decreased steady state production of no would in all likelihood increase the redox potential of hepatocytes , kcs and/or secs in favor of a more oxidative environment . it is known for example that l - name and l - nmma are nonspecific inhibitors of both enos and inos as well as potent vasoconstrictors in vivo . the potentially protective role of endogenous no in liver i / r injury is supported by studies demonstrating enhanced hepatocellular injury in post - ischemic animals rendered deficient in enos ( fig . in addition , investigators have demonstrated that no donors or over - expression of liver enos protects mice from liver i / r - induced injury . although enos has been shown by several groups of investigators to play a critical protective role in liver
studies from our laboratory suggest that i / r - induced liver injury is enhanced in inos - deficient mice however interpretation is complicated by the fact that we could not demonstrate upregulation of inos message in post - ischemic livers of wild type mice suggesting that the source of inos is not in the liver ( e.g. if this antioxidant / free radical scavenging activity of no is a major protective mechanism , then one would predict that administration of a long lived sod such as the fusion protein sod2/3 to enos - deficient mice would attenuate i / r - induced injury to the liver . these data suggested that the protective effect of sod2/3 in wild type mice is due to its ability to increase the bioavailability of no by preventing the o2 mediated inactivation of no . in addition , no - mediated s - nitrosation of a specific cysteine on the p50 and/or p65 subunits of nf-b results in inhibition of binding of this heterodimer to it consensus sequences upstream of the different pro - inflammatory genes . another possible mechanism may be that no - dependent activation of soluble guanylyl cyclase ( sgc ) with the subsequent production of the vasorelaxant cgmp may protect against reperfusion injury by enhancing blood flow thereby limiting the degree of ischemia to the liver . there is ample evidence demonstrating that enos - derived no as well as exogenous no - releasing compounds attenuate i / r - induced liver injury . there is now overwhelming evidence supporting the concept that i / r - induced tissue damage is associated with the o2-dependent decrease in steady - state levels of no . furthermore , accumulating evidence suggests that enos - dependent as well as enos - independent generation of no acts to limit post - ischemic tissue injury . therapeutic uses of no or nitrite are currently underway in several different laboratories to treat a variety of ischemic disorders . | [
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today , about half of the patients who have received pre - dialysis care choose peritoneal dialysis ( pd ) as their first dialysis modality .
besides personal preferences , in the first 2 years , pd preserves residual renal function ( rrf ) better and possibly has a better patient survival . however , long - term technique survival of pd is poor because of technical problems and peritoneal membrane failure ; the latter is considered to be a consequence of bioincompatible characteristics of pd fluids .
therefore , the most important challenge in pd is long - term preservation of peritoneal membrane structure and function .
several studies have shown that introduction of dialysis fluids into the peritoneal cavity induces changes , ultimately leading to
membrane failure. features of membrane failure include malfunction of local host defence mechanisms , signs of peritoneal sclerosis and neovascularization resulting in ultrafiltration failure ( uff ) .
the cumulative risk of uff was reported to be 3% after 1 year and 40% after 6 years .
the above - mentioned observations in humans have been studied in more detail in animals : chronic daily instillation of conventional pd fluids leads to an angiogenic response in the omentum , mesentery , liver surface and parietal peritoneal wall [ 58 ] .
in addition , detachment of the mesothelial cell layer , submesothelial extracellular matrix deposition and fibrotic alterations were described after exposure to these dialysis solutions .
these changes are related to duration of dialysis , occurrence of peritonitis and components of the dialysis solution .
although bioincompatibility , in general , is considered to be the major cause of the development of morphological changes of the peritoneal membrane ultimately resulting in uff , the major culprit is not known .
conventional dialysis fluids are acidic ( ph 5.5 ) , contain supraphysiologic concentrations of lactate ( 3540 mmol / l ) and have a high osmolality ( 347 486 mosmol / kg h2o ) as a result of high glucose concentrations ( 75215 mmol / l ) that , after heat sterilization , turn into glucose degradation products ( gdp ) . which of them is most harmful remains the question .
new pd fluid formulations have become available : they have different osmotic agents and buffers such as glucose polymer - based and amino acid - based pd fluids .
this report focuses on the effects of conventional pd fluids and glucose - based pd fluids with neutral ph .
cells that are continuously exposed to dialysis fluids are mesothelial cells and ( in part ) macrophages .
a majority of data on the in vivo effect of pd fluids are derived from ex vivo measurements of effects of pd fluids characteristics on cell function . in a number of studies with animal and human mesothelial cells ,
the characteristic or combination of characteristics in pd fluids that was responsible for the imbalance in these studies remained unclear .
the low ph and high osmolality in pd fluids have an inhibitory effect on proliferation of mesothelial cells and their functions because of the production of cytokines and prostaglandins . adding fresh pd fluid to residual pd fluids with normal ph within the peritoneal cavity resulted in a ph value of 7 after 10 min . in an ex vivo experiment
, the combination of low ph and high concentrations of sodium lactate in pd fluids did result in suppression of respiratory burst activation , a cell host defence mechanism , which is not because of either low ph or lactate concentration alone .
inhibition of the respiratory burst activation was related to the extreme sensitivity of nadph oxidase to low intracellular ph .
in this situation , the presence of lactate and glucose can lead to pseudo - hypoxia .
these findings suggest that the clinical importance of low ph alone might be overestimated and that other factors are more important .
other hyperosmotic solutes , which were used in the same concentrations as glucose ( mannitol and glycerol ) , did not decrease proliferation of the mesothelial cells as much as glucose did .
this implies that the toxicity on proliferation of the mesothelial cells depends not only on the hyperosmolality but also on some metabolic effects .
basal cytotoxicity in vitro tests on pd fluids show that all major commercial brands of pd fluids are cytotoxic to cell proliferation , ranging from 53 to 75% inhibition of cell growth .
this was found to be as a result of gdp and not because of interference with the pvc plastic of the bag as previously thought .
as the combination of low ph , lactate and the presence of gdp in pd fluids seems toxic to mesothelial cells , experiments were conducted with fluids that might be more biocompatible , such as isosmolar solutions with glucose polymers .
these solutions show better phagocytic capacity for common peritonitis - causing bacteria , and a significantly higher chemiluminescence response was found .
other studies demonstrated that exposure to solutions that consisted of a combination of lactate and bicarbonate buffer was less cytotoxic to the migratory capacity of normal human polymorphonuclear granulocytes .
bicarbonate / lactate - buffered pd fluids also improve ex vivo peritoneal macrophage tnf secretion , which might improve host defence status .
recent ex vivo findings suggest that human mesothelial cells not merely suffer , but are partly culprits for peritoneal injury . in long - term pd ,
new fibroblast - like cells arise from local conversion of mesothelial cells , which invade the submesothelial tissue and contribute to peritoneal fibrosis and angiogenesis .
thus , the combination of low ph , lactate and the presence of gdp in pd fluids seems cytoxic to and weakens defence mechanisms of mesothelial cells in ex vivo and in vitro studies . to avoid limitations of in vitro studies ,
rat , rabbit and occasionally mouse models have been used to examine physiological effects of pd fluids on mesothelium and the peritoneal membrane .
daily intraperitoneal injections of 3.86% glucose pd fluid in rats caused hyperplasia of mesothelial cells after 6 weeks .
di paolo and gotloib had similar results in rabbits and mice , respectively , and concluded that the alterations observed after long - term exposure of the mesothelium to pd fluid are mainly caused by the high concentration of glucose , whereas the eventual role of low ph seemed marginal .
the basement membrane of omental capillaries also shows marked lamination after 20-week daily exposure to hypertonic glucose ( 3.86% ) pd fluid .
this was in contrast to the same period of exposure to ringer 's lactate solution .
these data suggest a relation between glucose and damaging effects to the peritoneal mesothelial layer in long - term pd .
however , concerning this issue , animal studies differ in their results . in chronic peritoneal exposure studies in mice ,
mesothelial viability and integrity of the peritoneal membrane was examined by trypan blue staining and by assessing mesothelial denudation .
the mesothelial viability was better upon 30-day exposure to bicarbonate - buffered pd fluids or 12-week exposure to lactate / bicarbonate - buffered pd fluids .
other researchers , however , did not find any differences in mesothelial morphology between both types of fluid in rats and rabbits .
a rat experimental model in 10 nonuraemic rats , which were dialyzed twice daily for 4 weeks with high glucose concentration fluids , showed healing of the peritoneum after catheter implantation . still , hyaluronic acid levels in the dialysate increased and a tendency to thickening of the peritoneum was observed when compared to nondialyzed animals . in a long - term exposure model in rats by zareie et al .
, instillation of the current lactate - based pd fluid had deleterious effects on the peritoneal tissue .
exposition of the mesothelium to low ph lactate buffer solutions increased the number of mast cells , milky spots and milky spot areas , which are considered to be consequences of immune activation .
a clear thickening of the endothelial cell layer , suggestive of endothelial activation , and an increase in the number of blood vessels were also found .
addition of glucose to this buffer ( filter - sterilized pd fluid ) especially strengthened the induction of fibrosis and the number of omental vessels .
in addition to low ph and glucose , the presence of gdp ( heat - sterilized pd - fluids ) showed a further increase in the number and size of milky spots , parietal blood vessels , loss of mesothelial cell integrity , the number of fc - receptor positive cells , a thickening of the mesenteric submesothelial extracellular matrix and an increase in the number of rolling leucocytes .
interestingly , acidification of bicarbonate / lactate pd fluid did not contribute to peritoneal worsening , which confirms the findings earlier mentioned in ex vivo studies . during chronic exposition to bicarbonate / lactate solutions with fewer gdp and neutral ph , the increase in milky spots and the formation of new blood vessels were reduced . by means of a total histological score by light microscopy , reduced peritoneal fibrosis upon bicarbonate - buffered pd fluid dialysis has also been suggested , together with a diminished non - specific inflammatory response and increased production of inflammatory cytokines upon endotoxin challenge . in another pd model for rats ,
a standard lactate - buffered pd solution was compared with a bicarbonate / lactate - buffered solution during a 12-week study period . in rats treated with a standard low ph solution ,
an increase was demonstrated in vascular endothelial growth factor ( vegf ) , micro - vascular proliferation and submesothelial fibrosis .
furthermore , an accumulation of advanced glycation end products ( ages ) and an up - regulation of the receptor for ages were found .
the peritonitis rate was not different between rabbits that had been treated for 4 weeks by lactate / bicarbonate - buffered pd fluid , but the severity of peritonitis was significantly higher in the lactate group .
therefore , in animal models , mainly glucose and gdp are associated with submesothelial thickening , fibrosis , neoangiogenesis , the presence of ages and the poorer inflammatory response .
the difference in peritonitis incidence between the conventional and the more biocompatible pd fluids has not been established .
experiments have been performed in rabbits and rats to study the direct effects of neutral ph bicarbonate - buffered pd solutions on peritoneal transport , appetite and microcirculation .
these data point in the same direction , namely , an improved ultrafiltration , no vasodilation of peritoneal arterioles , improved appetite and leukocyte recruitment by bicarbonate - buffered pd fluids compared to lactate - buffered pd fluids . park et al
. demonstrated that in rats , peritoneal ultrafiltration had decreased after 12-week exposure to the conventional pd solution , but administration of a neutral bicarbonate / lactate - buffered pd fluid resulted in less loss of peritoneal ultrafiltration .
however , contrasting results were reported by suzuki et al . , who showed increased glucose absorption in rats along with decreased creatinine and protein clearance upon dialysis with bicarbonate - buffered pd fluid , which imply an increased peritoneal permeability compared to conventional lactate - buffered pd fluid .
suggested reduced transperitoneal protein transport after 4-week treatment in rats with a bicarbonate - buffered pd solution . in the study by pawlaczyk et al .
, no differences were seen in the transport characteristics between bicarbonate / lactate- and lactate - buffered pd fluids in rats .
therefore , at present , it is not clear whether bicarbonate - buffered pd fluids improve peritoneal transport physiology in chronic peritoneal exposure animal models .
however , this might be related to the animal model , as it is difficult to achieve statistically significant differences in peritoneal transport data in small laboratory animals such as the rat .
however , the data summarized so far indicate that at least in in vitro studies and in preclinical rat pd models , the new generation of ph neutral , two - chamber pd fluids are more biocompatible than conventional acidic pd fluids .
several clinical studies have been conducted to investigate parameters reflecting technique survival and peritoneum preservation in patients using conventional pd fluids versus the new more physiological , biocompatible ones .
designed a large randomized clinical trial comparing lactate - buffered pd fluids to bicarbonate / lactate solutions .
the previously mentioned study groups did not show differences in the occurrence of peritonitis between the two solutions .
with respect to ultrafiltration , a significant increase in ultrafiltration after 6 months was seen by tranaeus 's group , but these results were in contrast to those of rippe et al . , who did not find any increase in ultrafiltration over a period of 2 years .
the eurobalance study even found less ultrafiltration in the group that used the new pd fluids .
thus far , differences in peritoneal creatinine , urea clearance and kt / v have never been seen . in the eurobalance study ,
renal creatinine and urea clearances were higher when patients had undergone 3-month treatment with pd fluids that were ph neutral , lactate buffered and low in gdp .
several publications underline the importance of rrf for the morbidity and survival of pd patients [ 4749 ] .
. also showed that urine volume was higher in patients treated with the new pd fluids .
this was not shown in the nepp ( nutrineal , extraneal , physioneal , physioneal ) study , which compared conventional fluids with a regimen low in glucose and gdp with icodextrin and amino acids . in peritoneal biopsies and postmortal studies in long - term pd patients
, a predominant finding was revealed that was the development of peritoneal fibrosis that had a deleterious effect on membrane function . over the last 15 years
, interest has increased in the identification of potential markers that can be measured in the dialysis effluent that can provide information concerning the state of the peritoneum in vivo .
rippe et al . also investigated peritoneal mesothelial and interstitial integrity by analysing effluent dialysate .
after 2 years , a decrease in hyaluronan acid ( marker of inflammation ) and an increase in ca 125 were found in ph neutral pd fluid .
jones et al . had similar results after 6 months and found no changes in ca 125 and hyaluronan acid in the lactate group .
no differences were found in markers of fibrosis ( procollagen i peptide and tgf - 1 ) . in the nepp study , ca 125 levels in the effluents were higher in the group using nepp , suggesting a better preservation of the mesothelium .
however , levels of vegf , hyaluronic acid and il-6 were also higher . williams et al
. demonstrated higher levels of ca 125 in the effluent of patients using balance , a low gdp and neutral - ph pd fluid .
currently there are not many data on the effects of biocompatible solutions on survival . in a retrospective observational study , lee et al
. showed an advantage in survival rates in those who used balance compared to conventional fluids ( 74% versus 62% at 28 months ) .
a limitation in this study was the difficulty to interpret survival based on observational data . in conclusion , at present , the long - term effects of ph neutral pd fluids that are low in gdp are not clear .
they seem to better preserve the peritoneal membrane and have less systemic effects than the conventional ones .
none of the previously mentioned studies investigated local and systemic markers of inflammation , transport characteristics , rrf as well as technique survival in the same cohort of patients .
it is also not always clear whether patients had used biocompatible fluids before being included in the study .
furthermore , most of these studies , except for one , had a follow - up of only 612 months .
the effects on peritoneal transport , technique survival and patient survival remain unanswered . today , there is still a lack of quality prospective studies that directly compare these solutions to conventional glucose / lactate - based pd fluids . to answer questions that remain ,
cells that are continuously exposed to dialysis fluids are mesothelial cells and ( in part ) macrophages .
a majority of data on the in vivo effect of pd fluids are derived from ex vivo measurements of effects of pd fluids characteristics on cell function . in a number of studies with animal and human mesothelial cells ,
the characteristic or combination of characteristics in pd fluids that was responsible for the imbalance in these studies remained unclear .
the low ph and high osmolality in pd fluids have an inhibitory effect on proliferation of mesothelial cells and their functions because of the production of cytokines and prostaglandins . adding fresh pd fluid to residual pd fluids with normal ph within the peritoneal cavity resulted in a ph value of 7 after 10 min . in an ex vivo experiment
, the combination of low ph and high concentrations of sodium lactate in pd fluids did result in suppression of respiratory burst activation , a cell host defence mechanism , which is not because of either low ph or lactate concentration alone .
inhibition of the respiratory burst activation was related to the extreme sensitivity of nadph oxidase to low intracellular ph .
these findings suggest that the clinical importance of low ph alone might be overestimated and that other factors are more important .
other hyperosmotic solutes , which were used in the same concentrations as glucose ( mannitol and glycerol ) , did not decrease proliferation of the mesothelial cells as much as glucose did .
this implies that the toxicity on proliferation of the mesothelial cells depends not only on the hyperosmolality but also on some metabolic effects .
basal cytotoxicity in vitro tests on pd fluids show that all major commercial brands of pd fluids are cytotoxic to cell proliferation , ranging from 53 to 75% inhibition of cell growth .
this was found to be as a result of gdp and not because of interference with the pvc plastic of the bag as previously thought .
as the combination of low ph , lactate and the presence of gdp in pd fluids seems toxic to mesothelial cells , experiments were conducted with fluids that might be more biocompatible , such as isosmolar solutions with glucose polymers .
these solutions show better phagocytic capacity for common peritonitis - causing bacteria , and a significantly higher chemiluminescence response was found .
other studies demonstrated that exposure to solutions that consisted of a combination of lactate and bicarbonate buffer was less cytotoxic to the migratory capacity of normal human polymorphonuclear granulocytes .
bicarbonate / lactate - buffered pd fluids also improve ex vivo peritoneal macrophage tnf secretion , which might improve host defence status .
recent ex vivo findings suggest that human mesothelial cells not merely suffer , but are partly culprits for peritoneal injury . in long - term pd ,
new fibroblast - like cells arise from local conversion of mesothelial cells , which invade the submesothelial tissue and contribute to peritoneal fibrosis and angiogenesis .
thus , the combination of low ph , lactate and the presence of gdp in pd fluids seems cytoxic to and weakens defence mechanisms of mesothelial cells in ex vivo and in vitro studies .
to avoid limitations of in vitro studies , animal models of pd have been developed .
rat , rabbit and occasionally mouse models have been used to examine physiological effects of pd fluids on mesothelium and the peritoneal membrane .
daily intraperitoneal injections of 3.86% glucose pd fluid in rats caused hyperplasia of mesothelial cells after 6 weeks .
di paolo and gotloib had similar results in rabbits and mice , respectively , and concluded that the alterations observed after long - term exposure of the mesothelium to pd fluid are mainly caused by the high concentration of glucose , whereas the eventual role of low ph seemed marginal .
the basement membrane of omental capillaries also shows marked lamination after 20-week daily exposure to hypertonic glucose ( 3.86% ) pd fluid .
this was in contrast to the same period of exposure to ringer 's lactate solution .
these data suggest a relation between glucose and damaging effects to the peritoneal mesothelial layer in long - term pd .
however , concerning this issue , animal studies differ in their results . in chronic peritoneal exposure studies in mice ,
mesothelial viability and integrity of the peritoneal membrane was examined by trypan blue staining and by assessing mesothelial denudation .
the mesothelial viability was better upon 30-day exposure to bicarbonate - buffered pd fluids or 12-week exposure to lactate / bicarbonate - buffered pd fluids .
other researchers , however , did not find any differences in mesothelial morphology between both types of fluid in rats and rabbits .
a rat experimental model in 10 nonuraemic rats , which were dialyzed twice daily for 4 weeks with high glucose concentration fluids , showed healing of the peritoneum after catheter implantation . still , hyaluronic acid levels in the dialysate increased and a tendency to thickening of the peritoneum was observed when compared to nondialyzed animals . in a long - term exposure model in rats by zareie et al .
, instillation of the current lactate - based pd fluid had deleterious effects on the peritoneal tissue .
exposition of the mesothelium to low ph lactate buffer solutions increased the number of mast cells , milky spots and milky spot areas , which are considered to be consequences of immune activation .
a clear thickening of the endothelial cell layer , suggestive of endothelial activation , and an increase in the number of blood vessels were also found .
addition of glucose to this buffer ( filter - sterilized pd fluid ) especially strengthened the induction of fibrosis and the number of omental vessels .
in addition to low ph and glucose , the presence of gdp ( heat - sterilized pd - fluids ) showed a further increase in the number and size of milky spots , parietal blood vessels , loss of mesothelial cell integrity , the number of fc - receptor positive cells , a thickening of the mesenteric submesothelial extracellular matrix and an increase in the number of rolling leucocytes .
acidification of bicarbonate / lactate pd fluid did not contribute to peritoneal worsening , which confirms the findings earlier mentioned in ex vivo studies . during chronic exposition
to bicarbonate / lactate solutions with fewer gdp and neutral ph , the increase in milky spots and the formation of new blood vessels were reduced . by means of a total histological score by light microscopy , reduced peritoneal fibrosis upon bicarbonate - buffered pd fluid dialysis has also been suggested , together with a diminished non - specific inflammatory response and increased production of inflammatory cytokines upon endotoxin challenge . in another pd model for rats ,
a standard lactate - buffered pd solution was compared with a bicarbonate / lactate - buffered solution during a 12-week study period . in rats treated with a standard low ph solution ,
an increase was demonstrated in vascular endothelial growth factor ( vegf ) , micro - vascular proliferation and submesothelial fibrosis .
furthermore , an accumulation of advanced glycation end products ( ages ) and an up - regulation of the receptor for ages were found . the peritonitis rate was not different between rabbits that had been treated for 4 weeks by lactate / bicarbonate - buffered pd fluid , but the severity of peritonitis was significantly higher in the lactate group .
likewise , the dialysate leukocyte count had only declined significantly in the lactate group . therefore , in animal models , mainly glucose and gdp are associated with submesothelial thickening , fibrosis , neoangiogenesis , the presence of ages and the poorer inflammatory response .
the difference in peritonitis incidence between the conventional and the more biocompatible pd fluids has not been established .
experiments have been performed in rabbits and rats to study the direct effects of neutral ph bicarbonate - buffered pd solutions on peritoneal transport , appetite and microcirculation .
these data point in the same direction , namely , an improved ultrafiltration , no vasodilation of peritoneal arterioles , improved appetite and leukocyte recruitment by bicarbonate - buffered pd fluids compared to lactate - buffered pd fluids .
park et al . demonstrated that in rats , peritoneal ultrafiltration had decreased after 12-week exposure to the conventional pd solution , but administration of a neutral bicarbonate / lactate - buffered pd fluid resulted in less loss of peritoneal ultrafiltration .
however , contrasting results were reported by suzuki et al . , who showed increased glucose absorption in rats along with decreased creatinine and protein clearance upon dialysis with bicarbonate - buffered pd fluid , which imply an increased peritoneal permeability compared to conventional lactate - buffered pd fluid .
suggested reduced transperitoneal protein transport after 4-week treatment in rats with a bicarbonate - buffered pd solution . in the study by pawlaczyk et al .
, no differences were seen in the transport characteristics between bicarbonate / lactate- and lactate - buffered pd fluids in rats .
therefore , at present , it is not clear whether bicarbonate - buffered pd fluids improve peritoneal transport physiology in chronic peritoneal exposure animal models .
however , this might be related to the animal model , as it is difficult to achieve statistically significant differences in peritoneal transport data in small laboratory animals such as the rat .
however , the data summarized so far indicate that at least in in vitro studies and in preclinical rat pd models , the new generation of ph neutral , two - chamber pd fluids are more biocompatible than conventional acidic pd fluids .
several clinical studies have been conducted to investigate parameters reflecting technique survival and peritoneum preservation in patients using conventional pd fluids versus the new more physiological , biocompatible ones .
designed a large randomized clinical trial comparing lactate - buffered pd fluids to bicarbonate / lactate solutions .
the previously mentioned study groups did not show differences in the occurrence of peritonitis between the two solutions .
with respect to ultrafiltration , a significant increase in ultrafiltration after 6 months was seen by tranaeus 's group , but these results were in contrast to those of rippe et al . , who did not find any increase in ultrafiltration over a period of 2 years .
the eurobalance study even found less ultrafiltration in the group that used the new pd fluids .
thus far , differences in peritoneal creatinine , urea clearance and kt / v have never been seen . in the eurobalance study ,
renal creatinine and urea clearances were higher when patients had undergone 3-month treatment with pd fluids that were ph neutral , lactate buffered and low in gdp .
several publications underline the importance of rrf for the morbidity and survival of pd patients [ 4749 ] .
. also showed that urine volume was higher in patients treated with the new pd fluids .
this was not shown in the nepp ( nutrineal , extraneal , physioneal , physioneal ) study , which compared conventional fluids with a regimen low in glucose and gdp with icodextrin and amino acids . in peritoneal biopsies and postmortal studies in long - term pd patients
, a predominant finding was revealed that was the development of peritoneal fibrosis that had a deleterious effect on membrane function . over the last 15 years
, interest has increased in the identification of potential markers that can be measured in the dialysis effluent that can provide information concerning the state of the peritoneum in vivo .
rippe et al . also investigated peritoneal mesothelial and interstitial integrity by analysing effluent dialysate .
after 2 years , a decrease in hyaluronan acid ( marker of inflammation ) and an increase in ca 125 were found in ph neutral pd fluid .
jones et al . had similar results after 6 months and found no changes in ca 125 and hyaluronan acid in the lactate group .
no differences were found in markers of fibrosis ( procollagen i peptide and tgf - 1 ) . in the nepp study , ca 125 levels in the effluents were higher in the group using nepp , suggesting a better preservation of the mesothelium .
however , levels of vegf , hyaluronic acid and il-6 were also higher . williams et al
. demonstrated higher levels of ca 125 in the effluent of patients using balance , a low gdp and neutral - ph pd fluid .
currently there are not many data on the effects of biocompatible solutions on survival . in a retrospective observational study ,
lee et al . showed an advantage in survival rates in those who used balance compared to conventional fluids ( 74% versus 62% at 28 months ) .
a limitation in this study was the difficulty to interpret survival based on observational data . in conclusion , at present , the long - term effects of ph neutral pd fluids that are low in gdp are not clear .
they seem to better preserve the peritoneal membrane and have less systemic effects than the conventional ones .
none of the previously mentioned studies investigated local and systemic markers of inflammation , transport characteristics , rrf as well as technique survival in the same cohort of patients .
it is also not always clear whether patients had used biocompatible fluids before being included in the study .
furthermore , most of these studies , except for one , had a follow - up of only 612 months .
the effects on peritoneal transport , technique survival and patient survival remain unanswered . today , there is still a lack of quality prospective studies that directly compare these solutions to conventional glucose / lactate - based pd fluids . to answer questions that remain , | the most important challenge in peritoneal dialysis ( pd ) is long - term preservation of peritoneal membrane structure and function .
introduction of dialysis fluids into the peritoneal cavity induces changes .
these changes are related to duration of dialysis , occurrence of peritonitis and components of the dialysis solution .
bioincompatibility is considered to be the major cause of the development of morphological changes of the peritoneal membrane .
ph neutral pd fluids that are low in glucose degradation products ( gdp ) seem to better preserve the peritoneal membrane and have less systemic effects than the conventional ones . however , the long - term effects are not clear .
an overview of the effects of conventional pd fluids and glucose - based pd fluids with neutral ph in ex vivo and in vivo animal and clinical studies is presented . | Introduction
In vitro and ex vivo studies: effects of PD fluids characteristics on mesothelial cells and defence
Animal studies: effect of PD fluids on mesothelium and peritoneal membrane
Mesothelium
Peritoneum
Animal studies: peritoneal transport
Clinical studies | today , about half of the patients who have received pre - dialysis care choose peritoneal dialysis ( pd ) as their first dialysis modality . however , long - term technique survival of pd is poor because of technical problems and peritoneal membrane failure ; the latter is considered to be a consequence of bioincompatible characteristics of pd fluids . therefore , the most important challenge in pd is long - term preservation of peritoneal membrane structure and function . several studies have shown that introduction of dialysis fluids into the peritoneal cavity induces changes , ultimately leading to
membrane failure. the above - mentioned observations in humans have been studied in more detail in animals : chronic daily instillation of conventional pd fluids leads to an angiogenic response in the omentum , mesentery , liver surface and parietal peritoneal wall [ 58 ] . these changes are related to duration of dialysis , occurrence of peritonitis and components of the dialysis solution . although bioincompatibility , in general , is considered to be the major cause of the development of morphological changes of the peritoneal membrane ultimately resulting in uff , the major culprit is not known . conventional dialysis fluids are acidic ( ph 5.5 ) , contain supraphysiologic concentrations of lactate ( 3540 mmol / l ) and have a high osmolality ( 347 486 mosmol / kg h2o ) as a result of high glucose concentrations ( 75215 mmol / l ) that , after heat sterilization , turn into glucose degradation products ( gdp ) . new pd fluid formulations have become available : they have different osmotic agents and buffers such as glucose polymer - based and amino acid - based pd fluids . this report focuses on the effects of conventional pd fluids and glucose - based pd fluids with neutral ph . cells that are continuously exposed to dialysis fluids are mesothelial cells and ( in part ) macrophages . a majority of data on the in vivo effect of pd fluids are derived from ex vivo measurements of effects of pd fluids characteristics on cell function . in a number of studies with animal and human mesothelial cells ,
the characteristic or combination of characteristics in pd fluids that was responsible for the imbalance in these studies remained unclear . the low ph and high osmolality in pd fluids have an inhibitory effect on proliferation of mesothelial cells and their functions because of the production of cytokines and prostaglandins . adding fresh pd fluid to residual pd fluids with normal ph within the peritoneal cavity resulted in a ph value of 7 after 10 min . in an ex vivo experiment
, the combination of low ph and high concentrations of sodium lactate in pd fluids did result in suppression of respiratory burst activation , a cell host defence mechanism , which is not because of either low ph or lactate concentration alone . inhibition of the respiratory burst activation was related to the extreme sensitivity of nadph oxidase to low intracellular ph . in this situation , the presence of lactate and glucose can lead to pseudo - hypoxia . this was found to be as a result of gdp and not because of interference with the pvc plastic of the bag as previously thought . as the combination of low ph , lactate and the presence of gdp in pd fluids seems toxic to mesothelial cells , experiments were conducted with fluids that might be more biocompatible , such as isosmolar solutions with glucose polymers . bicarbonate / lactate - buffered pd fluids also improve ex vivo peritoneal macrophage tnf secretion , which might improve host defence status . in long - term pd ,
new fibroblast - like cells arise from local conversion of mesothelial cells , which invade the submesothelial tissue and contribute to peritoneal fibrosis and angiogenesis . thus , the combination of low ph , lactate and the presence of gdp in pd fluids seems cytoxic to and weakens defence mechanisms of mesothelial cells in ex vivo and in vitro studies . to avoid limitations of in vitro studies ,
rat , rabbit and occasionally mouse models have been used to examine physiological effects of pd fluids on mesothelium and the peritoneal membrane . di paolo and gotloib had similar results in rabbits and mice , respectively , and concluded that the alterations observed after long - term exposure of the mesothelium to pd fluid are mainly caused by the high concentration of glucose , whereas the eventual role of low ph seemed marginal . these data suggest a relation between glucose and damaging effects to the peritoneal mesothelial layer in long - term pd . however , concerning this issue , animal studies differ in their results . in chronic peritoneal exposure studies in mice ,
mesothelial viability and integrity of the peritoneal membrane was examined by trypan blue staining and by assessing mesothelial denudation . in a long - term exposure model in rats by zareie et al . , instillation of the current lactate - based pd fluid had deleterious effects on the peritoneal tissue . exposition of the mesothelium to low ph lactate buffer solutions increased the number of mast cells , milky spots and milky spot areas , which are considered to be consequences of immune activation . a clear thickening of the endothelial cell layer , suggestive of endothelial activation , and an increase in the number of blood vessels were also found . in addition to low ph and glucose , the presence of gdp ( heat - sterilized pd - fluids ) showed a further increase in the number and size of milky spots , parietal blood vessels , loss of mesothelial cell integrity , the number of fc - receptor positive cells , a thickening of the mesenteric submesothelial extracellular matrix and an increase in the number of rolling leucocytes . interestingly , acidification of bicarbonate / lactate pd fluid did not contribute to peritoneal worsening , which confirms the findings earlier mentioned in ex vivo studies . during chronic exposition to bicarbonate / lactate solutions with fewer gdp and neutral ph , the increase in milky spots and the formation of new blood vessels were reduced . furthermore , an accumulation of advanced glycation end products ( ages ) and an up - regulation of the receptor for ages were found . the peritonitis rate was not different between rabbits that had been treated for 4 weeks by lactate / bicarbonate - buffered pd fluid , but the severity of peritonitis was significantly higher in the lactate group . the difference in peritonitis incidence between the conventional and the more biocompatible pd fluids has not been established . experiments have been performed in rabbits and rats to study the direct effects of neutral ph bicarbonate - buffered pd solutions on peritoneal transport , appetite and microcirculation . these data point in the same direction , namely , an improved ultrafiltration , no vasodilation of peritoneal arterioles , improved appetite and leukocyte recruitment by bicarbonate - buffered pd fluids compared to lactate - buffered pd fluids . demonstrated that in rats , peritoneal ultrafiltration had decreased after 12-week exposure to the conventional pd solution , but administration of a neutral bicarbonate / lactate - buffered pd fluid resulted in less loss of peritoneal ultrafiltration . , no differences were seen in the transport characteristics between bicarbonate / lactate- and lactate - buffered pd fluids in rats . therefore , at present , it is not clear whether bicarbonate - buffered pd fluids improve peritoneal transport physiology in chronic peritoneal exposure animal models . however , this might be related to the animal model , as it is difficult to achieve statistically significant differences in peritoneal transport data in small laboratory animals such as the rat . however , the data summarized so far indicate that at least in in vitro studies and in preclinical rat pd models , the new generation of ph neutral , two - chamber pd fluids are more biocompatible than conventional acidic pd fluids . several clinical studies have been conducted to investigate parameters reflecting technique survival and peritoneum preservation in patients using conventional pd fluids versus the new more physiological , biocompatible ones . the previously mentioned study groups did not show differences in the occurrence of peritonitis between the two solutions . thus far , differences in peritoneal creatinine , urea clearance and kt / v have never been seen . in the eurobalance study ,
renal creatinine and urea clearances were higher when patients had undergone 3-month treatment with pd fluids that were ph neutral , lactate buffered and low in gdp . also showed that urine volume was higher in patients treated with the new pd fluids . this was not shown in the nepp ( nutrineal , extraneal , physioneal , physioneal ) study , which compared conventional fluids with a regimen low in glucose and gdp with icodextrin and amino acids . in peritoneal biopsies and postmortal studies in long - term pd patients
, a predominant finding was revealed that was the development of peritoneal fibrosis that had a deleterious effect on membrane function . over the last 15 years
, interest has increased in the identification of potential markers that can be measured in the dialysis effluent that can provide information concerning the state of the peritoneum in vivo . after 2 years , a decrease in hyaluronan acid ( marker of inflammation ) and an increase in ca 125 were found in ph neutral pd fluid . in the nepp study , ca 125 levels in the effluents were higher in the group using nepp , suggesting a better preservation of the mesothelium . currently there are not many data on the effects of biocompatible solutions on survival . in conclusion , at present , the long - term effects of ph neutral pd fluids that are low in gdp are not clear . they seem to better preserve the peritoneal membrane and have less systemic effects than the conventional ones . the effects on peritoneal transport , technique survival and patient survival remain unanswered . today , there is still a lack of quality prospective studies that directly compare these solutions to conventional glucose / lactate - based pd fluids . to answer questions that remain ,
cells that are continuously exposed to dialysis fluids are mesothelial cells and ( in part ) macrophages . a majority of data on the in vivo effect of pd fluids are derived from ex vivo measurements of effects of pd fluids characteristics on cell function . in a number of studies with animal and human mesothelial cells ,
the characteristic or combination of characteristics in pd fluids that was responsible for the imbalance in these studies remained unclear . the low ph and high osmolality in pd fluids have an inhibitory effect on proliferation of mesothelial cells and their functions because of the production of cytokines and prostaglandins . adding fresh pd fluid to residual pd fluids with normal ph within the peritoneal cavity resulted in a ph value of 7 after 10 min . in an ex vivo experiment
, the combination of low ph and high concentrations of sodium lactate in pd fluids did result in suppression of respiratory burst activation , a cell host defence mechanism , which is not because of either low ph or lactate concentration alone . inhibition of the respiratory burst activation was related to the extreme sensitivity of nadph oxidase to low intracellular ph . other hyperosmotic solutes , which were used in the same concentrations as glucose ( mannitol and glycerol ) , did not decrease proliferation of the mesothelial cells as much as glucose did . basal cytotoxicity in vitro tests on pd fluids show that all major commercial brands of pd fluids are cytotoxic to cell proliferation , ranging from 53 to 75% inhibition of cell growth . this was found to be as a result of gdp and not because of interference with the pvc plastic of the bag as previously thought . as the combination of low ph , lactate and the presence of gdp in pd fluids seems toxic to mesothelial cells , experiments were conducted with fluids that might be more biocompatible , such as isosmolar solutions with glucose polymers . bicarbonate / lactate - buffered pd fluids also improve ex vivo peritoneal macrophage tnf secretion , which might improve host defence status . recent ex vivo findings suggest that human mesothelial cells not merely suffer , but are partly culprits for peritoneal injury . in long - term pd ,
new fibroblast - like cells arise from local conversion of mesothelial cells , which invade the submesothelial tissue and contribute to peritoneal fibrosis and angiogenesis . thus , the combination of low ph , lactate and the presence of gdp in pd fluids seems cytoxic to and weakens defence mechanisms of mesothelial cells in ex vivo and in vitro studies . rat , rabbit and occasionally mouse models have been used to examine physiological effects of pd fluids on mesothelium and the peritoneal membrane . di paolo and gotloib had similar results in rabbits and mice , respectively , and concluded that the alterations observed after long - term exposure of the mesothelium to pd fluid are mainly caused by the high concentration of glucose , whereas the eventual role of low ph seemed marginal . these data suggest a relation between glucose and damaging effects to the peritoneal mesothelial layer in long - term pd . however , concerning this issue , animal studies differ in their results . in chronic peritoneal exposure studies in mice ,
mesothelial viability and integrity of the peritoneal membrane was examined by trypan blue staining and by assessing mesothelial denudation . the mesothelial viability was better upon 30-day exposure to bicarbonate - buffered pd fluids or 12-week exposure to lactate / bicarbonate - buffered pd fluids . other researchers , however , did not find any differences in mesothelial morphology between both types of fluid in rats and rabbits . a rat experimental model in 10 nonuraemic rats , which were dialyzed twice daily for 4 weeks with high glucose concentration fluids , showed healing of the peritoneum after catheter implantation . in a long - term exposure model in rats by zareie et al . , instillation of the current lactate - based pd fluid had deleterious effects on the peritoneal tissue . exposition of the mesothelium to low ph lactate buffer solutions increased the number of mast cells , milky spots and milky spot areas , which are considered to be consequences of immune activation . a clear thickening of the endothelial cell layer , suggestive of endothelial activation , and an increase in the number of blood vessels were also found . in addition to low ph and glucose , the presence of gdp ( heat - sterilized pd - fluids ) showed a further increase in the number and size of milky spots , parietal blood vessels , loss of mesothelial cell integrity , the number of fc - receptor positive cells , a thickening of the mesenteric submesothelial extracellular matrix and an increase in the number of rolling leucocytes . acidification of bicarbonate / lactate pd fluid did not contribute to peritoneal worsening , which confirms the findings earlier mentioned in ex vivo studies . during chronic exposition
to bicarbonate / lactate solutions with fewer gdp and neutral ph , the increase in milky spots and the formation of new blood vessels were reduced . furthermore , an accumulation of advanced glycation end products ( ages ) and an up - regulation of the receptor for ages were found . likewise , the dialysate leukocyte count had only declined significantly in the lactate group . therefore , in animal models , mainly glucose and gdp are associated with submesothelial thickening , fibrosis , neoangiogenesis , the presence of ages and the poorer inflammatory response . the difference in peritonitis incidence between the conventional and the more biocompatible pd fluids has not been established . experiments have been performed in rabbits and rats to study the direct effects of neutral ph bicarbonate - buffered pd solutions on peritoneal transport , appetite and microcirculation . these data point in the same direction , namely , an improved ultrafiltration , no vasodilation of peritoneal arterioles , improved appetite and leukocyte recruitment by bicarbonate - buffered pd fluids compared to lactate - buffered pd fluids . demonstrated that in rats , peritoneal ultrafiltration had decreased after 12-week exposure to the conventional pd solution , but administration of a neutral bicarbonate / lactate - buffered pd fluid resulted in less loss of peritoneal ultrafiltration . therefore , at present , it is not clear whether bicarbonate - buffered pd fluids improve peritoneal transport physiology in chronic peritoneal exposure animal models . however , this might be related to the animal model , as it is difficult to achieve statistically significant differences in peritoneal transport data in small laboratory animals such as the rat . however , the data summarized so far indicate that at least in in vitro studies and in preclinical rat pd models , the new generation of ph neutral , two - chamber pd fluids are more biocompatible than conventional acidic pd fluids . several clinical studies have been conducted to investigate parameters reflecting technique survival and peritoneum preservation in patients using conventional pd fluids versus the new more physiological , biocompatible ones . the previously mentioned study groups did not show differences in the occurrence of peritonitis between the two solutions . in the eurobalance study ,
renal creatinine and urea clearances were higher when patients had undergone 3-month treatment with pd fluids that were ph neutral , lactate buffered and low in gdp . also showed that urine volume was higher in patients treated with the new pd fluids . this was not shown in the nepp ( nutrineal , extraneal , physioneal , physioneal ) study , which compared conventional fluids with a regimen low in glucose and gdp with icodextrin and amino acids . in peritoneal biopsies and postmortal studies in long - term pd patients
, a predominant finding was revealed that was the development of peritoneal fibrosis that had a deleterious effect on membrane function . over the last 15 years
, interest has increased in the identification of potential markers that can be measured in the dialysis effluent that can provide information concerning the state of the peritoneum in vivo . after 2 years , a decrease in hyaluronan acid ( marker of inflammation ) and an increase in ca 125 were found in ph neutral pd fluid . in the nepp study , ca 125 levels in the effluents were higher in the group using nepp , suggesting a better preservation of the mesothelium . currently there are not many data on the effects of biocompatible solutions on survival . in conclusion , at present , the long - term effects of ph neutral pd fluids that are low in gdp are not clear . they seem to better preserve the peritoneal membrane and have less systemic effects than the conventional ones . the effects on peritoneal transport , technique survival and patient survival remain unanswered . today , there is still a lack of quality prospective studies that directly compare these solutions to conventional glucose / lactate - based pd fluids . | [
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the exploration of patterns and relationships in large health statistics and survey data is always a difficult data analysis task due to the volumes of data collected .
examples of widely used surveys include world bank 's living standards measurement survey ( lsm ) on economic aspects of well - being such as income and consumption , and the demographic and health survey ( dhs ) which measures health indicators .
these surveys result in useful and vast geographic data that can help analyze geographical trends on a number of socio - demographic , health and economic situations at community , national as well as international levels .
geographical analysis of these data is based on a combination of indicators usually forming a number of composites of attributes on health , poverty or demography . in such large multidimensional datasets , the extraction of patterns and the discovery of new knowledge may be difficult , as patterns may remain hidden .
new approaches in data analysis and visualization are needed to represent such data in a visual form that can better stimulate pattern recognition and hypothesis generation , and to allow for better understanding and knowledge construction .
a number of approaches are used to address the multidimensional aspect of the analysis of these datasets .
for example the human development index ( from undp ) , is a relative index based on measures of the life expectancy , education ( literacy ) , and income .
information visualization techniques can be used in combination with other data analysis techniques to provide alternative exploration techniques for such data .
a number of authors have proposed using artificial neural networks as part of a strategy to improve geographical analysis of large , complex datasets [ 2 - 5 ] .
artificial neural networks have the ability to perform pattern recognition and classification , and are especially useful in situations where the data volumes are large and the relationships are unclear or even hidden .
this is because of their ability to handle noisy data in difficult non - ideal contexts .
particular attention has been directed to using the self - organizing map ( som ) neural network as a means of organizing complex information spaces [ 8 - 10 ] , and for the creation of abstractions where conventional methods may be limited because underlying relationships are not clear or classes of interest are not obvious .
recent effort in knowledge discovery in databases ( kdd ) has also provided a window for geographic knowledge discovery .
data mining , knowledge discovery , and visualization methods are often combined to try to understand structures and patterns in complex geographical data [ 11 - 13 ] .
one way to integrate kdd framework in geographic data exploration is to combine the computational analysis methods with visual analysis in a process that can support exploratory and knowledge discovery tasks .
we explore the som in a framework for data mining , knowledge discovery , and spatial analysis , to uncover the structure , patterns , relationships and trends in the data .
some graphical representations based on information visualization techniques and cartographic methods , are then used to portray derived structures and patterns in a visual form that can allow for better understanding of the structures and the geographical processes .
the use of these graphical representations ( information spaces ) play a role by offering visual representations of data that bring the properties of human perception to bear .
we present a framework for combining effective pattern extraction with the som and graphical representations in an integrated visual - computational environment , to provide exploration of the data and to support knowledge acquisition through interactions .
this framework is informed by current understanding of effective application of visual variables for cartographic and information design , developing theories of interface metaphors for geospatial information displays , and previous empirical studies of map and information visualization effectiveness .
it suggests a visual exploration of the structure of the dataset in its multidimensional space to allow for orientations for the analysis of relationships and correlations .
a number of graphical interface options and exploratory task support are used to guide the user in his hypothesis testing , evaluation and interpretation of patterns from general patterns extracted to specific explorations of selected attributes and spatial locations . in this paper ,
an application of the method is explored for a health and demographic survey data to provide some understanding of the complex relationships between socio - economic indicators , geographical locations , and health and poverty status .
the ultimate goal is to support visual data mining and exploration , and gain insights on appropriate underlying distributions , patterns and trends , and therefore contribute to enhance the understanding of the geographical processes .
in this section , an example of exploration of ( potential ) patterns in data is presented using the different visualization techniques described in methodology section . for illustration , a dataset from the human development network ( world bank ) containing 30 basic indicators for health and living standard including health expenditure , health risk factors , mortality , reproductive health , for
although this is not a very large dataset , the approach can be applied for far larger datasets .
complex correlations in this kind of statistical data can be portrayed using the self - organizing map to visualize the complex joint effect of the factors related to health as contained in the dataset .
the proposed approach offers a number of visualizations to show the clustering structure and similarity ( patterns ) .
the neurons of the som network are represented here by rectangular cells ( see figure 1 ) .
a dark colouring between the neurons corresponds to a large distance and thus represents a gap between the values in the input space .
a light colouring between the neurons signifies that the vectors are close to each other in the input space .
thus light areas represent clusters and dark areas cluster separators ( a gap between the values in the input space ) .
this representation can be used to visualize the structure of the input space and to get an impression of otherwise invisible structures in a multidimensional data space .
this distance matrix representation shows not only the values at map units but also the distances between map units . in figure 1
countries having similar characteristics based on the multivariate attributes are positioned close to each other , and the distance between them represents the degree of similarity or dissimilarity .
the different clusters on the map can be automatically encoded with different colors , to allow a geographic view of the similarity mapping based on the multivariate attributes .
these common characteristics representation can be regarded as the health standard for these countries ( figure 2b ) . on the geographic map , each country
is assigned a color describing the its health standard type in relation to other countries in contrast to other projection methods in general , the som does not try to preserve the distances directly but rather the relations or local structure of the input data . while the distance matrix representation is a good method for visualizing clusters , it does not provide a very clear picture of the overall shape of the data space because the visualization is tied to the som grid .
alternative representations to the distance matrix representation can be used : 2d and 3d projections ( using projection methods such as the sammon 's mapping and pca to project som results ) , 2d and 3d surface plots , and component planes .
the projection of the som offers a view of the clustering of the data with data items depicted as coloured nodes .
size , position and colour of markers can be used to depict the relationships between the data items .
this gives an informative picture of the global shape and the overall smoothness of the som in 2d or 3d space .
representation of the general patterns and clustering in the input data with a distance matrix visualization the unified distance matrix showing clustering and distances between positions on the map .
countries having similar characteristics according to the multivariate attributes ( health and health care situation ) are found close to each other .
alternative representations of the som general clustering of patterns alternative representations of the som general clustering of the data with projection of the som results in 2d space ( a ) . this graphic can be interactively manipulated ( rotation , panning , zooming , walkthrough ) and be viewed in 3d space .
the output of the som ( the similarity coding extracted from the computational analysis ) can be projected on a geographic map , to display the common characteristics of the countries according to the multivariate attributes ( b ) .
the clustering structure can also be viewed as 2d or 3d surfaces representing the distance matrix ( figure 3 and figure 4 ) using color value to indicate the average distance to neighbouring map units .
this is a spatialization that uses a landscape metaphor to represent the density , shape , and size or volume of clusters . unlike the projection in figure 2a that shows only the position and clustering of map units , areas with uniform color are used in the surface plots to show the clustering structure and relationships among map units . in the 3d surface ( figure 4 ) ,
color value and height are used to represent the regionalization of map units according to the multidimensional attributes .
exploration of mortality in 2d surface the 2d surface shows a flat organizing of the clusters and indicates the average distance among the different countries according to several attributes related to mortality in the dataset ( life expectancy in 1980 , life expectancy in 2001 , infant mortality rate in 1980 , infant mortality rate in 2001 , under 5 mortality rate in 1980 , under 5 mortality rate in 2001 , adult male mortality in 2001 , adult female mortality rate in 2001 , percentage of male over 65 years old 2002 , percentage of female over 65 years in 2002 ) .
exploration of mortality in 3d surface the 3d surface plot shows a better view of the distance between the clusters for same selected of variables on mortality in the dataset shown in figure 3 ( life expectancy in 1980 , life expectancy in 2001 , infant mortality rate in 1980 , infant mortality rate in 2001 , under 5 mortality rate in 1980 , under 5 mortality rate in 2001 , adult male mortality in 2001 , adult female mortality rate in 2001 , percentage of male over 65 years old 2002 , percentage of female over 65 years in 2002 ) . here
distance between map units is represented by height value along the z axis and color .
it becomes easier to see the different clusters and view the distances between the different countries according to the select attributes related to mortality in the dataset .
to illustrate the use of the 2d and 3d surface plots , we present a selection of variables of the dataset related to mortality ( figure 3 and figure 4 ) .
the correlations and relationships in the input data space can be easily visualized using the component planes visualization ( figure 5 ) .
the component planes show the values of the map elements for different attributes and how each input vector varies over the space of the som units ( here representing countries )
comparatively with the maps , patterns and relationships among all the attributes can be easily examined in a signle visual represention using the som component planes visualization ( figure 5a ) .
since the som represents the similarity clustering of the multivariate attributes , the visual representation becomes more accessible and easy to use for exploratory analyses .
this kind of spatial clustering makes it possible to conduct exploratory analyses to help in identifying the causes and correlates of health problems when overlayed with environmental , social , transportantion , and facilities data .
these map overlays have been important hypothesis - generating tools in public health research and policy - making .
detail exploration of the dataset using the som component visualization all the components can be displayed to reveal the relationships between the variables and the spatial locations ( countries ) in ( a ) .
selected components related to a specific hypothesis can be further explored and to facilitate visual recognition of relationships among selected variables . here
geographic maps of components corresponding to hypothesis found in the exploration can be displayed ( c ) .
unlike standard choropleth maps , the position of the map units ( which is the same for all displays ) is determined during the training of the network , according to the characteristics of the data samples .
a cell here can represent one or several political units according to the similarity in the data .
two variables that are correlated will be represented by similar displays . in figure 5a , all the components are displayed and a selection of one example attribute is made more visible for the analysis in figure 5b . from the view in figure 5a , correlations and relationships can be explored , and hypotheses can be made . it becomes easy to see for example that that the hiv prevalence rate has had an impact on life expectancy for the countries most affected by the epidemic ( botswana , lesotho , swaziland , namibia , zimbabwe and south africa ) if we consider life expectancy in 1980 and that of 2001 ; or that the death rate is relatively higher in countries with less developed heath care ( number of hospital beds , number of physicians per 1000 inhabitants ) such as the african countries . to enhance visual detection of the relationships and correlations ,
the components can be ordered so that variables that are correlated are displayed next to each other . the kind of visual representation ( imagery cues ) provided in the som component planes visualization can be used as an effective tool to visually detect correlations among operating variables in a large volume of multivariate data , facilitates visual detection , and has an impact on knowledge construction .
new knowledge can be unearthed through this process of exploration , which can be followed by the identification of associations between attributes , and finally the formulation and ultimate testing of hypotheses .
geographic maps can be made to represent the result of this reasoning process for better geographical exploration and comparison ( figure 5c ) .
the proposed approach offers a number of visualizations to show the clustering structure and similarity ( patterns ) .
the neurons of the som network are represented here by rectangular cells ( see figure 1 ) .
a dark colouring between the neurons corresponds to a large distance and thus represents a gap between the values in the input space .
a light colouring between the neurons signifies that the vectors are close to each other in the input space .
thus light areas represent clusters and dark areas cluster separators ( a gap between the values in the input space ) .
this representation can be used to visualize the structure of the input space and to get an impression of otherwise invisible structures in a multidimensional data space .
this distance matrix representation shows not only the values at map units but also the distances between map units . in figure 1
countries having similar characteristics based on the multivariate attributes are positioned close to each other , and the distance between them represents the degree of similarity or dissimilarity .
the different clusters on the map can be automatically encoded with different colors , to allow a geographic view of the similarity mapping based on the multivariate attributes .
these common characteristics representation can be regarded as the health standard for these countries ( figure 2b ) . on the geographic map , each country
is assigned a color describing the its health standard type in relation to other countries in contrast to other projection methods in general , the som does not try to preserve the distances directly but rather the relations or local structure of the input data . while the distance matrix representation is a good method for visualizing clusters , it does not provide a very clear picture of the overall shape of the data space because the visualization is tied to the som grid .
alternative representations to the distance matrix representation can be used : 2d and 3d projections ( using projection methods such as the sammon 's mapping and pca to project som results ) , 2d and 3d surface plots , and component planes .
the projection of the som offers a view of the clustering of the data with data items depicted as coloured nodes .
size , position and colour of markers can be used to depict the relationships between the data items .
this gives an informative picture of the global shape and the overall smoothness of the som in 2d or 3d space .
representation of the general patterns and clustering in the input data with a distance matrix visualization the unified distance matrix showing clustering and distances between positions on the map . countries having similar characteristics according to the multivariate attributes ( health and health care situation ) are found close to each other .
alternative representations of the som general clustering of patterns alternative representations of the som general clustering of the data with projection of the som results in 2d space ( a ) . this graphic can be interactively manipulated ( rotation , panning , zooming , walkthrough ) and be viewed in 3d space .
the output of the som ( the similarity coding extracted from the computational analysis ) can be projected on a geographic map , to display the common characteristics of the countries according to the multivariate attributes ( b ) .
the clustering structure can also be viewed as 2d or 3d surfaces representing the distance matrix ( figure 3 and figure 4 ) using color value to indicate the average distance to neighbouring map units .
this is a spatialization that uses a landscape metaphor to represent the density , shape , and size or volume of clusters . unlike the projection in figure 2a that shows only the position and clustering of map units , areas with uniform color
are used in the surface plots to show the clustering structure and relationships among map units . in the 3d surface ( figure 4 ) ,
color value and height are used to represent the regionalization of map units according to the multidimensional attributes .
exploration of mortality in 2d surface the 2d surface shows a flat organizing of the clusters and indicates the average distance among the different countries according to several attributes related to mortality in the dataset ( life expectancy in 1980 , life expectancy in 2001 , infant mortality rate in 1980 , infant mortality rate in 2001 , under 5 mortality rate in 1980 , under 5 mortality rate in 2001 , adult male mortality in 2001 , adult female mortality rate in 2001 , percentage of male over 65 years old 2002 , percentage of female over 65 years in 2002 ) .
exploration of mortality in 3d surface the 3d surface plot shows a better view of the distance between the clusters for same selected of variables on mortality in the dataset shown in figure 3 ( life expectancy in 1980 , life expectancy in 2001 , infant mortality rate in 1980 , infant mortality rate in 2001 , under 5 mortality rate in 1980 , under 5 mortality rate in 2001 , adult male mortality in 2001 , adult female mortality rate in 2001 , percentage of male over 65 years old 2002 , percentage of female over 65 years in 2002 ) . here distance between map units
it becomes easier to see the different clusters and view the distances between the different countries according to the select attributes related to mortality in the dataset . to illustrate the use of the 2d and 3d surface plots , we present a selection of variables of the dataset related to mortality ( figure 3 and figure 4 ) .
the correlations and relationships in the input data space can be easily visualized using the component planes visualization ( figure 5 ) .
the component planes show the values of the map elements for different attributes and how each input vector varies over the space of the som units ( here representing countries )
comparatively with the maps , patterns and relationships among all the attributes can be easily examined in a signle visual represention using the som component planes visualization ( figure 5a ) .
since the som represents the similarity clustering of the multivariate attributes , the visual representation becomes more accessible and easy to use for exploratory analyses .
this kind of spatial clustering makes it possible to conduct exploratory analyses to help in identifying the causes and correlates of health problems when overlayed with environmental , social , transportantion , and facilities data .
these map overlays have been important hypothesis - generating tools in public health research and policy - making .
detail exploration of the dataset using the som component visualization all the components can be displayed to reveal the relationships between the variables and the spatial locations ( countries ) in ( a ) .
selected components related to a specific hypothesis can be further explored and to facilitate visual recognition of relationships among selected variables . here
geographic maps of components corresponding to hypothesis found in the exploration can be displayed ( c ) . unlike standard choropleth maps ,
the position of the map units ( which is the same for all displays ) is determined during the training of the network , according to the characteristics of the data samples .
a cell here can represent one or several political units according to the similarity in the data .
two variables that are correlated will be represented by similar displays . in figure 5a , all the components are displayed and a selection of one example attribute is made more visible for the analysis in figure 5b . from the view in figure 5a , correlations and relationships can be explored , and hypotheses can be made . it becomes easy to see for example that that the hiv prevalence rate has had an impact on life expectancy for the countries most affected by the epidemic ( botswana , lesotho , swaziland , namibia , zimbabwe and south africa ) if we consider life expectancy in 1980 and that of 2001 ; or that the death rate is relatively higher in countries with less developed heath care ( number of hospital beds , number of physicians per 1000 inhabitants ) such as the african countries . to enhance visual detection of the relationships and correlations ,
the components can be ordered so that variables that are correlated are displayed next to each other . the kind of visual representation ( imagery cues ) provided in the som component planes visualization can be used as an effective tool to visually detect correlations among operating variables in a large volume of multivariate data , facilitates visual detection , and has an impact on knowledge construction .
new knowledge can be unearthed through this process of exploration , which can be followed by the identification of associations between attributes , and finally the formulation and ultimate testing of hypotheses .
geographic maps can be made to represent the result of this reasoning process for better geographical exploration and comparison ( figure 5c ) .
in this paper we have presented an approach to combine visual and computational analysis for exploratory visualization intended to contribute to the analysis of large volumes of health survey data .
the approach focuses on the effective application of computational algorithms to extract patterns and relationships in large datasets , and visual representation of derived information that involves effective use of visual variables used in such complex information spaces to facilitate knowledge construction .
a number of visualization techniques were explored with the objective to support visual exploration and knowledge construction .
users can perform a number of exploratory tasks to understand the structure of the dataset as a whole and also to explore detailed information on individual or selected attributes of the dataset such as finding correlations and the relationships among attributes . with this respect , the som computational analysis can support exploratory visualization and the knowledge discovery process when integrated with appropriate visual exploration tools .
interactive manipulation ( zooming , rotation , panning , filtering , and brushing ) of the graphical representations can enhance user goal specific querying and selection from the general patterns extracted to more specific user selection of attributes and spatial locations for exploration , hypothesis testing , and ultimately knowledge construction . we have extended these alternative representations of the som results used to highlight different characteristics of the computational solution and integrated them with other graphics into multiple views to allow brushing and linking , for exploratory analysis and knowledge discovery purposes .
this link between the attribute space visualization based on the som , the geographic space with maps representing the som results , and other graphics such as parallel coordinate plots , in multiple views can provide alternative perspectives for better exploration , evaluation and interpretation of patterns and ultimately support for knowledge construction .
the proposed framework explores ways to effectively extract patterns using data mining based on the som and to represent the results using graphical representations for visual exploration .
as presented in figure 6 , the data mining stage allows to construct a clustering ( similarity matrix ) of the multidimensional input space using the som training algorithm tool ( som toolbox ) and graphics processing with matlab software . from this computational process , global structure and patterns
can be represented with graphical representations and maps ( geographic view ) of similarity results . more exploration can be made on relationships and correlations among the attributes .
the framework includes spatial analysis , data mining and knowledge discovery methods , supported by interactive tools that allow users to perform a number of exploratory tasks to understand the structure of the dataset as a whole and also explore detailed information on individual or selected attributes of the dataset .
different representation forms are integrated and support user interaction for exploratory tasks to facilitate the knowledge discovery process .
they include some graphical representations based on the som , maps and other graphics such as parallel coordinate plots .
cartographic methods support this design for effective use of visual variables with which the visualizations are depicted .
the graphical representations can be interactively manipulation in matlab graphical interface using rotation , zooming , panning , and brushing .
data exploration framework from the computational process , global structure and patterns can be visualized with graphical representations and maps of similarity results .
relationships and correlations among the attributes are presented with interactive graphical representations , maps and other graphics such as parallel coordinate plots .
the self - organizing map is an artificial neural network used to map multidimensional data onto a low dimensional space , usually a 2d representation space ( see figure 7 ) .
the network consists of a number of neural processing units ( neurons ) usually arranged on a rectangular or hexagonal grid , where each neuron is connected to the input .
the goal is to group nodes close together in certain areas of the data value range .
each of the units i is assigned an n - dimensional weight vector mi that has the same dimensionality as the input patterns .
selection of a node and adaptation of neighbouring nodes of the neural network to the input data .
each training iteration t starts with the random selection of one input pattern x(t ) . using euclidean distance between weight vector and input pattern ,
the unit with the lowest activation is referred to as the winner , c , of the training iteration : mc(t ) = mini { ||x(t ) - mi(t)|| } ( 1 ) finally the weight vector of the winner as well as the weight vectors of selected units in the neighbourhood of the winner are adapted to represent the input pattern . at each step t of the random sequence of the given x(t ) values , the values of mi are gradually and adaptively changed in the following adaptation process : mi(t + 1 ) = mi(t ) + hci(t)[x(t ) - mi(t ) ] ( 2 ) the degree of adaptation in the neighbourhood is characterized by a neighbourhood function h , which is a decreasing function of the units from the winning unit on the map lattice until no noticeable changes are observed . as a result of a general adaptation process
, a number of units in the neighbourhood of the winner lead to a spatial clustering of similar input patterns in neighbouring parts of the som .
the resultant maps ( soms ) are organized in such a way that similar data are mapped onto the same node or to neighboring nodes in the map .
this leads to a spatial clustering of similar input patterns in neighboring parts of the som and the clusters that appear on the map are themselves organized internally .
this arrangement of the clusters in the map reflects the attribute relationships of the clusters in the input space .
for example , the size of the clusters ( the number of nodes allotted to each cluster ) is reflective of the frequency distribution of the patterns in the input set .
actually , the som uses a distribution preserving property which has the ability to allocate more nodes to input patterns that appear more frequently during the training phase of the network configuration
. it also applies a topology preserving property , which comes from the fact that similar data are mapped onto the same node , or to neighboring nodes in the map . in other words ,
the topology of the dataset in its n - dimensional space is captured by the som and reflected in the ordering of its nodes .
this is an important feature of the som that allows the data to be projected onto the lower dimension space while roughly preserving the order of the data in its original space .
another important feature of the som for knowledge discovery in complex datasets is the fact that it is an unsupervised learning network meaning that the training patterns have no category information that accompany them .
unlike supervised methods which learn to associate a set of inputs with a set of outputs using a training data set for which both input and output are known , som adopts a learning strategy where the similarity relationships between the data and the clusters are used to classify and categorize the data .
the som can be useful as a knowledge discovery tool in database methodology since it follows the probability density function of underlying data .
one approach to analysis of large amount of data is by using data mining and knowledge discovery methods .
the main goal of data mining is identifying valid , novel , potentially useful and ultimately understanding patterns in data .
generally , three general categories of data mining goals can be identified : explanatory ( to explain some observed events ) , confirmatory ( to confirm a hypothesis ) , exploratory ( to analyze data for new or unexpected relationships ) .
typical tasks , for which data mining techniques are often used , include clustering , classification , generalization and prediction .
the most popular methods include decision trees ( tree induction ) , value prediction , and association rules often used for classification .
artificial neural networks are particularly used for exploratory analysis as non - linear clustering and classification techniques .
for example , unsupervised neural networks such as the self - organizing map are a type of neural clustering and neural architecture such as backpropagation and feedforward are neural induction methods used for classification ( supervised learning ) .
the algorithms used in data mining are often integrated into knowledge discovery in databases ( kdd ) , a larger framework that aims at finding new knowledge from large databases . while data mining deals with transforming data into information or facts , kdd is a higher - level process using information derived from data mining process to turn it into knowledge or integrate it into prior knowledge . in general
, kdd stands for discovering and visualizing the regularities , structures and rules from data , discovering useful knowledge from data and for finding new knowledge .
it consists of several generic steps , namely data pre - processing , transformation ( dimension reduction , projection ) , data mining ( structure mining ) and interpretation / evaluation .
new developments in data mining and kdd has provided a window for geographic data mining and knowledge discovery , which has become an established field in geographic visualization [ 11,12,23 - 27 ] .
this framework has been used in geospatial data exploration [ 11 - 13,23,28 ] to discover unexpected correlation and causal relationships , and understand structures and patterns in complex geographical data .
the promises inherent in the development of data mining and knowledge discovery processes for geographical analysis include the ability to yield unexpected correlation and causal relationships .
since the dimensionality of the dataset is very high , it is often ineffective to work in such high dimension space to search for patterns .
we use the som algorithm as a data mining tool to project input data into an alternative measurement space based on similarities and relationships in the input data that can aid the search for patterns .
it becomes possible to achieve better results in such similarity space rather than the original attribute space .
as described in the previous section , the som adapts its internal structures to structural properties of the multidimensional input such as regularities , similarities , and frequencies .
these properties of the som can be used to search for structures in the multidimensional input .
graphical representations are then used to enable visual data exploration allowing the user to get insight into the data , evaluate , filter , and map outputs .
this is intended to support visual data mining by allowing several variables and their interactions to be inspected simultaneously , and receive feedback from the knowledge discovery process by means of interaction techniques that support the process .
one of the advantages of the som over other types of neural network is that it is suitable for visualization .
the first level of the computational analysis described above provides a mechanism for extracting patterns from the data .
the output of this computational process is depicted using graphical representations ( information spaces ) to facilitate human perception and cognitive processes , by offering visualizations of the general structure of the dataset ( clustering ) , as well as the exploration of relationships among attributes .
several graphical representations provide ways for representing similarity ( patterns ) , relationships , including a distance matrix representation , 2d and 3d projections , 2d and 3d surfaces , and component planes visualization .
they highlight different characteristics of the computational solution and integrate them with other graphics into multiple views to allow brushing , linking , panning , zooming , rotation and 3d views for exploratory analysis and knowledge discovery purposes and enhance exploration .
the resulting information spaces suggest and take advantage of natural environment metaphor characteristics such as ' near = similar , far = different ' , which is epitomized by tobler 's first law of geography .
distance ( similarity between data items ) , regions ( aggregation of similar data items ) , and scale ( level of detail in a database ) are examples of spatial metaphors used in these representation spaces .
the scale allows exploration of the information space at multiple levels of detail , and provides the potential for hierarchical grouping of items , and revealing categories or classifications .
coordinate systems allow determining distance and direction , from which other spatial relationships ( size , shape , density , arrangement ) may be derived .
we integrate the graphical representations mentioned above and maps to represent the attribute space . using multiple views ,
interactions between several variables can be presented simultaneously over the space of the som , maps and parallel coordinate plots .
this can emphasize visual change detection and the monitoring of the variability through the attribute space .
these alternative and different views on the data can help stimulate the visual thinking process that is characteristic for visual exploration .
four goals of the exploration are emphasized : - patterns discovery ( through similarity representations ) - correlations and relationships exploration for hypothesis generation - exploration of the distribution of the dataset on the map - and the detection of irregularities in the data
the proposed framework explores ways to effectively extract patterns using data mining based on the som and to represent the results using graphical representations for visual exploration .
as presented in figure 6 , the data mining stage allows to construct a clustering ( similarity matrix ) of the multidimensional input space using the som training algorithm tool ( som toolbox ) and graphics processing with matlab software . from this computational process , global structure and patterns
can be represented with graphical representations and maps ( geographic view ) of similarity results . more exploration can be made on relationships and correlations among the attributes .
the framework includes spatial analysis , data mining and knowledge discovery methods , supported by interactive tools that allow users to perform a number of exploratory tasks to understand the structure of the dataset as a whole and also explore detailed information on individual or selected attributes of the dataset .
different representation forms are integrated and support user interaction for exploratory tasks to facilitate the knowledge discovery process .
they include some graphical representations based on the som , maps and other graphics such as parallel coordinate plots .
cartographic methods support this design for effective use of visual variables with which the visualizations are depicted .
the graphical representations can be interactively manipulation in matlab graphical interface using rotation , zooming , panning , and brushing .
data exploration framework from the computational process , global structure and patterns can be visualized with graphical representations and maps of similarity results .
relationships and correlations among the attributes are presented with interactive graphical representations , maps and other graphics such as parallel coordinate plots .
the self - organizing map is an artificial neural network used to map multidimensional data onto a low dimensional space , usually a 2d representation space ( see figure 7 ) .
the network consists of a number of neural processing units ( neurons ) usually arranged on a rectangular or hexagonal grid , where each neuron is connected to the input .
the goal is to group nodes close together in certain areas of the data value range .
each of the units i is assigned an n - dimensional weight vector mi that has the same dimensionality as the input patterns .
selection of a node and adaptation of neighbouring nodes of the neural network to the input data .
each training iteration t starts with the random selection of one input pattern x(t ) . using euclidean distance between weight vector and input pattern , the activation of the units
the unit with the lowest activation is referred to as the winner , c , of the training iteration : mc(t ) = mini { ||x(t ) - mi(t)|| } ( 1 ) finally the weight vector of the winner as well as the weight vectors of selected units in the neighbourhood of the winner are adapted to represent the input pattern . at each step t of the random sequence of the given x(t ) values , the values of mi are gradually and adaptively changed in the following adaptation process : mi(t + 1 ) = mi(t ) + hci(t)[x(t ) - mi(t ) ] ( 2 ) the degree of adaptation in the neighbourhood is characterized by a neighbourhood function h , which is a decreasing function of the units from the winning unit on the map lattice until no noticeable changes are observed . as a result of a general adaptation process ,
a number of units in the neighbourhood of the winner lead to a spatial clustering of similar input patterns in neighbouring parts of the som .
the resultant maps ( soms ) are organized in such a way that similar data are mapped onto the same node or to neighboring nodes in the map .
this leads to a spatial clustering of similar input patterns in neighboring parts of the som and the clusters that appear on the map are themselves organized internally .
this arrangement of the clusters in the map reflects the attribute relationships of the clusters in the input space .
for example , the size of the clusters ( the number of nodes allotted to each cluster ) is reflective of the frequency distribution of the patterns in the input set . actually , the som uses a distribution preserving property which has the ability to allocate more nodes to input patterns that appear more frequently during the training phase of the network configuration
. it also applies a topology preserving property , which comes from the fact that similar data are mapped onto the same node , or to neighboring nodes in the map . in other words ,
the topology of the dataset in its n - dimensional space is captured by the som and reflected in the ordering of its nodes .
this is an important feature of the som that allows the data to be projected onto the lower dimension space while roughly preserving the order of the data in its original space .
another important feature of the som for knowledge discovery in complex datasets is the fact that it is an unsupervised learning network meaning that the training patterns have no category information that accompany them . unlike supervised methods which learn to associate a set of inputs with a set of outputs using a training data set for which both input and output are known
, som adopts a learning strategy where the similarity relationships between the data and the clusters are used to classify and categorize the data .
the som can be useful as a knowledge discovery tool in database methodology since it follows the probability density function of underlying data .
one approach to analysis of large amount of data is by using data mining and knowledge discovery methods .
the main goal of data mining is identifying valid , novel , potentially useful and ultimately understanding patterns in data .
generally , three general categories of data mining goals can be identified : explanatory ( to explain some observed events ) , confirmatory ( to confirm a hypothesis ) , exploratory ( to analyze data for new or unexpected relationships ) .
typical tasks , for which data mining techniques are often used , include clustering , classification , generalization and prediction .
the most popular methods include decision trees ( tree induction ) , value prediction , and association rules often used for classification .
artificial neural networks are particularly used for exploratory analysis as non - linear clustering and classification techniques .
for example , unsupervised neural networks such as the self - organizing map are a type of neural clustering and neural architecture such as backpropagation and feedforward are neural induction methods used for classification ( supervised learning ) .
the algorithms used in data mining are often integrated into knowledge discovery in databases ( kdd ) , a larger framework that aims at finding new knowledge from large databases . while data mining deals with transforming data into information or facts , kdd is a higher - level process using information derived from data mining process to turn it into knowledge or integrate it into prior knowledge . in general
, kdd stands for discovering and visualizing the regularities , structures and rules from data , discovering useful knowledge from data and for finding new knowledge .
it consists of several generic steps , namely data pre - processing , transformation ( dimension reduction , projection ) , data mining ( structure mining ) and interpretation / evaluation .
new developments in data mining and kdd has provided a window for geographic data mining and knowledge discovery , which has become an established field in geographic visualization [ 11,12,23 - 27 ] .
this framework has been used in geospatial data exploration [ 11 - 13,23,28 ] to discover unexpected correlation and causal relationships , and understand structures and patterns in complex geographical data .
the promises inherent in the development of data mining and knowledge discovery processes for geographical analysis include the ability to yield unexpected correlation and causal relationships .
since the dimensionality of the dataset is very high , it is often ineffective to work in such high dimension space to search for patterns .
we use the som algorithm as a data mining tool to project input data into an alternative measurement space based on similarities and relationships in the input data that can aid the search for patterns .
it becomes possible to achieve better results in such similarity space rather than the original attribute space .
as described in the previous section , the som adapts its internal structures to structural properties of the multidimensional input such as regularities , similarities , and frequencies .
these properties of the som can be used to search for structures in the multidimensional input .
graphical representations are then used to enable visual data exploration allowing the user to get insight into the data , evaluate , filter , and map outputs .
this is intended to support visual data mining by allowing several variables and their interactions to be inspected simultaneously , and receive feedback from the knowledge discovery process by means of interaction techniques that support the process .
one of the advantages of the som over other types of neural network is that it is suitable for visualization .
the first level of the computational analysis described above provides a mechanism for extracting patterns from the data .
the output of this computational process is depicted using graphical representations ( information spaces ) to facilitate human perception and cognitive processes , by offering visualizations of the general structure of the dataset ( clustering ) , as well as the exploration of relationships among attributes .
several graphical representations provide ways for representing similarity ( patterns ) , relationships , including a distance matrix representation , 2d and 3d projections , 2d and 3d surfaces , and component planes visualization .
they highlight different characteristics of the computational solution and integrate them with other graphics into multiple views to allow brushing , linking , panning , zooming , rotation and 3d views for exploratory analysis and knowledge discovery purposes and enhance exploration .
the resulting information spaces suggest and take advantage of natural environment metaphor characteristics such as ' near = similar , far = different ' , which is epitomized by tobler 's first law of geography .
distance ( similarity between data items ) , regions ( aggregation of similar data items ) , and scale ( level of detail in a database ) are examples of spatial metaphors used in these representation spaces .
the scale allows exploration of the information space at multiple levels of detail , and provides the potential for hierarchical grouping of items , and revealing categories or classifications .
coordinate systems allow determining distance and direction , from which other spatial relationships ( size , shape , density , arrangement ) may be derived .
we integrate the graphical representations mentioned above and maps to represent the attribute space . using multiple views ,
interactions between several variables can be presented simultaneously over the space of the som , maps and parallel coordinate plots .
this can emphasize visual change detection and the monitoring of the variability through the attribute space .
these alternative and different views on the data can help stimulate the visual thinking process that is characteristic for visual exploration .
four goals of the exploration are emphasized : - patterns discovery ( through similarity representations ) - correlations and relationships exploration for hypothesis generation - exploration of the distribution of the dataset on the map - and the detection of irregularities in the data | backgroundsurvey data are increasingly abundant from many international projects and national statistics .
they are generally comprehensive and cover local , regional as well as national levels census in many domains including health , demography , human development , and economy .
these surveys result in several hundred indicators .
geographical analysis of such large amount of data is often a difficult task and searching for patterns is particularly a difficult challenge .
geovisualization research is increasingly dealing with the exploration of patterns and relationships in such large datasets for understanding underlying geographical processes .
one of the attempts has been to use artificial neural networks as a technology especially useful in situations where the numbers are vast and the relationships are often unclear or even hidden.resultswe investigate ways to integrate computational analysis based on a self - organizing map neural network , with visual representations of derived structures and patterns in a framework for exploratory visualization to support visual data mining and knowledge discovery .
the framework suggests ways to explore the general structure of the dataset in its multidimensional space in order to provide clues for further exploration of correlations and relationships.conclusionin this paper , the proposed framework is used to explore a demographic and health survey data .
several graphical representations ( information spaces ) are used to depict the general structure and clustering of the data and get insight about the relationships among the different variables .
detail exploration of correlations and relationships among the attributes is provided .
results of the analysis are also presented in maps and other graphics . | Background
Results and discussion
Exploration of the general patterns and clustering
Exploration of correlations and relationships
Conclusions
Methods
A framework to support visual exploration of large geographic data
The Self-Organizing Map algorithm
Visual data mining and knowledge discovery
Integrating the computational analysis and visualization
Authors' contributions | the exploration of patterns and relationships in large health statistics and survey data is always a difficult data analysis task due to the volumes of data collected . examples of widely used surveys include world bank 's living standards measurement survey ( lsm ) on economic aspects of well - being such as income and consumption , and the demographic and health survey ( dhs ) which measures health indicators . these surveys result in useful and vast geographic data that can help analyze geographical trends on a number of socio - demographic , health and economic situations at community , national as well as international levels . geographical analysis of these data is based on a combination of indicators usually forming a number of composites of attributes on health , poverty or demography . in such large multidimensional datasets , the extraction of patterns and the discovery of new knowledge may be difficult , as patterns may remain hidden . a number of approaches are used to address the multidimensional aspect of the analysis of these datasets . for example the human development index ( from undp ) , is a relative index based on measures of the life expectancy , education ( literacy ) , and income . a number of authors have proposed using artificial neural networks as part of a strategy to improve geographical analysis of large , complex datasets [ 2 - 5 ] . artificial neural networks have the ability to perform pattern recognition and classification , and are especially useful in situations where the data volumes are large and the relationships are unclear or even hidden . particular attention has been directed to using the self - organizing map ( som ) neural network as a means of organizing complex information spaces [ 8 - 10 ] , and for the creation of abstractions where conventional methods may be limited because underlying relationships are not clear or classes of interest are not obvious . data mining , knowledge discovery , and visualization methods are often combined to try to understand structures and patterns in complex geographical data [ 11 - 13 ] . one way to integrate kdd framework in geographic data exploration is to combine the computational analysis methods with visual analysis in a process that can support exploratory and knowledge discovery tasks . we explore the som in a framework for data mining , knowledge discovery , and spatial analysis , to uncover the structure , patterns , relationships and trends in the data . some graphical representations based on information visualization techniques and cartographic methods , are then used to portray derived structures and patterns in a visual form that can allow for better understanding of the structures and the geographical processes . the use of these graphical representations ( information spaces ) play a role by offering visual representations of data that bring the properties of human perception to bear . we present a framework for combining effective pattern extraction with the som and graphical representations in an integrated visual - computational environment , to provide exploration of the data and to support knowledge acquisition through interactions . it suggests a visual exploration of the structure of the dataset in its multidimensional space to allow for orientations for the analysis of relationships and correlations . in this paper ,
an application of the method is explored for a health and demographic survey data to provide some understanding of the complex relationships between socio - economic indicators , geographical locations , and health and poverty status . the ultimate goal is to support visual data mining and exploration , and gain insights on appropriate underlying distributions , patterns and trends , and therefore contribute to enhance the understanding of the geographical processes . in this section , an example of exploration of ( potential ) patterns in data is presented using the different visualization techniques described in methodology section . for illustration , a dataset from the human development network ( world bank ) containing 30 basic indicators for health and living standard including health expenditure , health risk factors , mortality , reproductive health , for
although this is not a very large dataset , the approach can be applied for far larger datasets . complex correlations in this kind of statistical data can be portrayed using the self - organizing map to visualize the complex joint effect of the factors related to health as contained in the dataset . this representation can be used to visualize the structure of the input space and to get an impression of otherwise invisible structures in a multidimensional data space . in figure 1
countries having similar characteristics based on the multivariate attributes are positioned close to each other , and the distance between them represents the degree of similarity or dissimilarity . on the geographic map , each country
is assigned a color describing the its health standard type in relation to other countries in contrast to other projection methods in general , the som does not try to preserve the distances directly but rather the relations or local structure of the input data . size , position and colour of markers can be used to depict the relationships between the data items . representation of the general patterns and clustering in the input data with a distance matrix visualization the unified distance matrix showing clustering and distances between positions on the map . alternative representations of the som general clustering of patterns alternative representations of the som general clustering of the data with projection of the som results in 2d space ( a ) . unlike the projection in figure 2a that shows only the position and clustering of map units , areas with uniform color are used in the surface plots to show the clustering structure and relationships among map units . exploration of mortality in 2d surface the 2d surface shows a flat organizing of the clusters and indicates the average distance among the different countries according to several attributes related to mortality in the dataset ( life expectancy in 1980 , life expectancy in 2001 , infant mortality rate in 1980 , infant mortality rate in 2001 , under 5 mortality rate in 1980 , under 5 mortality rate in 2001 , adult male mortality in 2001 , adult female mortality rate in 2001 , percentage of male over 65 years old 2002 , percentage of female over 65 years in 2002 ) . the component planes show the values of the map elements for different attributes and how each input vector varies over the space of the som units ( here representing countries )
comparatively with the maps , patterns and relationships among all the attributes can be easily examined in a signle visual represention using the som component planes visualization ( figure 5a ) . since the som represents the similarity clustering of the multivariate attributes , the visual representation becomes more accessible and easy to use for exploratory analyses . detail exploration of the dataset using the som component visualization all the components can be displayed to reveal the relationships between the variables and the spatial locations ( countries ) in ( a ) . unlike standard choropleth maps , the position of the map units ( which is the same for all displays ) is determined during the training of the network , according to the characteristics of the data samples . from the view in figure 5a , correlations and relationships can be explored , and hypotheses can be made . this representation can be used to visualize the structure of the input space and to get an impression of otherwise invisible structures in a multidimensional data space . the different clusters on the map can be automatically encoded with different colors , to allow a geographic view of the similarity mapping based on the multivariate attributes . the projection of the som offers a view of the clustering of the data with data items depicted as coloured nodes . size , position and colour of markers can be used to depict the relationships between the data items . representation of the general patterns and clustering in the input data with a distance matrix visualization the unified distance matrix showing clustering and distances between positions on the map . alternative representations of the som general clustering of patterns alternative representations of the som general clustering of the data with projection of the som results in 2d space ( a ) . the output of the som ( the similarity coding extracted from the computational analysis ) can be projected on a geographic map , to display the common characteristics of the countries according to the multivariate attributes ( b ) . unlike the projection in figure 2a that shows only the position and clustering of map units , areas with uniform color
are used in the surface plots to show the clustering structure and relationships among map units . exploration of mortality in 2d surface the 2d surface shows a flat organizing of the clusters and indicates the average distance among the different countries according to several attributes related to mortality in the dataset ( life expectancy in 1980 , life expectancy in 2001 , infant mortality rate in 1980 , infant mortality rate in 2001 , under 5 mortality rate in 1980 , under 5 mortality rate in 2001 , adult male mortality in 2001 , adult female mortality rate in 2001 , percentage of male over 65 years old 2002 , percentage of female over 65 years in 2002 ) . the component planes show the values of the map elements for different attributes and how each input vector varies over the space of the som units ( here representing countries )
comparatively with the maps , patterns and relationships among all the attributes can be easily examined in a signle visual represention using the som component planes visualization ( figure 5a ) . since the som represents the similarity clustering of the multivariate attributes , the visual representation becomes more accessible and easy to use for exploratory analyses . detail exploration of the dataset using the som component visualization all the components can be displayed to reveal the relationships between the variables and the spatial locations ( countries ) in ( a ) . unlike standard choropleth maps ,
the position of the map units ( which is the same for all displays ) is determined during the training of the network , according to the characteristics of the data samples . in this paper we have presented an approach to combine visual and computational analysis for exploratory visualization intended to contribute to the analysis of large volumes of health survey data . the approach focuses on the effective application of computational algorithms to extract patterns and relationships in large datasets , and visual representation of derived information that involves effective use of visual variables used in such complex information spaces to facilitate knowledge construction . a number of visualization techniques were explored with the objective to support visual exploration and knowledge construction . users can perform a number of exploratory tasks to understand the structure of the dataset as a whole and also to explore detailed information on individual or selected attributes of the dataset such as finding correlations and the relationships among attributes . with this respect , the som computational analysis can support exploratory visualization and the knowledge discovery process when integrated with appropriate visual exploration tools . interactive manipulation ( zooming , rotation , panning , filtering , and brushing ) of the graphical representations can enhance user goal specific querying and selection from the general patterns extracted to more specific user selection of attributes and spatial locations for exploration , hypothesis testing , and ultimately knowledge construction . we have extended these alternative representations of the som results used to highlight different characteristics of the computational solution and integrated them with other graphics into multiple views to allow brushing and linking , for exploratory analysis and knowledge discovery purposes . this link between the attribute space visualization based on the som , the geographic space with maps representing the som results , and other graphics such as parallel coordinate plots , in multiple views can provide alternative perspectives for better exploration , evaluation and interpretation of patterns and ultimately support for knowledge construction . the proposed framework explores ways to effectively extract patterns using data mining based on the som and to represent the results using graphical representations for visual exploration . as presented in figure 6 , the data mining stage allows to construct a clustering ( similarity matrix ) of the multidimensional input space using the som training algorithm tool ( som toolbox ) and graphics processing with matlab software . the framework includes spatial analysis , data mining and knowledge discovery methods , supported by interactive tools that allow users to perform a number of exploratory tasks to understand the structure of the dataset as a whole and also explore detailed information on individual or selected attributes of the dataset . they include some graphical representations based on the som , maps and other graphics such as parallel coordinate plots . data exploration framework from the computational process , global structure and patterns can be visualized with graphical representations and maps of similarity results . relationships and correlations among the attributes are presented with interactive graphical representations , maps and other graphics such as parallel coordinate plots . the self - organizing map is an artificial neural network used to map multidimensional data onto a low dimensional space , usually a 2d representation space ( see figure 7 ) . using euclidean distance between weight vector and input pattern ,
the unit with the lowest activation is referred to as the winner , c , of the training iteration : mc(t ) = mini { ||x(t ) - mi(t)|| } ( 1 ) finally the weight vector of the winner as well as the weight vectors of selected units in the neighbourhood of the winner are adapted to represent the input pattern . as a result of a general adaptation process
, a number of units in the neighbourhood of the winner lead to a spatial clustering of similar input patterns in neighbouring parts of the som . this leads to a spatial clustering of similar input patterns in neighboring parts of the som and the clusters that appear on the map are themselves organized internally . in other words ,
the topology of the dataset in its n - dimensional space is captured by the som and reflected in the ordering of its nodes . unlike supervised methods which learn to associate a set of inputs with a set of outputs using a training data set for which both input and output are known , som adopts a learning strategy where the similarity relationships between the data and the clusters are used to classify and categorize the data . one approach to analysis of large amount of data is by using data mining and knowledge discovery methods . the main goal of data mining is identifying valid , novel , potentially useful and ultimately understanding patterns in data . for example , unsupervised neural networks such as the self - organizing map are a type of neural clustering and neural architecture such as backpropagation and feedforward are neural induction methods used for classification ( supervised learning ) . new developments in data mining and kdd has provided a window for geographic data mining and knowledge discovery , which has become an established field in geographic visualization [ 11,12,23 - 27 ] . this framework has been used in geospatial data exploration [ 11 - 13,23,28 ] to discover unexpected correlation and causal relationships , and understand structures and patterns in complex geographical data . the promises inherent in the development of data mining and knowledge discovery processes for geographical analysis include the ability to yield unexpected correlation and causal relationships . since the dimensionality of the dataset is very high , it is often ineffective to work in such high dimension space to search for patterns . we use the som algorithm as a data mining tool to project input data into an alternative measurement space based on similarities and relationships in the input data that can aid the search for patterns . graphical representations are then used to enable visual data exploration allowing the user to get insight into the data , evaluate , filter , and map outputs . this is intended to support visual data mining by allowing several variables and their interactions to be inspected simultaneously , and receive feedback from the knowledge discovery process by means of interaction techniques that support the process . the output of this computational process is depicted using graphical representations ( information spaces ) to facilitate human perception and cognitive processes , by offering visualizations of the general structure of the dataset ( clustering ) , as well as the exploration of relationships among attributes . they highlight different characteristics of the computational solution and integrate them with other graphics into multiple views to allow brushing , linking , panning , zooming , rotation and 3d views for exploratory analysis and knowledge discovery purposes and enhance exploration . the scale allows exploration of the information space at multiple levels of detail , and provides the potential for hierarchical grouping of items , and revealing categories or classifications . four goals of the exploration are emphasized : - patterns discovery ( through similarity representations ) - correlations and relationships exploration for hypothesis generation - exploration of the distribution of the dataset on the map - and the detection of irregularities in the data
the proposed framework explores ways to effectively extract patterns using data mining based on the som and to represent the results using graphical representations for visual exploration . as presented in figure 6 , the data mining stage allows to construct a clustering ( similarity matrix ) of the multidimensional input space using the som training algorithm tool ( som toolbox ) and graphics processing with matlab software . the framework includes spatial analysis , data mining and knowledge discovery methods , supported by interactive tools that allow users to perform a number of exploratory tasks to understand the structure of the dataset as a whole and also explore detailed information on individual or selected attributes of the dataset . they include some graphical representations based on the som , maps and other graphics such as parallel coordinate plots . relationships and correlations among the attributes are presented with interactive graphical representations , maps and other graphics such as parallel coordinate plots . the self - organizing map is an artificial neural network used to map multidimensional data onto a low dimensional space , usually a 2d representation space ( see figure 7 ) . using euclidean distance between weight vector and input pattern , the activation of the units
the unit with the lowest activation is referred to as the winner , c , of the training iteration : mc(t ) = mini { ||x(t ) - mi(t)|| } ( 1 ) finally the weight vector of the winner as well as the weight vectors of selected units in the neighbourhood of the winner are adapted to represent the input pattern . as a result of a general adaptation process ,
a number of units in the neighbourhood of the winner lead to a spatial clustering of similar input patterns in neighbouring parts of the som . this leads to a spatial clustering of similar input patterns in neighboring parts of the som and the clusters that appear on the map are themselves organized internally . for example , the size of the clusters ( the number of nodes allotted to each cluster ) is reflective of the frequency distribution of the patterns in the input set . in other words ,
the topology of the dataset in its n - dimensional space is captured by the som and reflected in the ordering of its nodes . unlike supervised methods which learn to associate a set of inputs with a set of outputs using a training data set for which both input and output are known
, som adopts a learning strategy where the similarity relationships between the data and the clusters are used to classify and categorize the data . one approach to analysis of large amount of data is by using data mining and knowledge discovery methods . for example , unsupervised neural networks such as the self - organizing map are a type of neural clustering and neural architecture such as backpropagation and feedforward are neural induction methods used for classification ( supervised learning ) . new developments in data mining and kdd has provided a window for geographic data mining and knowledge discovery , which has become an established field in geographic visualization [ 11,12,23 - 27 ] . this framework has been used in geospatial data exploration [ 11 - 13,23,28 ] to discover unexpected correlation and causal relationships , and understand structures and patterns in complex geographical data . the promises inherent in the development of data mining and knowledge discovery processes for geographical analysis include the ability to yield unexpected correlation and causal relationships . since the dimensionality of the dataset is very high , it is often ineffective to work in such high dimension space to search for patterns . we use the som algorithm as a data mining tool to project input data into an alternative measurement space based on similarities and relationships in the input data that can aid the search for patterns . graphical representations are then used to enable visual data exploration allowing the user to get insight into the data , evaluate , filter , and map outputs . this is intended to support visual data mining by allowing several variables and their interactions to be inspected simultaneously , and receive feedback from the knowledge discovery process by means of interaction techniques that support the process . the output of this computational process is depicted using graphical representations ( information spaces ) to facilitate human perception and cognitive processes , by offering visualizations of the general structure of the dataset ( clustering ) , as well as the exploration of relationships among attributes . several graphical representations provide ways for representing similarity ( patterns ) , relationships , including a distance matrix representation , 2d and 3d projections , 2d and 3d surfaces , and component planes visualization . they highlight different characteristics of the computational solution and integrate them with other graphics into multiple views to allow brushing , linking , panning , zooming , rotation and 3d views for exploratory analysis and knowledge discovery purposes and enhance exploration . four goals of the exploration are emphasized : - patterns discovery ( through similarity representations ) - correlations and relationships exploration for hypothesis generation - exploration of the distribution of the dataset on the map - and the detection of irregularities in the data | [
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] |
tim catalyzes the conversion
of dihydroxyacetone phosphate ( dhap )
to ( r)-glyceraldehyde 3-phosphate ( gap ) , by proton
transfer reactions at carbon , through enzyme - bound cis - enediolate reaction intermediates ( scheme 1 ) .
classic studies by knowles and co - workers focused on determination
of the catalytic roles of the active site side chains , and were guided
by the knowledge that similar mechanisms are observed for catalysis
at enzyme active sites and in solution .
these studies
showed that intramolecular proton transfer between c-1 and c-2 is
performed by the carboxylate side - chain of glu165 , while the neutral electrophilic imidazole side - chain
of his-95 mediates proton transfer between o-1 and o-2 of the enediolate
intermediate ( scheme 1 ) .
the similarity in the mechanisms for nonenzymatic
isomerization in water and isomerization at the active site of tim
was highlighted in a classic review entitled
we want to understand why the
functional groups at the active site
of tim provide a greater stabilization of the transition state for
deprotonation of carbon than the same groups in water .
this demands an explanation for the role of enzyme - phosphodianion
interactions , which account for about 80% of the total transition
state stabilization .
knowles and associates identified
the 11-residue phosphodianion gripper loop 6 [ 166-pvwaigtgkta ]
as an enabling structural element of tim .
the binding of substrate dhap or transition
state analogues phosphoglycolate ( pga ) and phosphoglycolohydroxamate ( pgh ) to tim triggers the
closure of loop 6 over the ligand phosphodianion , and formation of caged enzyme - ligand complexes .
the deletion of residues 170173 from loop 6 of tim from
chicken muscle ( ctim ) , and introduction of a peptide
bond between a169 and k174 disrupts loop - dianion interactions .
this
results in a 10-fold falloff in kcat / km for catalysis of isomerization
due mainly to a decrease in kcat , so that the activating loop - dianion interactions
develop only on moving from the ground - state michaelis complex to
the transition state for tim - catalyzed isomerization . cutting the substrate for tim into carbon acid and dianion
pieces
( scheme 2 )
enables
the enzyme - activating dianion interactions to be studied separately
from anchoring interactions , which stabilize the michaelis complex
to the whole substrate .
the strong activation of tim - catalyzed deprotonation
of glycolaldehyde by phosphite dianion ( hpo3 = hpi ) shows that the tim active site is divided into
a catalytic domain , which contains the side chains that carry out
chemical isomerization of bound substrate , and a dianion activation
domain that uses enzyme - dianion interactions to promote the proton
transfer reaction at the catalytic site . the similar
organization of the binding pockets of yeast orotidine 5-monophosphate
decarboxylase
( scompdc ) and human l - glycerol
3-phosphate dehydrogenase ( hsgpdh ) into catalytic and dianion activation domains
is consistent with a common enzyme architecture that enables dianion
activation of these and other protein catalysts .
the activation
of tim , ompdc , and gpdh for catalysis is achieved
by sequestering their substrates at protein cages , which provide for
optimal stabilizing interactions with the different reaction transition
states .
their active sites are formed through energetically
demanding conformational changes , in which the binding
interactions between the protein and the substrate phosphodianion
are used to mold flexible protein loops into caged
structures .
flexible loop 7 [ 208-yggs-211 ] is part of the cage that activates
tim for catalysis of proton transfer .
closure of loop 6 is accompanied
by 90 and 180 rotations , respectively , in the planes defined
by the peptide bonds of g209 and g210 from loop 7 .
this swings the
ch2oh of s211 into position to hydrogen bond with
the backbone carbonyl oxygen and amide nitrogen from a169 and g173 ,
respectively ; and , the s211 backbone amide nh group into a position
to hydrogen bond with the ligand dianion .
an additional hydrogen bond forms between the side
chain of y208 and the backbone amide nitrogen from a176 .
knowles and sampson noticed
these new hydrogen
bonds between the hydroxyl groups of tyrosine-208 and serine-211 , and characterized the kinetic parameters
for y208f and s211a mutants of yeast tim ( ytim ) .
the large falloff in the activity observed for these mutant enzymes
was interesting , because the 89 separation between
y208 and s211 and the substrate ( figure 1 ) allows for minimal direct interactions
with the isomerization transition state .
knowles and sampson proposed
that this falloff in activity reflects destabilization of the active
closed form of tim with respect to the open enzyme . we have used our experimental protocol for the study of
tim - catalyzed reactions to probe the effects of site - directed mutations
on the activity of tim . we report here the results of experiments that were designed to
address the following questions : ( 1 ) is it possible to localize
the effects of mutations at loop
7 to the loss of a single hydrogen - bonding interaction ?
the dienolate
intermediate of the reaction catalyzed by bacterial ketosteroid isomerase
( ksi ) is stabilized by a hydrogen bond to the phenol side chain of
y16 .
the 5000-fold falloff in activity
of the y16f mutant of ksi from pseudomonas putida is ca .
100-fold larger than that for y16a , y16 g , y16s , and y16 t
mutants , so that large effect of the y16f mutation on enzyme activity
is not due only to the loss of a single hydrogen bond .
we have prepared y208f , y208 t , y208s , and y208a mutants of tim ,
and determined kinetic parameters that are up to 200-fold greater
than that for y208f .
we also find that the activity of s211 g tim is
20-fold greater than that for the s211a tim .
we conclude that the loss of hydrogen bonds to altered y208 and
s211 side chains is no more than partly responsible for these changes
in enzyme activity .
( 2 ) what is the role of y208 and s211 side
chains from loop 7 in
phosphite dianion activation of ytim for catalysis
of deprotonation of glycolaldehyde ? the large 9.1 and 8.1 ( figure 1 ) distance , respectively ,
between the side chains of y208 and s211 and c-2 of bound dhap prevents
direct interactions with the enzyme - bound ligand .
the observed effects
of these mutations on the enzyme kinetic parameters must therefore
reflect effects that arise from changes in the structure of the active
site cage .
our results suggest that the hydrogen bond to y208 fixes
the hydrophobic phenyl group over the active site cage , so as to minimize
the local dielectric constant and optimize electrostatic and h - bonding
interactions between tim and bound phosphite dianion .
( 3 ) what
is the role , if any , for protein dynamics in the process
of enzyme activation by phosphite dianion ?
transition state theory
provides a powerful framework for describing the activation barriers
to enzyme - catalyzed reactions determined by kinetic analyses , and
in rationalizing the changes in these activation barriers for mutant
enzymes .
we report that a two - state model that defines the activation
barrier g for tim - catalyzed
reactions as the difference in energy of the free reactants and enzyme - bound
transition state provides a satisfactory rationalization of these
results .
our results do not rigorously exclude effects that arise
from changes in protein dynamics , but sets conditions on models for
these dynamic effects .
representation of tim , which shows the distance between
loop 7
side chains y208 and s211 at the dianion activation site , and the
bound substrate dhap at the catalytic site ( pdb entry 1ney ] . the 89
separation between y208 and s211 and the substrate allows for
minimal direct interactions with the isomerization reaction transition
state .
an earlier communication reported
that five mutations of y208 ,
s211 yeast tim and of other side chains of tim from several organisms
result in nearly the same change in the activation barriers for the
catalyzed reactions of whole substrate and substrate pieces .
we concluded that that the two transition states
are stabilized by essentially the same interactions with the protein
catalyst .
we report here the full results from studies on a total
of seven mutants of y208 , s211 , and an internally consistent rationalization
for their effect on the catalytic activity of tim .
dihydroxyacetone phosphate
( magnesium salt ) ,
nadh ( disodium salt ) , dowex 50wx4 - 200r , triethanolamine hydrochloride
( teahcl ) , dithiothreitol ( dtt ) , rabbit muscle glycerol 3-phosphate
dehydrogenase , and glyceraldehyde 3-phosphate dehydrogenase were purchased
from sigma . nad ( free acid ) was purchased from mp biomedical .
hydrogen arsenate heptahydrate
and sodium phosphite ( dibasic , pentahydrate ) were purchased from fluka .
[ 1-c]-glycolaldehyde ( 99% enrichment
of c at c-1 , 0.09 m in water ) was purchased from omicron
biochemicals .
deuterium oxide ( 99% d ) and deuterium chloride ( 35%
w / w , 99.9% d ) were purchased from cambridge isotope laboratories .
all other chemicals were
reagent grade or better and were used without further purification .
the disodium salt of d - glyceraldehyde 3-phosphate diethyl
acetal was prepared by a literature procedure , and purified by column chromatography over deae - sephadex .
stock solutions of d - glyceraldehyde 3-phosphate ( gap ) were
prepared by hydrolysis of the corresponding diethyl acetal using dowex
50wx4 - 200r ( h form ) in boiling h2o .
the resulting solutions were stored at 20
c and adjusted to ph 7.5 by the addition of 1 m naoh prior to
use in enzyme assays .
stock solutions of phosphite dianion and [ 1-c]-glycolaldehyde ( [ 1-c]-ga ) in d2o were prepared as described in earlier work .
the plasmid containing the gene
for wildtype tim from saccharomyces cerevisiae was
a generous gift from professor nicole sampson .
the procedures for site - direct mutagenesis used to introduce
y208a , y208 t , y208s , y208f , and s211a mutations are described in the
supporting information to ref ( 28 ) .
the plasmids for the single s211 g and the double y208t / s211 g
mutants were prepared by introducing the s211 g mutation onto the wildtype
or y208t - encoding template using the primers 5-c - gaa - ttg - aga - atc - tta - tac - ggt - ggt - ggc - gct - aac - ggt - agc - aac - gc-3 and 5-gc - gaa - ttg - aga - atc - tta - acc - ggt - ggt - ggc - gct - aac - ggt - agc - aac - gcc-3 , respectively .
following amplification by pcr ,
the methylated parent dna was treated
with 20 units of the restriction enzyme dpni at 37
c for 1 h , and then transformed into the escherichia
coli strain k802 .
plasmid dna samples from several colonies
were purified using the qiaprep miniprep kit from qiagen .
the dna
sequence of the genes for the s211 g and y208t / s211 g mutants was verified
by sequencing at the roswell park cancer institute ( buffalo , ny ) .
wildtype and mutant ytims were expressed using the
tim - deficient tpia lde3 lysogenic
strain of e. coli , fb215471(de3 ) grown in lb medium at 37 c .
the enzymes were purified
by deae sepharose column chromatography after fractional purification
by polyethylenimine ( 0.33% ) and ammonium sulfate precipitation .
enzyme concentrations were determined based
upon their absorbance at 280 nm using the extinction coefficients
of 25440 mcm for wildtype
and s211 mutants and 23950 mcm for y208 mutants , calculated by the protparam tool available on
the expasy server .
all enzyme solutions were exhaustively
dialyzed against 20 mm triethanolamine buffer ( ph 7.5 ) at 7 c
prior to use ; and the enzyme assays were carried out in the same buffer
at 25 c and i = 0.1 ( nacl ) .
d - glycerol
3-phosphate dehydrogenase was assayed by monitoring the oxidation
of nadh by dhap ; and glyceraldehyde 3-phosphate dehydrogenase was
assayed by monitoring the enzyme - catalyzed reduction of nad by gap .
the tim - catalyzed isomerization of gap was monitored
by coupling the formation of the product dhap to the oxidation of
nadh catalyzed by l - glycerol 3-phosphate dehydrogenase .
the tim - catalyzed isomerization of dhap was monitored by coupling
the formation of the product gap to the reduction of nad catalyzed by glyceraldehyde 3-phosphate dehydrogenase in the presence
of 210 mm arsenate .
the values of kcat and km for mutant ytim - catalyzed isomerization of gap or dhap were determined
from the fit to the michaelis menten equation of initial velocities
( vi ) determined at varying concentrations
of substrate .
values for kcat , km , and ki for mutant ytim - catalyzed reaction of dhap were determined from the
nonlinear least - squares fit to eq 1 of initial velocities determined at varying concentrations
of arsenate dianion and dhap.1 h nmr spectra
at 500 mhz were recorded in d2o at 25 c using a varian
unity inova 500 spectrometer that was shimmed to give a line width
of 0.5 hz for the most downfield peak of the double triplet
due to the c-1 proton of the hydrate of [ 1-c]-ga .
spectra ( 1664 transients ) were obtained
using a sweep width of 6000 hz , a pulse angle of 90 and an acquisition
time of 6 s. a total relaxation delay of 120 s ( > 8t1 ) between
transients was used to ensure that accurate integrals were obtained
for the protons of interest .
baselines were subjected
to a first - order drift correction before determination of integrated
peak areas .
the
mutant ytim - catalyzed reactions of [ 1-c]-ga in the presence
or absence of hpo3 in d2o at 25 c were monitored by h nmr analyses as described
in earlier work .
the enzyme was first exhaustively
dialyzed at 7 c against 30 mm imidazole ( 20% free base , pd 7.0 )
in d2o ( i = 0.024 or 0.1 ( nacl ) ) .
the
reactions in the absence of phosphite dianion were initiated by adding
enzyme to give a final solution that contained 20 mm [ 1-c]-ga , 20 mm imidazole ( 20% free base , pd 7.0 , i = 0.1 ( nacl ) ) , and enzyme [ 0.40 mm , wildtype ytim ;
0.10 mm , y208 t ytim ; 0.058 mm , y208s ytim ; 0.068 mm , y208a ytim ; 0.24 mm , y208f ytim ; 0.47 mm , s211a ytim ; 0.26 mm , s211 g ytim ; 0.18 mm , y208t / s211 g ytim ] in a volume
of 850 l .
the reactions in the presence of hpo3 were initiated by adding enzyme to give a final solution
that contained 20 mm [ 1-c]-ga , 20 mm imidazole ( 20% free
base , pd 7.0 ) , 140 mm hpo3 ( 50%
dianion , pd 7.0 ) , and enzyme [ 1827 m , wildtype ytim ; 2061 m , y208 t ytim ;
2844 m , y208s ytim ; 4161 m ,
y208a ytim ; 130260 m , y208f ytim ; 50370 m , s211a ytim ;
1138 m , s211 g ytim ; 2568 m ,
y208t / s211 g ytim ] in a volume of 850 l ( i = 0.1 , nacl ) . in every case , 750 l of the reaction
mixture was transferred to an nmr tube , the h nmr spectrum
was recorded immediately , and spectra were then recorded at regular
intervals .
the remaining solution was incubated at 25 c and
the activity of tim for catalysis of isomerization of gap monitored .
no significant loss in activity of tim was observed during any of
these reactions . at the end of each nmr experiment ,
the observed h nmr peak areas for the reaction products
were normalized , as described in previous work .
the fraction of the substrate [ 1-c]-ga remaining
and the fractional yields of the identifiable reaction products [ 2-c]-ga , [ 2-c , 2-h]-ga , [ 1-c , 2-h]-ga , and [ 1-c , 2,2-di - h]-ga were determined from the integrated areas of the relevant h nmr signals for these compounds .
the disappearance of 3070% [ 1-c]-ga was monitored ,
and product yields were determined over the first ca .
2030%
of the reaction . observed first - order rate constants , kobs ( s ) , for the reactions of [ 1-c]-ga were determined from the slopes of linear semilogarithmic
plots of reaction progress against time ( eq 2 ) , where fs is
the fraction of [ 1-c]-ga remaining at time t. observed second - order rate constants , ( kcat / km)obs ( m s ) , for the tim - catalyzed reaction of [ 1-c]-ga were determined from the values of kobs using eq 3 , where fhyd = 0.94 is the fraction of
[ 1-c]-ga present as the hydrate.23
the kinetic parameters for the isomerization reactions of gap and
dhap catalyzed by wildtype and y208 and s211 mutants of ytim , determined at ph 7.5 and 25 c ( i = 0.1 ,
nacl ) , are reported in table 1 .
the kinetic parameters for the 208-tgag for 208-yggs loop
7 replacement mutation ( lrm ) at tim from chicken muscle ( table 1 ) were determined
in earlier work .
arsenate is a required
activator of glyceraldehyde 3-phosphate dehydrogenase , the coupling
enzyme in assays of tim - catalyzed isomerization of dhap .
table 1 also reports the
values of ki for arsenate inhibition of
the mutant tim - catalyzed reactions .
the values of kcat / km reported in table 1 were calculated using
the total concentration of gap in the enzyme assay .
however , gap exists
largely ( 96% ) in the hydrated form , and tim is specific for the carbonyl
form of this substrate ( 4% ) .
values for kcat / km are also
reported for catalysis of the reactive carbonyl form of gap .
the mutant ytim - catalyzed reactions of
[ 1-c]-ga in d2o were monitored by h nmr spectroscopy .
these experiments provide the observed kinetic
parameter ( kcat / km)obs ( eq 1 ) for the tim - catalyzed reaction of the carbonyl form of [ 1-c]-ga and the yield of the reaction products ( scheme 3 ) .
the product yields , determined
at four different times during the first 2030% of the reaction
of [ 1-c]-ga , are invariant ( 5% ) over this time .
the product yields for the reactions in the presence and absence of
phosphite dianion catalyzed by wildtype ytim , y208 t ytim , y208s ytim , y208a ytim , y208f ytim , and s211a ytim
are reported in tables s1s6 , respectively , of the supporting
information to ref ( 28 ) .
the product yields for the reactions in the presence and absence
of phosphite dianion catalyzed by s211 g ytim and
y208t / s211 g ytim are reported in tables s1 and s2 ,
respectively , of the supporting information to the present paper .
the value of ( kcat / km)obs for
the tim - catalyzed reactions of [ 1-c]-ga is equal to the
sum of the second - order rate constants
for the reactions that occur at the enzyme active site ( scheme 3a ) and for nonspecific protein - catalyzed
reactions ( scheme 3b ) .
the following procedures were followed to
determine the true values of ( kcat / km)tim for the unactivated and phosphite
dianion - catalyzed reactions at the enzyme active site . under standard
assay conditions
of 30 mm triethanolamine buffer at ph 7.5 , 25 c and i = 0.1 ( nacl ) .
the kinetic parameters have been calculated
using the total concentration of gap or dhap , unless indicated otherwise .
the errors for tim - catalyzed
isomerization
of gap were determined from the average of kinetic parameters determined
in three separate experiments .
the errors for tim - catalyzed isomerization
of dhap are the standard deviations determined from the nonlinear
least - squares fits of the kinetic data .
the upper value is calculated for
the total concentration of substrate gap , and the lower is calculated
for the reactive carbonyl form , which is 4% of total gap .
the initial velocity of the isomerization
of several concentrations of dhap was determined in the presence of
2 , 5 , and 10 mm arsenate .
loop replacement mutation of tim
from chicken muscle [ ref ( 29 ) ] .
the slow unactivated reaction of [ 1-c]-ga in the presence of wildtype and mutants of ytim gives the three products from reaction at the enzyme active site
( scheme 3a ) along with
significant yields of [ 1-c , 2,2-di - h]-ga
and [ 1-c , 2-h]-ga .
the latter products form
by nonspecific protein - catalyzed reactions of [ 1-c]-ga outside the enzyme active site .
this probably involves
deuterium exchange into iminium ions formed by reaction of [ 1-c]-ga with surface lysine side chains ( scheme 3b ) .
it is not possible to distinguish [ 1-c , 2-h]-ga formed in the specific and nonspecific reactions and we therefore
estimate the yield of [ 1-c , 2-h]-ga from scheme 3a by assuming that
the ratio of yields of [ 2-c , 2-h]-ga and
[ 1-c , 2-h]-ga from the unactivated reaction
is the same as for the phosphite dianion activated reaction , where
little or no [ 1-c , 2,2-di - h]-ga ( scheme 3b ) is generally observed.4 the true second - order
rate constants ( kcat / km)tim for unactivated wildtype and mutant tim - catalyzed
reactions of the carbonyl form of [ 1-c]-ga to give [ 2-c]-ga , [ 2-c , 2-h]-ga , and [ 1-c , 2-h]-ga ( scheme 3a ) were calculated using eq 4 , where fe is the fractional yield of the products from reactions at the active
site of tim that are reported in tables s1s6 of ref ( 28 ) and in tables s1 and s2
( supporting information ) of this paper .
the values of ( kcat / km)tim = ( kcat / km)e for the unactivated wildtype
and mutant tim - catalyzed reactions , determined at [ hpo3 ] = 0 m are summarized in table 2 .
the second - order rate
constants ( kcat / km)tim ( eq 4 ) for phosphite dianion activated wildtype and mutant ytim - catalyzed reactions of the carbonyl form of [ 1-c]-ga are reported in tables s1s6 of ref ( 28 ) and in tables s1 and s2
( supporting information ) of this paper .
values of fe = 1.0 were determined for
phosphite dianion activation of wildtype , y208 t , s211 g , and y208t / s211 g ytim catalyzed reactions of [ 1-c]-ga by 2.5
mm hpo3 .
the value of fe for the less active mutant enzymes increases with increasing
concentration of the phosphite dianion activator , as the velocity
of the activated reaction increases relative to the nearly constant
unactivated reaction velocity .
the following ranges of product yields
were determined for the different tims over a 2.520 mm range
of phosphite dianion concentrations : fe = 0.770.93 , s211a ytim ; fe = 0.610.83 , y208s ytim ; fe = 0.700.90 , y208a ytim ; and fe = 0.070.22 , y208f ytim .
figure 2 shows the dependence of the values of ( kcat / km)tim determined
for wildtype and four mutant [ s211 g , y208t / s211 g , y208 t , s211a ] ytim - catalyzed reactions of [ 1-c]-ga on the
concentration of added phosphite dianion . figure 2 from ref ( 28 ) shows similar plots for
reactions catalyzed by the y208a , y208s , and y208f mutant enzymes .
the plots for the reactions catalyzed by y208a , y208f and s211a mutant ytims are linear .
the values of [ ( kcat / km)ehpi]/kd determined as the slopes of these correlations
are reported in table 2 .
the solid lines through the experimental data for the y208 t , s211 g ,
y208t / s211 g , and y208s mutant enzymes show the nonlinear least - squares
fits to eq 5 , derived
for scheme 4 ( s = [ 1-c]-ga ) , and using the values of ( kcat / km)e from table 2 for the unactivated reaction
in the absence of phosphite dianion .
these fits give ( kcat / km)ehpi for turnover of [ 1-c]-ga by ehpo3 and kd for release of
phosphite dianion from ehpo3 ( table 2 ) .
we note the large
uncertainty in these kinetic parameters when kd 40 mm ( y208 t , s211 g , and y208s mutants ) , which is
substantially greater that the largest [ hpo3 ] = 20 mm used in these experiments.5 dependence
of second - order rate constants ( kcat / km)tim for the tim - catalyzed
turnover of the free carbonyl form of [ 1-c]-ga in d2o on [ hpo3 ] at pd 7.0 and 25
c at i = 0.1 , nacl .
the data were fitted to eq 5 derived for the model
shown in scheme 4 .
( c ) data for s211a ytim . determined by h nmr
analysis of the products of the reaction of 20 mm [ 1-c]-ga in d2o at pd 7.0 ( 20 mm imidazole ) , 25 c and i = 0.1 ( nacl ) .
second - order rate constant for the
unactivated tim - catalyzed reaction of [ 1-c]-ga in the
absence of phosphite dianion .
second - order rate constant for the
reaction of [ 1-c]-ga catalyzed by the phosphite - liganded
enzyme obtained from the fits of experimental data to eq 5 derived for scheme 4 .
dissociation constant for release
of phosphite dianion from the free enzyme obtained from the fits of
experimental data to eq 5 derived for scheme 4 .
dissociation constant
for release
of phosphite dianion from the transition state complex , calculated
as described in the text .
third - order rate constant
for the
phosphite - activated tim - catalyzed reaction of [ 1-c]-ga
obtained from the fits of linear plots of ( kcat / km)tim against [ hpo3 ] .
loop replacement mutation of tim
from chicken muscle [ ref ( 29 ) ] .
the kinetic parameters for isomerization of gap catalyzed by y208f
and s211a mutants of ytim ( table 1 ) are in good agreement with previously published
values .
the effects of these mutations
on the activation barrier g for the isomerization of gap catalyzed by wildtype enzyme are shown
on figure 3 .
the simplest
interpretation of the effect of a y208f mutation , which eliminates
the intraloop hydrogen bond between the hydroxyl group of y208 and
the amide nitrogen of a176 , is that this hydrogen bond provides a
4.4 kcal / mol stabilization of the isomerization reaction transition
state .
however , this
hydrogen bond is eliminated by the y208a mutation , whose smaller effect
on g compared with y208f
shows that the phenyl group of y208 is held in an active conformation
by the hydrogen bond to the phenol hydroxyl , and relaxes to a second
conformation at the y208f mutant , where the enzyme activity is reduced
by ca .
these results
are consistent with the notion that the binding energy from the hydrogen
bond to the phenol hydroxyl is utilized to hold the phenyl group in
a conformation that reduces the dielectric constant for the active
site cage , and optimizes stabilizing
electrostatic interactions between the transition state and the protein
catalyst .
the s211a and s211 g mutations of tim each eliminate intraloop
hydrogen bonds to the side - chain hydroxyl , yet show different effects
on enzyme activity ( figure 3 ) .
the explanation for the effects of y208 and s211 mutations
will be considered in greater detail in later sections of this paper .
there are two additional recent examples of the difficulty in obtaining
reliable estimates , from the effect of a single point mutation , of
the contribution of side - chain hydrogen bonds to stabilization of
the transition states for enzyme - catalyzed proton transfer and decarboxylation
reactions .
( 1 ) the y16f mutation at ketosteroid isomerase from p.
putida(53,54 ) eliminates the side - chain hydrogen
bond to the dienolate oxyanion intermediate .
100-fold larger
decrease in kcat / km for y16f , compared with y16a , y16 g , y16s , and y16 t mutations
was proposed to reflect the relief of destabilizing hydrophobic interactions
between the phenyl side chain and the intermediate oxyanion at the
latter four mutants , along with formation of a hydrogen bond to a
water molecule at the cavity created by removal of the side - chain
phenyl ring .
( 2 ) the caged complex between yeast orotidine 5-monophosphate
decarboxylase ( scompdc ) and omp is stabilized by
an interaction between the side chain of y217 and the substrate phosphodianion ,
but now the 60-fold reduction in kcat / km determined for the y217f mutant is smaller than the 4400-fold effect of a y217a
mutation .
these and many other experimental results
are consistent with minimal destabilizing hydrophobic interactions
between the phosphodianion and the phenyl side chain of the y217f
mutant , so that the contribution of the phenol hydrogen bond to catalysis
may be estimated from the effect of the y217f mutation on ompdc - catalyzed
decarboxylation of omp . the side - chain of y217
lies in a flexible loop that folds over the substrate phosphodianion .
the y217a mutation results in a large decrease in the rate of loop
closure of the phosphodianion , so that this conformational change
becomes rate - determining for decarboxylation of omp .
bar graph which compares the effect of mutations of y208 and s211
on the activation barrier to tim - catalyzed isomerization of gap , calculated
from values kcat / km for the wildtype and mutant enzymes ( table 1 ) .
rate constants
for reactions that proceed through structurally related transition
states often show linear logarithmic correlations that are referred
to as linear free energy relationships ( lfer ) .
the slopes of these linear relationships provide a detailed characterization
of the structures of transition states for enzymatic reactions that
is difficult or impossible to obtain by other methods .
the tim - catalyzed isomerization of gap , with second - order rate constant
( kcat / km)gap , differs from the tim - catalyzed reaction of the substrate
pieces ga + hpi , with third - order rate constant kcat / khpikga ( scheme 5 ) , by the presence of the covalent connection between
the pieces .
figure 4 presents the excellent linear logarithmic free energy correlation
between these rate constants , with the slope 1.04 0.03 . a large
positive deviation from this correlation
the l232 mutation results in a large increase in kcat / khpikga , but there can be no similar increase in ( kcat / km)gap , because
the wildtype tim - catalyzed reaction of gap is partly diffusion limited . a smaller positive deviation is observed for the 208-tgag for 208-yggs
loop replacement mutation ( lrm ) . this point is arbitrarily excluded
in order to emphasize the excellent quality of the correlation of
data for the single and double mutant enzymes .
linear free energy relationship ,
with slope 1.04 0.03 , between
the second - order rate constant log ( kcat / km)gap for wildtype and mutant
tim - catalyzed isomerization of gap and the corresponding third - order
rate constant log ( kcat / khpikga ) for the enzyme - catalyzed
reactions of the substrate pieces ga and hpi . key : green
symbols , mutants of tim from trypanosoma brucei ( tbbtim ) ; blue symbols , mutants of tim from chicken muscle ( ctim ) ; red symbols , mutants of ytim .
the linear correlation from figure 4 shows that most mutations result in the same destabilization of the transition states for the catalyzed
reactions
of the whole substrate [ ggap ] and substrate pieces [ gga+hpi ] .
we conclude that these two
transition states show strikingly similar interactions with loop 6
and 7 of tim , and are remarkably similar by this criterion .
the slope
of 1.0 for figure 4 requires a constant difference in activation barriers for the two
reactions of g =
6.6 0.3 kcal / mol ( scheme 5 ) .
this energetic advantage was recently noted in other
systems , and represents partly or entirely
the entropic advantage to the binding of the transition state for
the reaction of the whole substrate , compared with the reaction of
the substrate in pieces .
inspection
of x - ray crystal structures of the complexes between
tim and dhap ( figure 5a ) or the intermediate analogues phosphoglycolate and phosphoglycolohydroxamate , suggests that
elimination of the linkage that connects the substrate pieces ( figure 5b ) might not have
a large effect on interactions between the enzyme and reaction transition
states .
however , these phosphodianion interactions were implicitly
assumed to form fully at the michaelis complex , so that examination
of these x - ray crystal structures failed to provide the insight necessary
to predict enzyme activation by phosphite dianion and the third - order
tim - catalyzed reaction of the substrate pieces .
we suggest that further
critical evaluations of implicit assumptions made when examining x - ray
crystal structures might provide the necessary insight for the design
of important experimental studies on enzyme mechanisms .
( a ) representation
showing the active site of ytim , in a complex with
dhap , from the x - ray crystal structure of
mcdermott and co - workers ( pdb entry 1ney ) .
this structure provides an optimal orientation of the carboxylate side chain of the
catalytic base ( e165 ) , the imidazole side chain of the active site
electrophile ( h95 ) , and the alkyl ammonium side chain of k12 .
( b )
hypothetical representation showing ytim in complex
with phosphite dianion and glycolaldehyde , generated in silico from
( a ) by eliminating the covalent connection at dhap .
we conclude that there is similar strong activation
of tim by interactions
with the phosphodianion of whole substrate and the phosphite dianion
piece .
the question is how are these interactions utilized
for the purpose of enzyme activation ?
the open conformation
of tim ( eo , scheme 6 ) is essentially inactive , so that activation reflects the
high specificity of both the dianion and the transition state for
binding the high - energy active closed form of tim ( ec , scheme 6 ) .
the 1000 fold activation of tim by phosphite dianion
reflects the utilization of 4-kcal / mol out of a total 12 kcal / mol
dianion binding energy to drive the unfavorable protein conformational
change from eo to ec .
the barrier to formation
of the catalytically active caged complexes [ ecs , echpo3 , and ecshpo3 ) is due , minimally , to the enthalpic cost of
desolvation of the active site and the entropic cost of freezing the
motion of flexible protein loops and the catalytic side chains .
the binding energy of the whole substrate gap is also utilized to
drive the same activating conformational change .
the difference between
the modest observed binding energy gobs for gap , calculated from the michaelis constant km , and the larger intrinsic substrate binding energy gint , is equal to the binding energy that is
utilized to drive the enzyme conformational change [ rt ln(1/kc ) ] .
the
ligand - driven closure of loops 6 [ residues 166176 ] and 7 [ 208-yggs-211 ]
moves the s211 hydroxyl into position to hydrogen bond with the backbone
carbonyl oxygen and amide nitrogen from a169 and g173 , respectively ;
the backbone nh group of s211 into a position to hydrogen bond with
the ligand phosphodianion ; and the y208 hydroxyl into a position to
hydrogen bond to the backbone amide nitrogen from a176 ( figure 6 ) .
knowles and sampson proposed that the side chains
of y208 and s211 stabilize the loop closed form of tim ec ( scheme 6 ) relative
to the open form eo , and concluded on the basis of the
large effects of y208f and s211a mutations on catalysis ( see figure 3 ) that these intraloop
interactions play a role in catalysis .
representation
of the structure of the closed form of tim liganded
with pgh ( pdb entry 1tph ) , an analogue of the enediolate reaction
intermediate .
the hydrogen bonds between the hydroxyl of y208 ( loop
7 ) and the amide of a176 ( loop 6 ) , and between the hydroxyl of s211
and the carbonyl oxygen and amide nitrogen from a169 and g173 , respectively
are shown .
cutting the substrate into dianion and carbon acid pieces
allows
separate determination of the effects of mutations on transition state
stabilization from interactions at the dianion activation site and
the catalytic site .
( i ) mutations that
alter the structure of the dianion activation site will cause a decrease
in kd for release of
the dianion at the ternary transition state complex ( eq 6 , derived for scheme 7 ) .
( ii ) mutations
that alter the substrate reactivity at the catalytic site will cause
a decrease in ( kcat / km)e for deprotonation of glycolaldehyde .
( iii ) mutations that cause a change in kc ( scheme 6 ) by changing the stability of ec relative
to eo will result in equal changes in ( kcat / km)e = ( kcat / km)e(kc ) and ( kd ) = ( kd)/(kc ) ( scheme 6 ) .
this has been observed in studies on the l232a mutant of tbbtim.6 an examination of
the kinetic parameters for wildtype , y208 or
s211 mutant enzyme - catalyzed reactions of the substrate pieces shows
the following effects , which are summarized in table 3 .
( 1 ) the mutations of y208 cause a
decrease in kd for
release of the dianion from the
ternary transition state complex , which correspond to 0.42.4
kcal / mol decreases in the intrinsic phosphite dianion binding energy
g ( table 2 ) .
this provides direct evidence that y208
is required for robust enzyme activation by dianions . with the exception
of y208f
, these mutations do not cause large changes ( kcat / km)e for unactivated
deprotonation of ga .
( 2 ) the s211a mutation results in a 60-fold decrease in ( kcat / km)e for deprotonation of glycolaldehyde , increases
in the
values of kd and km for release of phosphite dianion and gap from the free enzyme ,
but little change in kd for release of phosphite dianion from the transition state complex
( table 2 and scheme 6 ) .
these changes
are consistent with a decrease in kc for
the y211a mutant enzyme conformational change ( scheme 6 ) .
this results in utilization of a greater
fraction of the total dianion binding energy , which remains nearly
constant ( table 2 ) ,
to drive the conformational change .
by contrast , the s211 g mutation
eliminates the same hydrogen bond contact as s211a , and results in
only small changes in the kinetic parameters for the tim - catalyzed
reactions of whole substrate and the substrate in pieces .
the s211
side chain lies at the surface of tim , where it is exposed to solvent .
we suggest that loop-6 hydrogen - bonding interactions with water substitute
for interactions with the excised ch2oh side chain
to provide a significant stabilization of the loop - closed enzyme .
( 3 ) the y208f mutation results in a decrease in the total dianion
binding energy and in ( kcat / km)e = ( kcat / km)ekc for carbon deprotonation at the catalytic site ( scheme 7 ) .
there is only
a small increase in km for isomerization
of gap , so that the mutation does not cause a large change in kc for the enzyme conformational change .
we conclude
that this mutation results in a decrease in ( kcat / km)e for
deprotonation of ga ( see following section ) .
the intrinsic
dianion binding energy ,
calculated from data reported in table 2 using eq 6 derived for scheme 7 .
the observed rate constant
for enzyme - catalyzed
reactions of the truncated substrate glycolaldehyde ; [ ( kcat / km)e]obs = ( kcat / km)e(kc ) where ( kcat / km)e is the second - order rate constant for the reactions of glycolaldehyde
catalyzed by the closed form of tim ( scheme 6 ) .
it is assumed when there is no change
in [ ( kcat / km)e]obs for a mutant enzyme , that there is no
effect of the mutation on either ( kcat / km)e or ( kc ) .
the true second - order rate constant
for the reactions of glycolaldehyde catalyzed by the closed form of
tim ( scheme 6 ) .
the equilibrium constant for conversion
of the inactive open form of tim ( eo ) to the active closed
enzyme ( ec , scheme 6 ) .
the p168a mutation of tim from t. brucei.(29,83 ) it is interesting
that mutations of both y208 and s211 eliminate
intraloop hydrogen bonds , which appear to stabilize the closed form
of tim , but only the s211a mutation shows effects on kinetic parameters
for reactions of the substrate pieces that are consistent with a decrease
in kc for loop closure . the minimal two - state
model shown in scheme 6 provides a satisfactory rationalization of the effects of a wide
range of mutations on the kinetic parameters for reactions catalyzed
by tim , ompdc and gpdh .
however , in this case , the model is consistent with the implausible
conclusion that y208 mutations , which eliminate a hydrogen bond that
stabilizes ec ( figure 6 ) , have no effect on kc but result in a reduction in the intrinsic dianion binding energy
g = rt ln(kd ) .
we
propose , as a more plausible explanation for these results , that this
dianion binding energy is used to stabilize an alternative closed
conformation ec for the y208 mutant enzymes , which
is more stable than ec , but for which there is a smaller
total dianion binding energy -g . in any case , the minimal model from scheme 6 will break down in cases where there are
two conformations of the closed enzyme ( ec and ec ) , which each show catalytic activity , and where site directed
mutations stabilize ec relative to the ec , the active form for wildtype tim . we also note that in contrast
with the solvent exposed side chain of s211 , the side chain of y208
is buried beneath loop 6 at ec .
we suggest that the movement
of 208 mutant side chains is constrained and/or coupled to shifts
in the position of its neighbors in a manner that we are unable to
deal with in this paper .
the activation
of tim by interactions with bound dianions is reflected in the values
of kd and kcat , respectively , for reactions of the pieces ( scheme 7 ) and whole substrate .
figure 7 shows the logarithmic
linear free energy relationship between kd for activation by phosphite dianion ( table 2 ) and kcat for catalysis of the reaction of gap ( table 1 ) catalyzed by wildtype and
many loop 7 mutant enzymes .
50% of the phosphite dianion binding energy lost
due to mutations at loop 7 is expressed as a reduction in the enzyme - activating
phosphodianion binding energy for mutant tim - catalyzed isomerization
of the whole substrate gap . the remaining ca .
50%
is expressed at the ground state michaelis complex of wildtype ytim to gap .
logarithmic correlation
between values of kcat for ytim catalyzed isomerization of gap
and the intrinsic dianion binding energy for phosphite dianion activation
of wildtype and mutant tim - catalyzed reactions of [ 1-c]-ga .
key : green symbols , mutants of tim from t. brucei
( tbbtim ) ; blue symbols , mutants of tim from chicken muscle
( ctim ) ; red symbols , mutants of ytim .
large negative deviations from
the linear correlation shown in figure 7 are observed for
three mutants : y208f , p168a , and the
208-tgag for 208-yggs lrm .
these negative
deviations reflect the substantial effects of these mutation on substrate
reactivity at the catalytic site , which result in decreases in both ( kcat / km)e for catalysis of the unactivated reaction
and in kcat for catalysis of the reaction
of the whole substrate , but which do not affect the dianion binding
energy rt
for example , the 20-fold smaller value
of ( kcat / km)e for y208f mutant compared to the wildtype ytim - catalyzed reaction of glycolaldehyde , is similar to the 40-fold
reduction in kcat implied by the 1.6 log
unit negative deviation of this mutant enzyme from the correlation
in figure 7 .
by contrast ,
the s211a mutation results in a 30-fold reduction of the observed
kinetic parameter ( kcat / km)e compared with wildtype tim , but much smaller
changes in kcat and kd , so that the data for this mutant
appear to lie on the correlation shown in figure 7 .
this is consistent with the above conclusion
that the change in the observed kinetic parameter
( kcat / km)e is due to a decrease in kc ( scheme 6 ) for the enzyme
conformational change , rather than a decrease in the microscopic rate constant ( kcat / km)e for the reaction of glycolaldehyde
catalyzed by the active closed form of tim ( table 3 ) . the exclusion of s211a from the linear
correlation shown in scheme 7 results in an increase in the slope of the correlation from
0.53 0.16 to 0.57 0.17 .
the large effect of the
y208f mutations on the kinetic parameters
for ytim catalyzed reactions of whole substrates
and substrate pieces represents the sum of the effects of this mutation
on dianion activation ( kd ) and on catalysis ( [ kcat / km)e ] , table 3 ) . by comparison
, the effect of the p168a mutation
of tbbtim is localized to the catalytic site .
it
is the result of the loss of a steric clash between the side chain
of p168 and the carbonyl oxygen of g211 , which drives the 2
shift in the position of the carboxylate side chain of e167 from the
low activity swung - out position for open unliganded tim to the active
swung - in position at the closed wildtype enzyme .
the catalytic base is observed to lie at the swung - out
position for the p168a mutant enzyme .
this change in the position
of this base affects mainly the kinetic parameters ( kcat / km)e and kcat for deprotonation of glycolaldehye and gap
respectively , but results in little change in kd for phosphite dianion binding and in km for the isomerization of gap .
the large negative deviation from figure 7 observed for the 208-tgag
for 208-yggs loop
7 replacement mutation ( lrm ) at tim also reflects the large effect
of the mutation on the values of ( kcat / km)e and kcat for reaction at the catalytic site compared to the
small effect on kd ( table 2 ) .
our data show that complex changes in the kinetic parameters
for the wildtype tim - catalyzed reaction of the substrate pieces are
observed when the 208-tgag for 208-yggs loop 7 replacement is carried
out as consecutive point mutations ( tables 2 and 3 ) .
the initial
y208 t mutation affects mainly kd for dianion binding , the second y208t / s211 g mutation is conservative ,
and the final g210a substitution at this double mutant results in
a large decrease in ( kcat / km)e , while restoring most of the transition
state dianion binding energy [ rt
they represent an interesting benchmark against which
to test the ability of computational methods to model experimental
data on tim , and the opportunity to use computational methods to provide
genuine insight into the relationship between tim structure and function .
an important
goal of this
research is to characterize the similarities and differences in the
active - site architecture of three enzymes , which catalyze chemically
distinct reactions , but show similar strong activation for catalysis
by dianions ; tim , orotidine 5-monophosphate
decarboxylase ( ompdc ) , and glycerol 3-phosphate dehydrogenase
( gpdh ) .
we find that the effects of most tim mutations
may be localized to either effects on the intrinsic dianion binding
energy or effects on catalysis ( table 3 ) .
this is consistent with the division of the substrate
binding pocket into distinct dianion activation and catalytic sites .
however , the close proximity of these sites
allows : ( 1 ) the cationic side chain of lys-12 to interact with both
the bound phosphodianion , through an intervening water molecule , and
the carbonyl oxygen of dhap . ( 2 ) the effect of some mutations , such asy208f ( table 3 ) , spans these two sites . by
comparison
, the greater separation between the substrate phosphodianion
and orotate ring of orotidine 5-monophosphate has allowed
for evolution of well - separated dianion activation and catalytic sites
at orotidine 5-monophosphate decarboxylase .
now the effects of mutations of amino acid side - chains ,
which interact with the substrate phosphodianion , are expressed exclusively
as changes in the intrinsic dianion binding energy . the classic tim - barrel
( )8 fold has a plastic , or floppy , structure
conferred by 16 flexible loops , which connect 16 rigid ( eight )
and ( eight ) secondary structural elements .
these flexible loops enable tim barrel proteins to access
the inestimable number of conformations needed to obtain catalysis
of a broad range of reactions .
we propose that the conformational
change required to mold floppy loops 6 and 7 of the
eponymous tim barrel to the rigid caged catalytic complex is uphill
thermodynamically , in part because of the intrinsically disordered
state of the unliganded protein .
this role for nonreacting substrate fragments is
generalizable to any tim barrel - catalyzed reaction where a ligand - induced
conformational change is observed . in the case
of tim ,
once the essential function of driving the conformational
change has been performed , the dianion becomes a spectator , and the
catalyzed reaction proceeds with equal efficiency for the whole substrate
and substrate pieces ( figures 4 and 5a , b ) .
transition state theory
provides a useful framework for the interpretation of the complex
effects of loop 7 mutations on the tim - catalyzed reactions of whole
and truncated substrates .
however , a role for dynamics must also be considered , since almost anything done to a protein
changes protein dynamics , so that an explanation for any experimental
result is immediately at hand .
loop 7 [ 208-yggs-211 ] participates in the phosphodianion driven
conformational change from an open enzyme eo to the closed
form ec where the side chains from y208 and s211 form hydrogen
bonds to the backbone of loop 6 ( figure 6 ) .
the dynamics of the motion of loop 6 has
been probed in computational and experimental studies .
we consider whether our experimental results provide any evidence
to support the hypothesis that motions of loop 6 and 7 promote proton
transfer reaction at the catalytic site , which is 89
removed from the altered loop 7 side chains .
the major apparent effect of mutations of y208 is to cause a reduction
in the intrinsic dianion binding energy ( table 2 ) .
this is consistent with a role for the
hydrophobic side chain of y208 in maintaining a low effective dielectric
constant at the dianion activation site , which enables optimal interactions
between the protein catalyst and the polar isomerization reaction
transition state .
mutations of y208 might interfere with a vibrational
motion that promotes catalysis , but this effect could only be important
when phosphite dianion is bound to the enzyme , because most the mutations
cause no change in ( kcat / km)e for the reaction in the absence of this
dianion ( table 2 ) .
the excellent linear free energy correlation between the rate constants
for reactions of the whole substrate and pieces ( figure 4 ) shows there is no transmission
of the energy of a hypothetical promoting vibrational motion through
the covalent bond that connects these pieces in the whole substrate .
the major effect of the s211a mutation is to reduce the phosphodianion
binding energy expressed at michaelis complexes , while maintaining
a nearly constant intrinsic dianion binding energy ( tables 1 and 2 ) .
these results are consistent with stabilization of eo relative to ec at the s211a mutant , so that a larger
fraction of the total dianion binding energy is required to drive
the protein conformational change .
it difficult or impossible to rationalize
the effects of mutations at both y208 and s211 as originating from
changes in promoting vibrational modes , since in one case ( y208 ) the
mode only promotes the reaction at the complex which contains the
dianion [ ( kcat / km)e is invariant , table 2 ] , while in the other ( s211 ) the mode would
act to cause similar increases in ( kcat / km)e and in ( kcat / km)hpi / kd for reactions in the absence and presence
of phosphite dianion , respectively ( table 2 ) .
sampson and knowles observed a solvent
viscosity dependence on kcat / km for y208f
mutant tim - catalyzed isomerization of gap , and a large primary deuterium
kinetic isotope effect of kh / kd = 6 on kcat / km for tim - catalyzed isomerization of [ l - h]-dihydroxyacetone
phosphate .
the first result is consistent
with a viscosity - dependent rate limiting conversion of open eo to closed ec , while the second requires that substrate
deprotonation be rate determining for the mutant enzyme - catalyzed
proton transfer reaction .
sampson and knowles proposed either that ,
loop closure and deprotonation are coupled and occur
in the same rate - limiting step or that these two processes happen
sequentially but interdependently .
y208f is the only
mutation at position 208 that results in a large decrease in the reactivity
of glycolaldehyde at the catalytic site ( table 3 ) .
this is consistent either with the notion
that the barrier to conversion of eo to ec somehow
contributes to the overall activation barrier for the y208a mutant
enzyme - catalyzed deprotonation of ga , or with an effect of the mutation
on enzyme structure that extends to the catalytic site . | the side chains of
y208 and s211 from loop 7 of triosephosphate
isomerase ( tim ) form hydrogen bonds to backbone amides and carbonyls
from loop 6 to stabilize the caged enzyme substrate complex .
the effect of seven mutations [ y208 t , y208s , y208a , y208f , s211 g ,
s211a , y208t / s211 g ] on the kinetic parameters for tim catalyzed reactions
of the whole substrates dihydroxyacetone phosphate and d - glyceraldehyde
3-phosphate [ ( kcat / km)gap and ( kcat / km)dhap ] and of the substrate pieces
glycolaldehyde and phosphite dianion ( kcat / khpikga )
are reported . the linear logarithmic correlation between these kinetic
parameters , with slope of 1.04 0.03 , shows that most mutations
of tim result in an identical change in the activation barriers for
the catalyzed reactions of whole substrate and substrate pieces , so
that the transition states for these reactions are stabilized by similar
interactions with the protein catalyst . the second linear logarithmic
correlation [ slope = 0.53 0.16 ] between kcat for isomerization of gap and kd for phosphite dianion binding to the transition
state for wildtype and many mutant tim - catalyzed reactions of substrate
pieces shows that ca .
50% of the wildtype tim dianion binding energy ,
eliminated by these mutations , is expressed at the wildtype michaelis
complex , and ca .
50% is only expressed at the wildtype transition
state .
negative deviations from this correlation are observed when
the mutation results in a decrease in enzyme reactivity at the catalytic
site
. the main effect of y208 t , y208s , and y208a mutations is to cause
a reduction in the total intrinsic dianion binding energy , but the
effect of y208f extends to the catalytic site . | Introduction
Experimental Procedures
Results
Discussion | this
results in a 10-fold falloff in kcat / km for catalysis of isomerization
due mainly to a decrease in kcat , so that the activating loop - dianion interactions
develop only on moving from the ground - state michaelis complex to
the transition state for tim - catalyzed isomerization . the strong activation of tim - catalyzed deprotonation
of glycolaldehyde by phosphite dianion ( hpo3 = hpi ) shows that the tim active site is divided into
a catalytic domain , which contains the side chains that carry out
chemical isomerization of bound substrate , and a dianion activation
domain that uses enzyme - dianion interactions to promote the proton
transfer reaction at the catalytic site . an earlier communication reported
that five mutations of y208 ,
s211 yeast tim and of other side chains of tim from several organisms
result in nearly the same change in the activation barriers for the
catalyzed reactions of whole substrate and substrate pieces . the
reactions in the absence of phosphite dianion were initiated by adding
enzyme to give a final solution that contained 20 mm [ 1-c]-ga , 20 mm imidazole ( 20% free base , pd 7.0 , i = 0.1 ( nacl ) ) , and enzyme [ 0.40 mm , wildtype ytim ;
0.10 mm , y208 t ytim ; 0.058 mm , y208s ytim ; 0.068 mm , y208a ytim ; 0.24 mm , y208f ytim ; 0.47 mm , s211a ytim ; 0.26 mm , s211 g ytim ; 0.18 mm , y208t / s211 g ytim ] in a volume
of 850 l . the reactions in the presence of hpo3 were initiated by adding enzyme to give a final solution
that contained 20 mm [ 1-c]-ga , 20 mm imidazole ( 20% free
base , pd 7.0 ) , 140 mm hpo3 ( 50%
dianion , pd 7.0 ) , and enzyme [ 1827 m , wildtype ytim ; 2061 m , y208 t ytim ;
2844 m , y208s ytim ; 4161 m ,
y208a ytim ; 130260 m , y208f ytim ; 50370 m , s211a ytim ;
1138 m , s211 g ytim ; 2568 m ,
y208t / s211 g ytim ] in a volume of 850 l ( i = 0.1 , nacl ) . observed first - order rate constants , kobs ( s ) , for the reactions of [ 1-c]-ga were determined from the slopes of linear semilogarithmic
plots of reaction progress against time ( eq 2 ) , where fs is
the fraction of [ 1-c]-ga remaining at time t. observed second - order rate constants , ( kcat / km)obs ( m s ) , for the tim - catalyzed reaction of [ 1-c]-ga were determined from the values of kobs using eq 3 , where fhyd = 0.94 is the fraction of
[ 1-c]-ga present as the hydrate.23
the kinetic parameters for the isomerization reactions of gap and
dhap catalyzed by wildtype and y208 and s211 mutants of ytim , determined at ph 7.5 and 25 c ( i = 0.1 ,
nacl ) , are reported in table 1 . the product yields for the reactions in the presence and absence of
phosphite dianion catalyzed by wildtype ytim , y208 t ytim , y208s ytim , y208a ytim , y208f ytim , and s211a ytim
are reported in tables s1s6 , respectively , of the supporting
information to ref ( 28 ) . the value of ( kcat / km)obs for
the tim - catalyzed reactions of [ 1-c]-ga is equal to the
sum of the second - order rate constants
for the reactions that occur at the enzyme active site ( scheme 3a ) and for nonspecific protein - catalyzed
reactions ( scheme 3b ) . it is not possible to distinguish [ 1-c , 2-h]-ga formed in the specific and nonspecific reactions and we therefore
estimate the yield of [ 1-c , 2-h]-ga from scheme 3a by assuming that
the ratio of yields of [ 2-c , 2-h]-ga and
[ 1-c , 2-h]-ga from the unactivated reaction
is the same as for the phosphite dianion activated reaction , where
little or no [ 1-c , 2,2-di - h]-ga ( scheme 3b ) is generally observed.4 the true second - order
rate constants ( kcat / km)tim for unactivated wildtype and mutant tim - catalyzed
reactions of the carbonyl form of [ 1-c]-ga to give [ 2-c]-ga , [ 2-c , 2-h]-ga , and [ 1-c , 2-h]-ga ( scheme 3a ) were calculated using eq 4 , where fe is the fractional yield of the products from reactions at the active
site of tim that are reported in tables s1s6 of ref ( 28 ) and in tables s1 and s2
( supporting information ) of this paper . the second - order rate
constants ( kcat / km)tim ( eq 4 ) for phosphite dianion activated wildtype and mutant ytim - catalyzed reactions of the carbonyl form of [ 1-c]-ga are reported in tables s1s6 of ref ( 28 ) and in tables s1 and s2
( supporting information ) of this paper . values of fe = 1.0 were determined for
phosphite dianion activation of wildtype , y208 t , s211 g , and y208t / s211 g ytim catalyzed reactions of [ 1-c]-ga by 2.5
mm hpo3 . figure 2 shows the dependence of the values of ( kcat / km)tim determined
for wildtype and four mutant [ s211 g , y208t / s211 g , y208 t , s211a ] ytim - catalyzed reactions of [ 1-c]-ga on the
concentration of added phosphite dianion . the solid lines through the experimental data for the y208 t , s211 g ,
y208t / s211 g , and y208s mutant enzymes show the nonlinear least - squares
fits to eq 5 , derived
for scheme 4 ( s = [ 1-c]-ga ) , and using the values of ( kcat / km)e from table 2 for the unactivated reaction
in the absence of phosphite dianion . we note the large
uncertainty in these kinetic parameters when kd 40 mm ( y208 t , s211 g , and y208s mutants ) , which is
substantially greater that the largest [ hpo3 ] = 20 mm used in these experiments.5 dependence
of second - order rate constants ( kcat / km)tim for the tim - catalyzed
turnover of the free carbonyl form of [ 1-c]-ga in d2o on [ hpo3 ] at pd 7.0 and 25
c at i = 0.1 , nacl . these results
are consistent with the notion that the binding energy from the hydrogen
bond to the phenol hydroxyl is utilized to hold the phenyl group in
a conformation that reduces the dielectric constant for the active
site cage , and optimizes stabilizing
electrostatic interactions between the transition state and the protein
catalyst . bar graph which compares the effect of mutations of y208 and s211
on the activation barrier to tim - catalyzed isomerization of gap , calculated
from values kcat / km for the wildtype and mutant enzymes ( table 1 ) . the tim - catalyzed isomerization of gap , with second - order rate constant
( kcat / km)gap , differs from the tim - catalyzed reaction of the substrate
pieces ga + hpi , with third - order rate constant kcat / khpikga ( scheme 5 ) , by the presence of the covalent connection between
the pieces . a large
positive deviation from this correlation
the l232 mutation results in a large increase in kcat / khpikga , but there can be no similar increase in ( kcat / km)gap , because
the wildtype tim - catalyzed reaction of gap is partly diffusion limited . linear free energy relationship ,
with slope 1.04 0.03 , between
the second - order rate constant log ( kcat / km)gap for wildtype and mutant
tim - catalyzed isomerization of gap and the corresponding third - order
rate constant log ( kcat / khpikga ) for the enzyme - catalyzed
reactions of the substrate pieces ga and hpi . the linear correlation from figure 4 shows that most mutations result in the same destabilization of the transition states for the catalyzed
reactions
of the whole substrate [ ggap ] and substrate pieces [ gga+hpi ] . this energetic advantage was recently noted in other
systems , and represents partly or entirely
the entropic advantage to the binding of the transition state for
the reaction of the whole substrate , compared with the reaction of
the substrate in pieces . however , these phosphodianion interactions were implicitly
assumed to form fully at the michaelis complex , so that examination
of these x - ray crystal structures failed to provide the insight necessary
to predict enzyme activation by phosphite dianion and the third - order
tim - catalyzed reaction of the substrate pieces . knowles and sampson proposed that the side chains
of y208 and s211 stabilize the loop closed form of tim ec ( scheme 6 ) relative
to the open form eo , and concluded on the basis of the
large effects of y208f and s211a mutations on catalysis ( see figure 3 ) that these intraloop
interactions play a role in catalysis . ( ii ) mutations
that alter the substrate reactivity at the catalytic site will cause
a decrease in ( kcat / km)e for deprotonation of glycolaldehyde . this has been observed in studies on the l232a mutant of tbbtim.6 an examination of
the kinetic parameters for wildtype , y208 or
s211 mutant enzyme - catalyzed reactions of the substrate pieces shows
the following effects , which are summarized in table 3 . ( 1 ) the mutations of y208 cause a
decrease in kd for
release of the dianion from the
ternary transition state complex , which correspond to 0.42.4
kcal / mol decreases in the intrinsic phosphite dianion binding energy
g ( table 2 ) . ( 2 ) the s211a mutation results in a 60-fold decrease in ( kcat / km)e for deprotonation of glycolaldehyde , increases
in the
values of kd and km for release of phosphite dianion and gap from the free enzyme ,
but little change in kd for release of phosphite dianion from the transition state complex
( table 2 and scheme 6 ) . by contrast , the s211 g mutation
eliminates the same hydrogen bond contact as s211a , and results in
only small changes in the kinetic parameters for the tim - catalyzed
reactions of whole substrate and the substrate in pieces . ( 3 ) the y208f mutation results in a decrease in the total dianion
binding energy and in ( kcat / km)e = ( kcat / km)ekc for carbon deprotonation at the catalytic site ( scheme 7 ) . there is only
a small increase in km for isomerization
of gap , so that the mutation does not cause a large change in kc for the enzyme conformational change . the observed rate constant
for enzyme - catalyzed
reactions of the truncated substrate glycolaldehyde ; [ ( kcat / km)e]obs = ( kcat / km)e(kc ) where ( kcat / km)e is the second - order rate constant for the reactions of glycolaldehyde
catalyzed by the closed form of tim ( scheme 6 ) . (29,83 ) it is interesting
that mutations of both y208 and s211 eliminate
intraloop hydrogen bonds , which appear to stabilize the closed form
of tim , but only the s211a mutation shows effects on kinetic parameters
for reactions of the substrate pieces that are consistent with a decrease
in kc for loop closure . 50% of the phosphite dianion binding energy lost
due to mutations at loop 7 is expressed as a reduction in the enzyme - activating
phosphodianion binding energy for mutant tim - catalyzed isomerization
of the whole substrate gap . logarithmic correlation
between values of kcat for ytim catalyzed isomerization of gap
and the intrinsic dianion binding energy for phosphite dianion activation
of wildtype and mutant tim - catalyzed reactions of [ 1-c]-ga . these negative
deviations reflect the substantial effects of these mutation on substrate
reactivity at the catalytic site , which result in decreases in both ( kcat / km)e for catalysis of the unactivated reaction
and in kcat for catalysis of the reaction
of the whole substrate , but which do not affect the dianion binding
energy rt
for example , the 20-fold smaller value
of ( kcat / km)e for y208f mutant compared to the wildtype ytim - catalyzed reaction of glycolaldehyde , is similar to the 40-fold
reduction in kcat implied by the 1.6 log
unit negative deviation of this mutant enzyme from the correlation
in figure 7 . by contrast ,
the s211a mutation results in a 30-fold reduction of the observed
kinetic parameter ( kcat / km)e compared with wildtype tim , but much smaller
changes in kcat and kd , so that the data for this mutant
appear to lie on the correlation shown in figure 7 . this is consistent with the above conclusion
that the change in the observed kinetic parameter
( kcat / km)e is due to a decrease in kc ( scheme 6 ) for the enzyme
conformational change , rather than a decrease in the microscopic rate constant ( kcat / km)e for the reaction of glycolaldehyde
catalyzed by the active closed form of tim ( table 3 ) . the large effect of the
y208f mutations on the kinetic parameters
for ytim catalyzed reactions of whole substrates
and substrate pieces represents the sum of the effects of this mutation
on dianion activation ( kd ) and on catalysis ( [ kcat / km)e ] , table 3 ) . this change in the position
of this base affects mainly the kinetic parameters ( kcat / km)e and kcat for deprotonation of glycolaldehye and gap
respectively , but results in little change in kd for phosphite dianion binding and in km for the isomerization of gap . the large negative deviation from figure 7 observed for the 208-tgag
for 208-yggs loop
7 replacement mutation ( lrm ) at tim also reflects the large effect
of the mutation on the values of ( kcat / km)e and kcat for reaction at the catalytic site compared to the
small effect on kd ( table 2 ) . our data show that complex changes in the kinetic parameters
for the wildtype tim - catalyzed reaction of the substrate pieces are
observed when the 208-tgag for 208-yggs loop 7 replacement is carried
out as consecutive point mutations ( tables 2 and 3 ) . the initial
y208 t mutation affects mainly kd for dianion binding , the second y208t / s211 g mutation is conservative ,
and the final g210a substitution at this double mutant results in
a large decrease in ( kcat / km)e , while restoring most of the transition
state dianion binding energy [ rt
they represent an interesting benchmark against which
to test the ability of computational methods to model experimental
data on tim , and the opportunity to use computational methods to provide
genuine insight into the relationship between tim structure and function . in the case
of tim ,
once the essential function of driving the conformational
change has been performed , the dianion becomes a spectator , and the
catalyzed reaction proceeds with equal efficiency for the whole substrate
and substrate pieces ( figures 4 and 5a , b ) . transition state theory
provides a useful framework for the interpretation of the complex
effects of loop 7 mutations on the tim - catalyzed reactions of whole
and truncated substrates . loop 7 [ 208-yggs-211 ] participates in the phosphodianion driven
conformational change from an open enzyme eo to the closed
form ec where the side chains from y208 and s211 form hydrogen
bonds to the backbone of loop 6 ( figure 6 ) . the major apparent effect of mutations of y208 is to cause a reduction
in the intrinsic dianion binding energy ( table 2 ) . mutations of y208 might interfere with a vibrational
motion that promotes catalysis , but this effect could only be important
when phosphite dianion is bound to the enzyme , because most the mutations
cause no change in ( kcat / km)e for the reaction in the absence of this
dianion ( table 2 ) . it difficult or impossible to rationalize
the effects of mutations at both y208 and s211 as originating from
changes in promoting vibrational modes , since in one case ( y208 ) the
mode only promotes the reaction at the complex which contains the
dianion [ ( kcat / km)e is invariant , table 2 ] , while in the other ( s211 ) the mode would
act to cause similar increases in ( kcat / km)e and in ( kcat / km)hpi / kd for reactions in the absence and presence
of phosphite dianion , respectively ( table 2 ) . this is consistent either with the notion
that the barrier to conversion of eo to ec somehow
contributes to the overall activation barrier for the y208a mutant
enzyme - catalyzed deprotonation of ga , or with an effect of the mutation
on enzyme structure that extends to the catalytic site . | [
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] |
in the next 30 years , the number of people over 65 in the world will almost double to 1.3 billion .
the percentage of those 80 and older will be 31% , while the number was 18% in 1980 .
this huge shift to an older population globally has given rise to the notions of the third and fourth ages in the lifespan development literature .
this differentiation of the last part of the lifespan into two separate phases is important because of the characteristic patterns of gains ( growth ) and losses ( decline ) seen in the young old and the old old . for
many of those in the fourth age ( 80 + ) issues such as physical illness and approaching mortality decimate their functioning and subsequently lead to the need for nursing home ( nh ) care .
depression is one of the most prevalent mentally ill health problems facing european citizens today , and it is considered one of the most frequent mental disorders in the elderly population .
moreover , depression is particularly common among these living in long - term care facilities .
reported rates of major depression in older adults , depending on the location and characteristics of the sample , have ranged from 1% to 42% . in general , the prevalence of late - life depression has been found to be higher in those patients with more medical illness .
specifically , rates of depression in nhs are three to four times higher than those in community - dwelling older adults . also , elderly who lack
a review that included 36 studies from various countries reported a prevalence rate for major depression ranging from 6% to 26% and for minor depression from 11% to 50% . the prevalence rate of depressive symptoms ranged from 36% to 49% .
the three strongest explanatory factors on the age - effect of depression found are impairment , diagnosis , and somatic symptoms , respectively [ 11 , 12 ] .
worse general medical health is seen as the strongest factor associated with depression among nh patients . in general ,
nh patients are characterized by high age , frailty , mortality , disability , powerlessness , and dependency .
moreover , studies in nursing homes report a large overlap of depression and anxiety [ 1417 ] .
the course of major depression and significant depressive symptoms in nh patients tends to be chronic , with a persistence rate of more than 50% of depressed patients still depressed after 612 months [ 18 , 19 ] .
depression in nh patients is associated with negative outcomes such as impaired quality of life , impaired activities of daily living , substantial caregiver burden and worsened medical outcomes , increased risk of hospital admission , and a higher mortality rate .
a recent study reports that the presence of depression has a statistically and clinically significant negative impact on well - being .
the nh patients ' sense of loss of independence and privacy , feelings of isolation and loneliness , the ever present death and grief , and lack of meaningful in - house activities are identified as risk factors for depression .
conversely , nh in - house activities and different kinds of mental health services have significant benefit for nh patients at risk for depression [ 2527 ] .
accordingly , efforts to prevent and decrease depression are of great importance for nh patients ' well - being and quality of life .
the world health organization has estimated that by 2020 , depression is expected to be the highest ranking cause of disease in the developed world .
therefore , finding new and alternative approaches to weaken this detrimental development is highly warranted .
self - transcendence is found to be such a correlate and a resource for well - being among vulnerable populations and at the end of life [ 2931 ] .
self - transcendence includes a psycho - social - spiritual force toward personal maturity that is distinct from the more self - absorbed strivings for self - esteem and intimacy typical in earlier developmental phases .
hence , self - transcendence enhances an individual 's searching for new perspectives , meaning , and well - being and allows him or her to overcome ego concerns .
the idea of self - transcendence is inspired by human developmental theory emphasizing maturity as the developmental task in later life . in accordance with this theory
, the developmental crisis in old age entails integrity versus despair and is resolved by the development of inner resources as personal maturity and wisdom , qualities providing well - being .
the developmental process of self - transcendence is stimulated by the challenges of age and coming to terms with death .
adults with higher levels of self - transcendence do not seek absolute answers to questions in life but seek meaning in life events as integrated within a moral , social , and historical context .
the central core of self - transcendence is described as connection between the individual , environment , and a transcendent being .
self - transcendence refers to a multidimensional expansion of the self - boundaries intrapersonally ( through self - acceptance and finding meaning in life ) , interpersonally ( by reaching out to others or connecting with nature ) , transpersonally ( by reaching out to a higher entity , being of purpose ) , and temporality ( by integrating one 's past and future into the present ; a dynamic process involving adaption to physical , emotional , and/or spiritual distress ) . within a holistic framework of the body , mind , and spirit as a whole
, self - transcendence has been considered a central aspect of human spirituality . as a general human dimension of personal maturity , self - transcendence has been related to spiritual and nonspiritual factors .
human spirituality is expressed and experienced in the context of caring connections with the self , others , nature , and a life force or god [ 3539 ] .
accordingly , spirituality seems closely related to connectedness , the essence of self - transcendence .
a recent study of the relationships between spiritual well - being and self - transcendence revealed that both interpersonal and intrapersonal self - transcendence significantly affect spiritual well - being in cognitively intact nh patients .
self - transcendence has been studied in various disciplines , but it is of particular interest to nursing .
the perspective of promoting health and well - being is fundamental and a major nursing concern in long - term care [ 4143 ]
. studies on various populations demonstrate an inverse association between depression and self - transcendence [ 30 , 31 , 4449 ] .
previous research demonstrates the significance of self - transcendence to older people well - being [ 31 , 45 , 50 , 51 ] .
empirical support for the importance of self - transcendence for those at the end of life and facing their own mortality has been well documented [ 33 , 47 , 5255 ] . in summary ,
the literature suggests depression as a common mental disorder among older people characterized by high age , impairment , and somatic symptoms .
the patients ' sense of loss of independence and privacy , feelings of isolation and loneliness , and lack of meaningful activities are risk factors for depression in nh patients .
self - transcendence is a resource for well - being among vulnerable individuals and at the end of life .
a previous investigation of the dimensionality of the self - transcendence scale revealed that self - transcendence comprises one interpersonal and one intrapersonal factor .
therefore , this study investigates the associations between interpersonal and intrapersonal self - transcendence and depression among cognitively intact nh patients . to the authors ' knowledge
, previous research has not examined the relationships between interpersonal and intrapersonal self - transcendence and depression in nhs by means of structural equation modelling ( sem ) .
our research question was does self - transcendence affect depression in cognitively intact nh patients ?
based on the theoretical and empirical knowledge of depression and self - transcendence , the following hypotheses were formulated .
hypothesis 1intrapersonal self - transcendence has negative effect on depression among cognitively intact nh patients .
intrapersonal self - transcendence has negative effect on depression among cognitively intact nh patients .
hypothesis 2interpersonal self - transcendence has negative effect on depression among cognitively intact nh patients .
the data were collected in 2008 - 2009 from 250 nh patients who met the inclusion criteria : ( 1 ) local authority 's decision of long - term nh care , ( 2 ) residential time 6 months or longer , ( 3 ) informed consent competency recognized by responsible doctor and nurse , and ( 4 ) capable of being interviewed .
the nurse presented them with oral and written information about their rights as participants and their right to withdraw at any moment .
this population has difficulties completing a questionnaire on their own ; therefore one - on - one interviews were conducted by three trained researchers and completed in the nh patients ' private rooms .
researchers with identical professional background were chosen ( rn , ma , trained and experienced in communication with elderly , as well as teaching gerontology at an advanced level ) and trained to conduct the interviews as similarly as possible .
the questionnaires relevant for the present study were part of a battery of nine questionnaires comprising 130 items .
interviewers held a large - print copy of questions and possible responses in front of the participants to avoid misunderstandings .
approval by the norwegian social science data services was obtained for a license to maintain a register containing personal data ( ref .
16443 ) , and likewise we attained approval from the regional committee for medical and health research ethics in central norway ( ref .
the total sample consisted of 202 ( 80.8% ) of 250 long - term nh patients representing 44 nhs .
long - term nh care was defined as 24-hour care ; short - term care patients , rehabilitations patients , and cognitively impaired patients were not included .
participants ' ages ranged from 65 to 104 years , with a mean of 86 years ( sd = 7.65 ) .
the sample consisted of 146 women ( 72.3% ) and 56 men ( 27.7% ) , where the mean age for women was 87.3 years and 82 years for the men . in total , 38 ( 19% )
were married / cohabitant , 135 ( 67% ) widows / widowers , 11 ( 5.5% ) divorced , and 18 ( 19% ) were single .
the mean residential time when interviewed was 2.6 years for both sexes ( range 0.513 years ) ; 117 stayed in rural nhs , while 85 stayed in urban nhs . in all , 26.1% showed mild to moderate depression , only one woman scored 15 + indicating severe depression , 70.4% was not depressed , and nearly 88% had no anxiety disorder .
missing data was low in frequency : depression 5.0% and self - transcendence scale 5.9% and were handled by means of the listwise procedure .
depression was assessed by the depression subscale of the hospital anxiety and depression scale ( hads ) , comprising seven items .
each item is scored from 03 , and the maximum score is 21 on the subscale .
the ranges of scores for cases are 07 normal , 810 mild disorder , 1114 moderate disorder , and 1521 severe disorder .
hads has been tested extensively and has well - established psychometric properties . in order to increase acceptability and avoid individuals feeling
as though they are being tested for mental disorders , symptoms of severe psychopathology have been excluded .
hads is translated into norwegian and found to be valid for older people [ 11 , 12 ] . since the present study focuses on the associations between self - transcendence and depression , only the depression - items ( subscale hads - d ) were included in the sem model to be tested here .
i still enjoy the things i used to enjoy , i can laugh and see the funny side of things ,
i have lost interest in my appearance , and i look forward with enjoyment to things . the item i can enjoy a good book or television - program
loaded very low ( = 0.20 , r = 0.05 ) , indicating less relevance for depression in this nh population and was dismissed .
as the in - house activities in nhs are scarce , tv programs might be the only activity available , while reading books is difficult due to impairments .
the items were scored on a four - point scale ranging from totally disagree to totally agree .
the internal consistence of the construct ( table 1 ) was satisfactory ( = .68 ) .
interpersonal and intrapersonal self - transcendence were assessed by items from the self - transcendence scale ( sts ) .
the sts was developed from the 36-item developmental resources of later adulthood scale [ 33 , 59 ] , which intended to identify intrapersonal , interpersonal , transpersonal , and temporal experiences characteristic of later life , reflecting expanded boundaries of self .
the sts comprises 15 items measuring characteristics of a matured view of life representing the extent to which a person expands personal boundaries .
each item is rated on a four - point likert - type scale from 1.0 ( not at all ) to 4.0 ( very much ) , with higher scores indicating higher st ( see the supplemental information in supplementary material available online at doi:10.5402/2012/301325 ) . in previous studies , cronbach 's ranged from.80 to.88 [ 31 , 43 , 48 ] .
content validity is adequate , based on a thorough review of empirical and theoretical literature .
support for construct validity has been found in the relationships of self - transcendence scores to other measures [ 29 , 60 , 61 ] .
the sts was translated into swedish and back into english , and the back - translated version was then approved by the instrument constructor .
the swedish version demonstrated internal consistency of.70.85 ( op.cit . ) and was translated into norwegian for the purposes of the present study .
the swedish and norwegian language and culture are nearly identical in all aspects that matter this study .
the sts is practically unchanged in the norwegian version , but the words are spelled in norwegian and the meanings have been checked .
the two - factor construct of self - transcendence was used , but the number of items was reduced ; the item letting go of my past losses ( st15 ) , which is reversed scored , loaded extremely low ( = 0.11 ) and demonstrated r = 0.02 .
this item was uncorrelated ; therefore , there might be some translation problems and this item was dismissed .
the items having hobbies and interests i can enjoy , being involved with other people ,
sharing my wisdom or experience with others , helping others in some way , having an ongoing interest in learning , able to move beyond things that once seemed so important , and finding meaning in my spiritual beliefs were indicators for interpersonal self - transcendence , while the items accepting myself as growing older , adjusting well to my present life situation , adjusting well to changes in my physical abilities , finding meaning in my past experiences , accepting death as a part of life , letting others help me when i may need it , and enjoying my pace of life constituted the intrapersonal st construct .
cronbach 's in current study was 0.72 ( all 15 items ) , while for interpersonal self - transcendence ( seven items ) was .76 and .65 for intrapersonal self - transcendence ( seven items ) ( table 1 ) .
reliability for the latent constructs in this study is further investigated inside the confirmatory factor analysis ( cfa ) .
composite reliability ( c ) is reported in table 2 displaying values between 0.65 and 0.75 ; values greater than 0.60 are desirable . a structural equation model ( sem ) of the hypothesized relations between the latent constructs of depression and self - transcendence was tested by means of lisrel 8.8 . using sem accounts for random measurement error , and the psychometric properties of the scales in the model are more accurately derived .
since the standard errors are estimated under nonnormality , the satorra - bentler - scaled chi - square statistic was applied as a goodness - of - fit statistic , which is the correct asymptotic mean even under nonnormality . in line with the rules of thumb of conventional cut - off criteria ,
the following fit indices were used to evaluate model fit : chi - square ( )a small
and a nonsignificant p value corresponds to good fit . also , we used the root mean square error of approximation ( rmsea ) and the standardized root mean square residual ( srms ) with values below 0.05 indicating good fit , while values smaller than 0.08 are interpreted as acceptable [ 66 , 67 ] . the comparative fit index ( cfi ) and the nonnormed fit index ( nnfi ) with an acceptable fit at 0.95 and good fit at 0.97 and above were used , and the normed fit index ( nfi ) and the goodness - of - fit index ( gfi ) with an acceptable fit at 0.90 , while a good fit was set to 0.95 . for the adjusted gfi ( agfi ) ,
acceptable fit was set to 0.85 and good fit at 0.90 ( op.cit . ) . before examining the hypothesized relationships in the sem analysis
the cfa provided a good fit to the observed data for the interpersonal self - transcendence ( = 22.28 , p < 0.073 , rmsea = 0.055 , srmr = 0.047 , nfi = 0.95 , nnfi / cfi = 0.97/0.98 , gfi / agfi = 0.96/0.92 ) , intrapersonal self - transcendence ( = 15.77 , p < 0.0033 , rmsea = 0.026 , srmr = 0.056 , nfi = 0.93 , nnfi / cfi = 0.99/0.99 , gfi / agfi = 0.96/0.92 ) , and the depression scale ( = 9.43 , p < 0.46 , rmsea = 0.016 , srmr = 0.039 , nfi = 0.96 , nnfi / cfi = 1.00/1.00 , gfi / agfi = 0.98/0.95 ) .
all parameter estimates were significant ( p < 0.05 ) and loaded positively and clearly on their intended latent variable with standardized factor loadings between 0.22 and 0.77 . for scaling the variances of the dependent latent variable was set at 1 .
table 1 displays the means ( m ) , standard deviations ( sd ) , cronbach 's alpha , and correlations matrix for the constructs of self - transcendence ( interpersonal and intrapersonal ) and depression .
moderate correlations were found between the latent constructs included in the sem model ( table 1 ) .
the alpha levels for the various measures indicate an acceptable level of inter - item consistency in the measures with cronbach 's alpha coefficients of .65 or higher . in order to investigate how interpersonal self - transcendence ( st-1 ) and intrapersonal self - transcendence ( st-2 ) relate to depression ,
model 1 comprising six depression , seven interpersonal self - transcendence and seven intrapersonal self - transcendence items was estimated .
all estimates were significant ( p < 0.05 ) , and the factor loadings ranged 0.220.72 and r - values 0.050.59 .
model 1 fitted well ( = 201.64 , p = 0.035 , df = 167 , rmsea = 0.034 , p value = 0.96 , nnfi = 0.96 , cfi = 0.97 , srmr = 0.067 , and agfi = 0.85 ) .
however , nfi = 0.84 and gfi = 0.88 were low . traditionally , a cut - off point of 0.90 has been recommended for the gfi and nfi .
however , the agfi , which adjusts the gfi by degrees of freedom , is acceptable .
also the nfi is sensitive to sample size , underestimating fit for samples less than 200 ; therefore researchers should not rely solely on the gfi and nfi .
table 3 shows the standardized regression coefficients of the directional relationships and the total effects between the latent constructs in model 1 .
as hypothesized , both directional paths were significant displaying an inverse relationship , from interpersonal self - transcendence to depression ( 1,1 = 0.30 * ) and from intrapersonal self - transcendence to depression ( 1,2 = 0.41 * ) ( table 3 ) .
a scrutiny of the total effects of interpersonal self - transcendence and intrapersonal self - transcendence on depression revealed statistical significant total effects from both interpersonal and intrapersonal self - transcendence on all the depression items ( table 3 ) .
the world health organization estimated that , by 2020 , depression will be the highest ranking cause of disease in the developed world .
moreover , a huge shift to an older population worldwide is highlighted , and the most rapidly growing segment is that of people over 80 years old .
simultaneously , depression is one of the most frequent mental disorders in the elderly population and specifically among nh patients .
therefore , finding new interventions to increase well - being and decrease depression among nh patients is highly warranted .
the aim of this study was to explore the association between interpersonal and intrapersonal self - transcendence and depression in cognitively intact nursing home patients . by doing
so we sought to contribute to a holistic nursing perspective of promoting well - being in nh patients in two ways : first , this study supplies empirical knowledge to the growing body of self - transcendence knowledge by exploring self - transcendence among nh patients ; second , by applying a two - factor construct of self - transcendence that has been shown to be psychometrically superior the one - factor construct , the present study allows a more complex examination of the associations between self - transcendence and depression .
the present study examines the relationships and the influences of interpersonal and intrapersonal self - transcendence on depression in nh patients . by means of advanced statistical analysis such as structural equation modeling ( sem )
, this study provides more specific guidelines to nursing interventions promoting well - being in nh patients and suggests that finding ways to enhance an individual 's intrapersonal and interpersonal self - transcendence might be beneficial in this matter .
more specifically , we found that the hypothesized relationship between self - transcendence ( intrapersonal and interpersonal ) and depression was fully supported .
it is reasonable that interpersonal self - transcendence including personal interests , learning , involvement with others , connectedness , sharing one 's wisdom , and helping others is associated with low depression .
therefore , nursing interventions facilitating nh patients ' involvement and connectedness , sharing wisdom , and possibilities helping others might decrease and prevent depression . also , the intrapersonal self - transcendence comprising nh patients ' self - acceptance as growing older , adjusting well to the changes in physical abilities and the present situation , and finding meaning in one 's past experiences related negatively to depression .
the statistical tests suggested that the intrapersonal self - transcendence affects depression more than the interpersonal self - transcendence among cognitively intact nh patients .
therefore , staff nurses ' awareness of nh patients ' self - acceptance and adjustment to the life situation is essential for well - being as well as enjoyment , humor , and laughter . supporting nh patients ' effort to accept themselves and adjust well to the life situation and one 's declines is significant to decrease and prevent depression . at 86 years old ( mean age in the present study ) , representing the old old
living in a nh represents a life perspective where openness to keep learning , enjoying hobbies and interests , and involving with others might seem less important than accepting oneself and adjusting well to the here - and - now in the nh environment .
loss of functions , relationships , privacy , self - determination , and connectedness and the forthcoming death , together with searching for meaning in the in - house institutionalized nh life , seem in general as the main challenges .
generally nh patients are in great need of care and assistance because of physical declines and other limitations , and they represent a vulnerable group . from this particular point of view
therefore , these individuals need initiative and vitalization from others and the environment . elderly who lack social / emotional support
consequently , the nurse - patient relationships might be an overriding figure in the nh patients ' daily life and might serve as a vital resource of human involvement , interaction , connectedness , and a facilitator of meaningful in - house activities for nh patients .
few social relations for dialogue , self - reflection , and connectedness are left ; in our sample only 19% had a partner .
the social climate in a nh environment is largely determined by the staff - patient interactions that take place within it .
the potentials for self - transcendence and well - being are important considerations in nh care ; the participants had stayed in nh for 6 months or longer . because the length of stay is long , much time is available to enter into meaningful relationships and communication with patients , pursuing appropriate interventions to promote self - transcendence . within erikson 's model of human psychosocial development ,
therefore , self - transcendence as an individual developmental process toward personal maturity and a resource for well - being might serve as an indicator of successful aging among nh patients .
some of the intrapersonal self - transcendence indicators loaded low and displayed low r - values .
these items involve finding meaning in past experiences , accepting death as a part of life , letting others help , and enjoying one 's pace of life .
being in the fourth age , living in a nh , represents a life situation where letting others help and accepting death are quite obvious .
nh patients ' daily life is strongly influenced by the routines and availability of help and support from the staff nurses . in this regard ,
nh patients might find that they do not have their own pace of life , and enjoying one 's pace of life seems less relevant .
also the items i feel as i 'm slowed down and i have lost interest in my appearance loaded low , but the composite reliability was good . the low values might indicate that these items are less relevant for this particular sample .
this study expands previous studies by testing the associations between the two - factor construct of self - transcendence and depression among nh patients by using structural equation modeling . using sem accounts for random measurement error and the psychometric properties of the scales in the model
nevertheless , the findings of this study must be discussed with some limitations in mind .
first , model 1 comprises 20 variables , indicating a desirable n = 200 , while in the present study n = 185 .
a larger sample would significantly increase the statistical power of the tests . a second limitation concerns the use of self - reported data , which implies a certain risk that the findings are based on common - method variance .
moreover , the fact that the researchers visited the participants to help fill in the questionnaires might have introduced some bias into the respondents ' reporting .
therefore , frail older nh patients might become exhausted when completing the questionnaires , which may cause a possible bias . to avoid such a bias ,
experienced researchers were carefully picked and trained in conducting the interviews following a standardised procedure , including small breaks on specific points during the interview process .
this procedure worked out very well ; in just three cases the interviews had to be completed the next day due to respondent 's fatigue .
the nfi and gfi were lower than the recommended value of 0.90 for both indices , indicating uncertainty regarding the associations in model 1 .
however , the nfi and gfi are sensitive to small samples , underestimating fit for samples less than 200 ; therefore they should not be relied on solely . nevertheless , the cfi is a revised form of the nfi that considers the sample size and performs well even when sample size is small .
in addition , agfi is an adjusted form of gfi that accounts for the degrees of freedom and was inside the recommended cut - off value .
according to the european commission 's green paper on mental health , depression is one of the most prevalent mental health problems facing european citizens today . taking into account the highly chronic nature of these psychological states , we consider our findings noteworthy in their suggestion that self - transcendence might impair the level of depression .
knowledge of how self - transcendence and depression relate to each other in this respect is important for researchers , nurses , and clinicians .
accordingly , facilitating nursing intervention to provide patients ' self - transcendence would promote integrity and well - being and prevent despair and depression .
the results suggest that the intrapersonal self - transcendence affects depression stronger than the interpersonal self - transcendence among cognitively intact nh patients .
this might indicate that facilitating self - acceptance and helping patients adjust well are crucial to decreasing depression among nh patients .
furthermore , connecting with others , sharing wisdom , and helping others when possible appear as vital to preventing depression and increasing well - being in cognitively intact nh patients .
due to a combination of factors such as patients ' communication impairment , clinicians ' focus on treating medical conditions , normalization of depression in later life , and a lack of training in mental health among staff in nhs , depression can easily go undetected among the nh population [ 75 , 76 ] .
therefore , self - transcendence might serve as a framework for staff nurses ' awareness , assessing patients ' mood , and connectedness resources .
staff nurses are increasingly aware that good nursing care consists of more than the competent performance of a number of nursing activities . however , for many nurses
, it is much less clear what this more means and what importance it has in nursing .
nurses may encourage self - transcendence by facilitating patients ' connections with others , stimulating inner reflections and connection to their inner thoughts and emotions , and by promoting spiritual faith , facilitating hobbies , helping others , and sharing wisdom .
enhancing acceptance of the self , death , and one 's life situation might prevent and decrease depression among nh patients . as connectedness is described as the core of self - transcendence , offering connectedness might be a central aspect of nh care .
the interpersonal relationship in patient - nurse interactions has been found to be an essential factor of quality of care , as perceived by long - term care patients [ 71 , 77 , 78 ] .
nurse - patient interaction can help nh patients preserve their dignity , identity , and integrity . by means of listening to the patients , communicating , and treating the patients with respect , by using empathic understanding , and acknowledging him / her as a person who is to be taken seriously and attended to , staff nurses might promote self - transcendence and therefore personal maturity , decreased depression , and enhanced well - being [ 71 , 8084 ] . nursing research and education should pay more attention to interventions promoting self - transcendence and well - being in order to develop a more comprehensive and practice - based view of good nursing care that inspires nh staff nurses as they perform their daily care practices . more research of the effectiveness of such strategies is highly needed .
educational nursing curricula should underline and facilitate nurse - patient interaction and interventions to expand nh patients ' personal self - boundaries . advancing the staff nurses ' presence with the patient
the present study suggests that self - transcendence negatively and significantly affects depression in cognitively intact nh patients .
consequently , nursing interventions and in - house activities aiming to increase patients ' self - transcendence might have a great impact on nh patients ' depression and their well - being . |
aims . this study 's aim was to test the effects of self - transcendence on depression among cognitively intact nursing home patients .
background .
depression is considered the most frequent mental disorder among the elderly population . specifically , the depression rate among nursing home patients is three to four times higher than that among community - dwelling elderly .
therefore , finding new and alternative ways to prevent and decrease depression is of great importance for nursing home patients ' well - being .
self - transcendence is related to spiritual as well as nonspiritual factors , and it is described as a correlate and resource for well - being among vulnerable populations and at the end of life . methods .
a two - factor construct of the self - transcendence scale ( interpersonal and intrapersonal ) and the hospital anxiety and depression scale ( hads ) was applied . a sample of 202 cognitively intact nursing home patients in central norway was selected to respond to the questionnaires in 2008/2009 .
results .
a hypothesized sem model demonstrated significant direct relationships and total effects of self - transcendence on depression .
conclusion and implication for practice . facilitating
patients ' self - transcendence , both interpersonally and intrapersonally , might decrease depression among cognitively intact nursing home patients . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Limitations
6. Relevance to Clinical Nursing
7. Conclusion | this huge shift to an older population globally has given rise to the notions of the third and fourth ages in the lifespan development literature . this differentiation of the last part of the lifespan into two separate phases is important because of the characteristic patterns of gains ( growth ) and losses ( decline ) seen in the young old and the old old . for
many of those in the fourth age ( 80 + ) issues such as physical illness and approaching mortality decimate their functioning and subsequently lead to the need for nursing home ( nh ) care . depression is one of the most prevalent mentally ill health problems facing european citizens today , and it is considered one of the most frequent mental disorders in the elderly population . specifically , rates of depression in nhs are three to four times higher than those in community - dwelling older adults . depression in nh patients is associated with negative outcomes such as impaired quality of life , impaired activities of daily living , substantial caregiver burden and worsened medical outcomes , increased risk of hospital admission , and a higher mortality rate . the nh patients ' sense of loss of independence and privacy , feelings of isolation and loneliness , the ever present death and grief , and lack of meaningful in - house activities are identified as risk factors for depression . accordingly , efforts to prevent and decrease depression are of great importance for nh patients ' well - being and quality of life . therefore , finding new and alternative approaches to weaken this detrimental development is highly warranted . self - transcendence is found to be such a correlate and a resource for well - being among vulnerable populations and at the end of life [ 2931 ] . hence , self - transcendence enhances an individual 's searching for new perspectives , meaning , and well - being and allows him or her to overcome ego concerns . the idea of self - transcendence is inspired by human developmental theory emphasizing maturity as the developmental task in later life . in accordance with this theory
, the developmental crisis in old age entails integrity versus despair and is resolved by the development of inner resources as personal maturity and wisdom , qualities providing well - being . the developmental process of self - transcendence is stimulated by the challenges of age and coming to terms with death . adults with higher levels of self - transcendence do not seek absolute answers to questions in life but seek meaning in life events as integrated within a moral , social , and historical context . the central core of self - transcendence is described as connection between the individual , environment , and a transcendent being . self - transcendence refers to a multidimensional expansion of the self - boundaries intrapersonally ( through self - acceptance and finding meaning in life ) , interpersonally ( by reaching out to others or connecting with nature ) , transpersonally ( by reaching out to a higher entity , being of purpose ) , and temporality ( by integrating one 's past and future into the present ; a dynamic process involving adaption to physical , emotional , and/or spiritual distress ) . within a holistic framework of the body , mind , and spirit as a whole
, self - transcendence has been considered a central aspect of human spirituality . as a general human dimension of personal maturity , self - transcendence has been related to spiritual and nonspiritual factors . human spirituality is expressed and experienced in the context of caring connections with the self , others , nature , and a life force or god [ 3539 ] . accordingly , spirituality seems closely related to connectedness , the essence of self - transcendence . a recent study of the relationships between spiritual well - being and self - transcendence revealed that both interpersonal and intrapersonal self - transcendence significantly affect spiritual well - being in cognitively intact nh patients . self - transcendence has been studied in various disciplines , but it is of particular interest to nursing . the perspective of promoting health and well - being is fundamental and a major nursing concern in long - term care [ 4143 ]
. previous research demonstrates the significance of self - transcendence to older people well - being [ 31 , 45 , 50 , 51 ] . empirical support for the importance of self - transcendence for those at the end of life and facing their own mortality has been well documented [ 33 , 47 , 5255 ] . in summary ,
the literature suggests depression as a common mental disorder among older people characterized by high age , impairment , and somatic symptoms . self - transcendence is a resource for well - being among vulnerable individuals and at the end of life . a previous investigation of the dimensionality of the self - transcendence scale revealed that self - transcendence comprises one interpersonal and one intrapersonal factor . therefore , this study investigates the associations between interpersonal and intrapersonal self - transcendence and depression among cognitively intact nh patients . to the authors ' knowledge
, previous research has not examined the relationships between interpersonal and intrapersonal self - transcendence and depression in nhs by means of structural equation modelling ( sem ) . our research question was does self - transcendence affect depression in cognitively intact nh patients ? based on the theoretical and empirical knowledge of depression and self - transcendence , the following hypotheses were formulated . hypothesis 1intrapersonal self - transcendence has negative effect on depression among cognitively intact nh patients . intrapersonal self - transcendence has negative effect on depression among cognitively intact nh patients . hypothesis 2interpersonal self - transcendence has negative effect on depression among cognitively intact nh patients . researchers with identical professional background were chosen ( rn , ma , trained and experienced in communication with elderly , as well as teaching gerontology at an advanced level ) and trained to conduct the interviews as similarly as possible . 16443 ) , and likewise we attained approval from the regional committee for medical and health research ethics in central norway ( ref . missing data was low in frequency : depression 5.0% and self - transcendence scale 5.9% and were handled by means of the listwise procedure . depression was assessed by the depression subscale of the hospital anxiety and depression scale ( hads ) , comprising seven items . each item is scored from 03 , and the maximum score is 21 on the subscale . since the present study focuses on the associations between self - transcendence and depression , only the depression - items ( subscale hads - d ) were included in the sem model to be tested here . the internal consistence of the construct ( table 1 ) was satisfactory ( = .68 ) . interpersonal and intrapersonal self - transcendence were assessed by items from the self - transcendence scale ( sts ) . the sts was developed from the 36-item developmental resources of later adulthood scale [ 33 , 59 ] , which intended to identify intrapersonal , interpersonal , transpersonal , and temporal experiences characteristic of later life , reflecting expanded boundaries of self . support for construct validity has been found in the relationships of self - transcendence scores to other measures [ 29 , 60 , 61 ] . the two - factor construct of self - transcendence was used , but the number of items was reduced ; the item letting go of my past losses ( st15 ) , which is reversed scored , loaded extremely low ( = 0.11 ) and demonstrated r = 0.02 . the items having hobbies and interests i can enjoy , being involved with other people ,
sharing my wisdom or experience with others , helping others in some way , having an ongoing interest in learning , able to move beyond things that once seemed so important , and finding meaning in my spiritual beliefs were indicators for interpersonal self - transcendence , while the items accepting myself as growing older , adjusting well to my present life situation , adjusting well to changes in my physical abilities , finding meaning in my past experiences , accepting death as a part of life , letting others help me when i may need it , and enjoying my pace of life constituted the intrapersonal st construct . cronbach 's in current study was 0.72 ( all 15 items ) , while for interpersonal self - transcendence ( seven items ) was .76 and .65 for intrapersonal self - transcendence ( seven items ) ( table 1 ) . a structural equation model ( sem ) of the hypothesized relations between the latent constructs of depression and self - transcendence was tested by means of lisrel 8.8 . using sem accounts for random measurement error , and the psychometric properties of the scales in the model are more accurately derived . since the standard errors are estimated under nonnormality , the satorra - bentler - scaled chi - square statistic was applied as a goodness - of - fit statistic , which is the correct asymptotic mean even under nonnormality . also , we used the root mean square error of approximation ( rmsea ) and the standardized root mean square residual ( srms ) with values below 0.05 indicating good fit , while values smaller than 0.08 are interpreted as acceptable [ 66 , 67 ] . the comparative fit index ( cfi ) and the nonnormed fit index ( nnfi ) with an acceptable fit at 0.95 and good fit at 0.97 and above were used , and the normed fit index ( nfi ) and the goodness - of - fit index ( gfi ) with an acceptable fit at 0.90 , while a good fit was set to 0.95 . before examining the hypothesized relationships in the sem analysis
the cfa provided a good fit to the observed data for the interpersonal self - transcendence ( = 22.28 , p < 0.073 , rmsea = 0.055 , srmr = 0.047 , nfi = 0.95 , nnfi / cfi = 0.97/0.98 , gfi / agfi = 0.96/0.92 ) , intrapersonal self - transcendence ( = 15.77 , p < 0.0033 , rmsea = 0.026 , srmr = 0.056 , nfi = 0.93 , nnfi / cfi = 0.99/0.99 , gfi / agfi = 0.96/0.92 ) , and the depression scale ( = 9.43 , p < 0.46 , rmsea = 0.016 , srmr = 0.039 , nfi = 0.96 , nnfi / cfi = 1.00/1.00 , gfi / agfi = 0.98/0.95 ) . table 1 displays the means ( m ) , standard deviations ( sd ) , cronbach 's alpha , and correlations matrix for the constructs of self - transcendence ( interpersonal and intrapersonal ) and depression . in order to investigate how interpersonal self - transcendence ( st-1 ) and intrapersonal self - transcendence ( st-2 ) relate to depression ,
model 1 comprising six depression , seven interpersonal self - transcendence and seven intrapersonal self - transcendence items was estimated . all estimates were significant ( p < 0.05 ) , and the factor loadings ranged 0.220.72 and r - values 0.050.59 . table 3 shows the standardized regression coefficients of the directional relationships and the total effects between the latent constructs in model 1 . as hypothesized , both directional paths were significant displaying an inverse relationship , from interpersonal self - transcendence to depression ( 1,1 = 0.30 * ) and from intrapersonal self - transcendence to depression ( 1,2 = 0.41 * ) ( table 3 ) . a scrutiny of the total effects of interpersonal self - transcendence and intrapersonal self - transcendence on depression revealed statistical significant total effects from both interpersonal and intrapersonal self - transcendence on all the depression items ( table 3 ) . moreover , a huge shift to an older population worldwide is highlighted , and the most rapidly growing segment is that of people over 80 years old . simultaneously , depression is one of the most frequent mental disorders in the elderly population and specifically among nh patients . therefore , finding new interventions to increase well - being and decrease depression among nh patients is highly warranted . the aim of this study was to explore the association between interpersonal and intrapersonal self - transcendence and depression in cognitively intact nursing home patients . by doing
so we sought to contribute to a holistic nursing perspective of promoting well - being in nh patients in two ways : first , this study supplies empirical knowledge to the growing body of self - transcendence knowledge by exploring self - transcendence among nh patients ; second , by applying a two - factor construct of self - transcendence that has been shown to be psychometrically superior the one - factor construct , the present study allows a more complex examination of the associations between self - transcendence and depression . the present study examines the relationships and the influences of interpersonal and intrapersonal self - transcendence on depression in nh patients . by means of advanced statistical analysis such as structural equation modeling ( sem )
, this study provides more specific guidelines to nursing interventions promoting well - being in nh patients and suggests that finding ways to enhance an individual 's intrapersonal and interpersonal self - transcendence might be beneficial in this matter . more specifically , we found that the hypothesized relationship between self - transcendence ( intrapersonal and interpersonal ) and depression was fully supported . it is reasonable that interpersonal self - transcendence including personal interests , learning , involvement with others , connectedness , sharing one 's wisdom , and helping others is associated with low depression . therefore , nursing interventions facilitating nh patients ' involvement and connectedness , sharing wisdom , and possibilities helping others might decrease and prevent depression . also , the intrapersonal self - transcendence comprising nh patients ' self - acceptance as growing older , adjusting well to the changes in physical abilities and the present situation , and finding meaning in one 's past experiences related negatively to depression . the statistical tests suggested that the intrapersonal self - transcendence affects depression more than the interpersonal self - transcendence among cognitively intact nh patients . therefore , staff nurses ' awareness of nh patients ' self - acceptance and adjustment to the life situation is essential for well - being as well as enjoyment , humor , and laughter . at 86 years old ( mean age in the present study ) , representing the old old
living in a nh represents a life perspective where openness to keep learning , enjoying hobbies and interests , and involving with others might seem less important than accepting oneself and adjusting well to the here - and - now in the nh environment . loss of functions , relationships , privacy , self - determination , and connectedness and the forthcoming death , together with searching for meaning in the in - house institutionalized nh life , seem in general as the main challenges . from this particular point of view
therefore , these individuals need initiative and vitalization from others and the environment . elderly who lack social / emotional support
consequently , the nurse - patient relationships might be an overriding figure in the nh patients ' daily life and might serve as a vital resource of human involvement , interaction , connectedness , and a facilitator of meaningful in - house activities for nh patients . few social relations for dialogue , self - reflection , and connectedness are left ; in our sample only 19% had a partner . the potentials for self - transcendence and well - being are important considerations in nh care ; the participants had stayed in nh for 6 months or longer . because the length of stay is long , much time is available to enter into meaningful relationships and communication with patients , pursuing appropriate interventions to promote self - transcendence . within erikson 's model of human psychosocial development ,
therefore , self - transcendence as an individual developmental process toward personal maturity and a resource for well - being might serve as an indicator of successful aging among nh patients . some of the intrapersonal self - transcendence indicators loaded low and displayed low r - values . these items involve finding meaning in past experiences , accepting death as a part of life , letting others help , and enjoying one 's pace of life . in this regard ,
nh patients might find that they do not have their own pace of life , and enjoying one 's pace of life seems less relevant . this study expands previous studies by testing the associations between the two - factor construct of self - transcendence and depression among nh patients by using structural equation modeling . using sem accounts for random measurement error and the psychometric properties of the scales in the model
nevertheless , the findings of this study must be discussed with some limitations in mind . moreover , the fact that the researchers visited the participants to help fill in the questionnaires might have introduced some bias into the respondents ' reporting . nevertheless , the cfi is a revised form of the nfi that considers the sample size and performs well even when sample size is small . according to the european commission 's green paper on mental health , depression is one of the most prevalent mental health problems facing european citizens today . knowledge of how self - transcendence and depression relate to each other in this respect is important for researchers , nurses , and clinicians . accordingly , facilitating nursing intervention to provide patients ' self - transcendence would promote integrity and well - being and prevent despair and depression . the results suggest that the intrapersonal self - transcendence affects depression stronger than the interpersonal self - transcendence among cognitively intact nh patients . this might indicate that facilitating self - acceptance and helping patients adjust well are crucial to decreasing depression among nh patients . furthermore , connecting with others , sharing wisdom , and helping others when possible appear as vital to preventing depression and increasing well - being in cognitively intact nh patients . due to a combination of factors such as patients ' communication impairment , clinicians ' focus on treating medical conditions , normalization of depression in later life , and a lack of training in mental health among staff in nhs , depression can easily go undetected among the nh population [ 75 , 76 ] . therefore , self - transcendence might serve as a framework for staff nurses ' awareness , assessing patients ' mood , and connectedness resources . nurses may encourage self - transcendence by facilitating patients ' connections with others , stimulating inner reflections and connection to their inner thoughts and emotions , and by promoting spiritual faith , facilitating hobbies , helping others , and sharing wisdom . enhancing acceptance of the self , death , and one 's life situation might prevent and decrease depression among nh patients . as connectedness is described as the core of self - transcendence , offering connectedness might be a central aspect of nh care . by means of listening to the patients , communicating , and treating the patients with respect , by using empathic understanding , and acknowledging him / her as a person who is to be taken seriously and attended to , staff nurses might promote self - transcendence and therefore personal maturity , decreased depression , and enhanced well - being [ 71 , 8084 ] . nursing research and education should pay more attention to interventions promoting self - transcendence and well - being in order to develop a more comprehensive and practice - based view of good nursing care that inspires nh staff nurses as they perform their daily care practices . educational nursing curricula should underline and facilitate nurse - patient interaction and interventions to expand nh patients ' personal self - boundaries . advancing the staff nurses ' presence with the patient
the present study suggests that self - transcendence negatively and significantly affects depression in cognitively intact nh patients . consequently , nursing interventions and in - house activities aiming to increase patients ' self - transcendence might have a great impact on nh patients ' depression and their well - being . | [
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numerous beneficial effects of an adequate vitamin d status on a wide range of clinical conditions have been proposed particularly since the turn of the 21st century .
these claims have generated an unprecedented level of interest in the medical media and lay press alike translating into great interest in assessing vitamin d status particularly amongst patients presenting to family or general practitioners .
this growth in requests for serum 25-hydroxyvitamin d levels and the consequent issues for the clinical laboratory have recently been well addressed in this journal ( 1 ) . a major factor generating this marked interest in vitamin d
has been the reports of simple associations between a particular disease state or condition and low vitamin d status .
it is clearly apparent that an improved vitamin d status can be associated with many attributes of good health relating to mobility and activities promoting sunlight exposure with out the involvement of a direct biological action of vitamin d. however during the 21st century there has been a flowering of knowledge of metabolism of vitamin d within a range of tissues including synthesis of the active metabolite 1,25-dihydroxyvitamin d ( 1,25d ) and activation of the vitamin d receptor ( vdr ) within the tissue of synthesis .
such a mechanism now provides plausible physiological and molecular mechanisms for a diverse array of activities and tissue responses as has been reviewed recently ( 2 ) . in this article
i will review the current knowledge for the action of vitamin d on plasma calcium and phosphate homeostasis and bone mineral homeostasis with particular focus on the knowledge gaps .
data published in the 21st century are challenging the concept of a single paradigm for the actions of vitamin d. an alternate paradigm involving local bone tissue metabolism of vitamin d with actions different from those observed with the endocrine source of 1,25d provides a plausible mechanism for activities observed in population studies and from randomised controlled trials of vitamin d supplementation to reduce the risk of fracture in the elderly .
the well characterised endocrine pathway of vitamin d metabolism and action is summarised in figure 1 and is considered to be solely responsible for the contribution of vitamin d to regulate plasma calcium and phosphate .
new findings of the interaction between fgf23 and 1,25d and plasma phosphate levels are coming to light providing further insight in this area ( 3 ) .
the identification of the endocrine pathway for activation of vitamin d in the 1970 s was a major achievement .
the biologically active metabolite , 1,25d in plasma arises from sequential hydroxylations of vitamin d , firstly by the liver to form 25d and then by the kidney to form 1,25d ( 4 ) .
the catabolism of vitamin d metabolites through the action of the 25-hydroxyvitamin-24-hydroxylase ( cyp24 ) has also been demonstrated to be an essential regulatory pathway for vitamin d homeostasis . under conditions
when this enzyme activity is reduced or ablated the serum half - lives of both 25d and 1,25d are increased and contribute to the development of hypercalcaemia , particularly following vitamin d supplementation ( 5 ) . this mechanism has recently been identified to account for idiopathic infantile hypercalcemia and other toxic effects of vitamin d ( 6 ) the binding of 1,25d to its highly specific nuclear receptor , vdr , modulates transcriptional activity of vitamin d responsive genes through binding to vitamin d response elements ( vdre s ) located within the promoter regions of these genes .
most organs and tissues in the body express the vdr with one study demonstrating that the vdr occupied some 2776 genomic positions modulating the expression of at least 229 genes ( 7 ) .
another study indicated that approximately 913 genes were responsive to vdr in a squamous cell cancer cell line with 80% of genes being up - regulated and that these effects take some 6 to 12 hours to reach measurable levels ( 8) .
1,25d also initiates rapid responses resulting from activation of mitogen activated protein ( map ) kinases and other intracellular signalling pathways ( 9 ) .
this latter activity demonstrates the rapid ( within minutes ) activation of various pathways for 1,25d and data are also published that implicate a membrane - specific receptor for 1,25d contributing to these rapidly acting activities .
these pathways are considered to operate whether 1,25d arises from the plasma as a result of synthesis by the kidney and acting as an endocrine agent or whether it arises from endogenous synthesis by extra - renal tissues and acts as an autocrine or paracrine agent . the endocrine mechanism for plasma 1,25d
was demonstrated to contribute to maintaining calcium homeostasis in the 1960 s ( 10 ) and continues to dominate thinking in the field today .
the current controversial issue in this area is what is the critical level for serum 25d to maintain plasma calcium homeostasis ?
an extensive hormonal interaction operates to maintain calcium homeostasis regulated by plasma ionised calcium level acting through the calcium - sensing receptor .
the action of this receptor directly modulates the levels of parathyroid hormone ( pth ) and calcitonin which in turn act on the kidney , bone and gut ( 4 ) .
low calcium increases pth secretion and synthesis whereas high calcium increases calcitonin secretion and synthesis .
pth acts rapidly on the kidney to stimulate renal calcium reabsorption from the glomerular filtrate and in concert with 1,25d it stimulates bone resorption to enhance the flow of calcium from bone into plasma .
1,25d in association with pth increases the number of osteoclasts by way of an indirect mechanism through increased expression of the osteoblast - derived cytokine , rankl , which promotes osteoclastic differentiation ( 11 ) .
expression of the vdr gene is required for 1,25d to stimulate the expression of rankl in osteoblasts as those derived from vdr knockout mice are unable to stimulate the differentiation of osteoclasts ( 12 ) .
alternatively it is important to note that in vitamin d deficiency or when vitamin d activity is ablated through gene mutations , hypocalcaemia develops despite a high pth in humans or rodents ( 13 , 14 ) .
these findings suggest that pth requires 1,25d in order to increase osteoclast number and stimulate bone resorption to normalise extracellular fluid ( ecf ) calcium .
each of these activities operates to restore the low plasma ionised calcium to its homeostatic level .
pth also acts on the kidney to stimulate transcription of the gene coding for the enzyme which converts 25d to 1,25d , 25-hydroxyvitamin d-1-hydroxylase ( cyp27b1 ) increasing the levels in frank hypocalcaemia with secondary hyperparathyroidism by some 100 fold ( 15 ) .
it is this mechanism that can maintain serum 1,25d in the normal range or at adequate levels to maintain intestinal calcium absorption in the face of significant falls in serum 25d .
thus evidence from postmenopausal women with various levels of serum 25d considered to be in the depleted range ( ie 40
nmol / l or lower ) indicate that blood ionised calcium , serum 1,25d levels and intestinal radiocalcium absorption do not fall significantly until serum 25d is below 20 nmol / l ( 16 ) .
these data suggest that the critical level of serum 25d to maintain the endocrine actions of 1,25d to maintain plasma calcium homeostasis is 20 nmol / l or greater .
the level of bone mineral at any one time is the result of at least two opposing cellular actions , bone formation by osteoblasts and bone resorption by osteoclasts .
rickets in children ( or osteomalacia in adults when bone growth has ceased ) is the index disease for vitamin d deficiency .
strong data have been generated from rodent models and human disease to indicate that dietary deficiency or genetic ablation of vitamin d activity results in hypocalcaemia , hypophosphataemia , hyperparathyroidism and rickets ( 17 ) .
correction of the hypocalcaemia , hypophosphataemia and secondary hyperparathyroidism corrects the rickets . such a result
therefore it has been concluded that the action of vitamin d on bone mineral can be completely accounted for by its actions to maintain plasma calcium and phosphate homeostasis with no requirement for direct actions of vitamin d on bone cells to maintain or increase bone mineral status .
the calcaemic action of 1,25d particularly in conjunction with pth to stimulate osteoclastogenesis and bone resorption as discussed above is a direct endocrine action of 1,25d on bone cells to reduce bone mineral in order to maintain plasma calcium levels .
in contrast to these findings a large body of data have been and continue to be reported on the expression of vdr by each of the major types bone cells and on effects of 1,25d in vitro to inhibit proliferation of osteoblast - like cells and to stimulate osteoblast maturation and mineralisation ( 18 ) .
osteoblasts as well as osteocytes and osteoclasts express the vdr and the cyp27b1 and cyp24 enzyme genes as well as a number of vitamin d responsive genes associated with mineralisation in osteoblasts including type i collagen , alkaline phosphatase and osteocalcin and other genes necessary for osteoblast and osteocyte maturation amongst a much larger number of proteins .
furthermore there is evidence including clinical and rodent model studies suggesting that vitamin d is likely to exert activities directly on bone cells to improve or maintain bone mineral status ( 19 ) .
clinical studies since the early 1980 s have reported that an increased risk of hip fracture amongst the elderly occurs at mean levels of serum 25d of some 40 nmol / l ( 20 ) , a level now considered as adequate for maintaining plasma calcium homeostasis .
furthermore population studies from the us have demonstrated that bone mineral density increases with increasing serum 25d levels reaching a plateau at serum 25d levels of approximately 75 nmol / l ( 21 ) .
data from randomised clinical trials of vitamin d supplementation and fracture incidence indicate that anti - fracture efficacy at the hip or other non - vertebral sites is not achieved until serum 25d levels of 75 nmol / l are achieved ( 22 ) .
interesting confirmatory data for a direct anabolic action of vitamin d on bone have arisen from animal model studies .
mouse models in which vitamin d activity has been ablated either by knocking out the gene for the vdr or the cyp27b1 enzyme , as stated previously develop rickets when fed a normal calcium diet . when the dietary calcium and the phosphate levels are markedly increased sufficient to normalise plasma calcium and phosphate levels , the rickets phenotype is rescued and as mentioned above , many researchers have described the bone as
one research group has extended the feeding of the rescue diet to these genetically modified mice for a longer period ( 16 weeks instead of 10 weeks ) ( 23 ) . on careful examination of their skeleton
however these mice had lower levels of normally mineralised bone than wild type mice ; that is they had osteoporosis . as well the gene knockout mice had fewer osteogenic bone marrow cells although there was no difference in the number of osteoclasts in the bone of wild type and gene knockout mice .
thus these data support the concept that vitamin d activity is necessary for normal bone mineral levels at least in adult mice due to the production of an optimal number of osteoblast forming cells and osteoblast activity .
further independent data from other mouse models support this concept that vitamin d activity within mature osteoblast cells exerts an anabolic action on bone tissue .
the osvdr transgenic mouse line was prepared to over express the human gene for vdr only in mature osteoblasts and some osteocytes by utilising the promoter region of the human osteocalcin gene to regulate vdr transgene expression ( 24 ) .
adult osvdr mice demonstrate a strong bone phenotype compared with wild type mice with an increase of both cortical and trabecular bone volumes of some 15% at a number of sites in the skeleton .
the increased bone volume is a result of both an increase in bone formation and decreased bone resorption ( 24 ) . a similar but distinct transgenic mouse line ( osc mice )
has recently been prepared in which the human cyp27b1 gene is expressed under the control of the human osteocalcin promoter .
these osc mice demonstrate increased synthesis of 1,25d from 25d only in mature osteoblasts and some osteocytes .
these mice demonstrate increased trabecular bone volume in females at 20 weeks of age ( 25 ) .
these latest results have only been published as abstracts and are only preliminary at this time .
another line of inquiry has utilised dietary studies in rodent models to demonstrate that at levels of serum 25d between 20 and 80 nmol / l , trabecular bone volume is reduced over 3 months as a result of increased bone resorption from increased osteoclastogenesis due to increased bone expression of the key osteoclastogenic cytokine , rankl ( figure 2 ) , ( 26 ) . at 25d levels below 20 nmol / l and dietary calcium of 4% , osteomalacia is observed . at values above 20 nmol / l the mineralisation lag time is normal , which therefore excludes osteomalacia . in these animals it was only the serum 25d level and
not serum 1,25d or pth which significantly correlated with trabecular bone volume , osteoclast surface or bone rankl mrna levels .
all of these data are consistent with a model that local metabolism of vitamin d by bone cells to form 1,25d and its local action within bone cells can increase bone mineral status ( 27 ) .
, pth does not increase the synthesis of 1,25d in bone ( 28 ) and there is direct evidence that pth treatment of bone cells does not activate the promoter region of the human gene for cyp27b1(29 ) .
in fact high pth levels are associated with decreased synthesis expression of the cyp27b1 gene in bone tissue ( 28 ) .
perhaps the most important finding in this area is that increased dietary calcium intake increases the expression of the cyp27b1 gene in bone by three - fold and reduces the expression of this gene in the kidney ( 30 ) .
certainly rodent studies have found that optimal bone status is dependent on adequate vitamin d and dietary calcium status rather than either one alone ( 21 ) .
when rats are fed either an adequate vitamin d level but low dietary calcium or vice versa they develop osteoporosis and when fed both a low vitamin d and calcium diet they develop osteomalacia ( figure 3 ) .
the well characterised endocrine pathway of vitamin d metabolism and action is summarised in figure 1 and is considered to be solely responsible for the contribution of vitamin d to regulate plasma calcium and phosphate .
new findings of the interaction between fgf23 and 1,25d and plasma phosphate levels are coming to light providing further insight in this area ( 3 ) .
the identification of the endocrine pathway for activation of vitamin d in the 1970 s was a major achievement .
the biologically active metabolite , 1,25d in plasma arises from sequential hydroxylations of vitamin d , firstly by the liver to form 25d and then by the kidney to form 1,25d ( 4 ) .
the catabolism of vitamin d metabolites through the action of the 25-hydroxyvitamin-24-hydroxylase ( cyp24 ) has also been demonstrated to be an essential regulatory pathway for vitamin d homeostasis . under conditions
when this enzyme activity is reduced or ablated the serum half - lives of both 25d and 1,25d are increased and contribute to the development of hypercalcaemia , particularly following vitamin d supplementation ( 5 ) . this mechanism has recently been identified to account for idiopathic infantile hypercalcemia and other toxic effects of vitamin d ( 6 ) the binding of 1,25d to its highly specific nuclear receptor , vdr , modulates transcriptional activity of vitamin d responsive genes through binding to vitamin d response elements ( vdre s ) located within the promoter regions of these genes .
most organs and tissues in the body express the vdr with one study demonstrating that the vdr occupied some 2776 genomic positions modulating the expression of at least 229 genes ( 7 ) .
another study indicated that approximately 913 genes were responsive to vdr in a squamous cell cancer cell line with 80% of genes being up - regulated and that these effects take some 6 to 12 hours to reach measurable levels ( 8) .
1,25d also initiates rapid responses resulting from activation of mitogen activated protein ( map ) kinases and other intracellular signalling pathways ( 9 ) .
this latter activity demonstrates the rapid ( within minutes ) activation of various pathways for 1,25d and data are also published that implicate a membrane - specific receptor for 1,25d contributing to these rapidly acting activities .
these pathways are considered to operate whether 1,25d arises from the plasma as a result of synthesis by the kidney and acting as an endocrine agent or whether it arises from endogenous synthesis by extra - renal tissues and acts as an autocrine or paracrine agent . the endocrine mechanism for plasma 1,25d
was demonstrated to contribute to maintaining calcium homeostasis in the 1960 s ( 10 ) and continues to dominate thinking in the field today .
the current controversial issue in this area is what is the critical level for serum 25d to maintain plasma calcium homeostasis ?
an extensive hormonal interaction operates to maintain calcium homeostasis regulated by plasma ionised calcium level acting through the calcium - sensing receptor .
the action of this receptor directly modulates the levels of parathyroid hormone ( pth ) and calcitonin which in turn act on the kidney , bone and gut ( 4 ) .
low calcium increases pth secretion and synthesis whereas high calcium increases calcitonin secretion and synthesis .
pth acts rapidly on the kidney to stimulate renal calcium reabsorption from the glomerular filtrate and in concert with 1,25d it stimulates bone resorption to enhance the flow of calcium from bone into plasma .
1,25d in association with pth increases the number of osteoclasts by way of an indirect mechanism through increased expression of the osteoblast - derived cytokine , rankl , which promotes osteoclastic differentiation ( 11 ) .
expression of the vdr gene is required for 1,25d to stimulate the expression of rankl in osteoblasts as those derived from vdr knockout mice are unable to stimulate the differentiation of osteoclasts ( 12 ) .
alternatively it is important to note that in vitamin d deficiency or when vitamin d activity is ablated through gene mutations , hypocalcaemia develops despite a high pth in humans or rodents ( 13 , 14 ) .
these findings suggest that pth requires 1,25d in order to increase osteoclast number and stimulate bone resorption to normalise extracellular fluid ( ecf ) calcium .
each of these activities operates to restore the low plasma ionised calcium to its homeostatic level .
pth also acts on the kidney to stimulate transcription of the gene coding for the enzyme which converts 25d to 1,25d , 25-hydroxyvitamin d-1-hydroxylase ( cyp27b1 ) increasing the levels in frank hypocalcaemia with secondary hyperparathyroidism by some 100 fold ( 15 ) .
it is this mechanism that can maintain serum 1,25d in the normal range or at adequate levels to maintain intestinal calcium absorption in the face of significant falls in serum 25d .
thus evidence from postmenopausal women with various levels of serum 25d considered to be in the depleted range ( ie 40 nmol / l or lower ) indicate that blood ionised calcium , serum 1,25d levels and intestinal radiocalcium absorption do not fall significantly until serum 25d is below 20 nmol / l ( 16 ) .
these data suggest that the critical level of serum 25d to maintain the endocrine actions of 1,25d to maintain plasma calcium homeostasis is 20 nmol / l or greater .
the level of bone mineral at any one time is the result of at least two opposing cellular actions , bone formation by osteoblasts and bone resorption by osteoclasts .
rickets in children ( or osteomalacia in adults when bone growth has ceased ) is the index disease for vitamin d deficiency .
strong data have been generated from rodent models and human disease to indicate that dietary deficiency or genetic ablation of vitamin d activity results in hypocalcaemia , hypophosphataemia , hyperparathyroidism and rickets ( 17 ) .
correction of the hypocalcaemia , hypophosphataemia and secondary hyperparathyroidism corrects the rickets . such a result
therefore it has been concluded that the action of vitamin d on bone mineral can be completely accounted for by its actions to maintain plasma calcium and phosphate homeostasis with no requirement for direct actions of vitamin d on bone cells to maintain or increase bone mineral status .
the calcaemic action of 1,25d particularly in conjunction with pth to stimulate osteoclastogenesis and bone resorption as discussed above is a direct endocrine action of 1,25d on bone cells to reduce bone mineral in order to maintain plasma calcium levels .
in contrast to these findings a large body of data have been and continue to be reported on the expression of vdr by each of the major types bone cells and on effects of 1,25d in vitro to inhibit proliferation of osteoblast - like cells and to stimulate osteoblast maturation and mineralisation ( 18 ) .
osteoblasts as well as osteocytes and osteoclasts express the vdr and the cyp27b1 and cyp24 enzyme genes as well as a number of vitamin d responsive genes associated with mineralisation in osteoblasts including type i collagen , alkaline phosphatase and osteocalcin and other genes necessary for osteoblast and osteocyte maturation amongst a much larger number of proteins .
furthermore there is evidence including clinical and rodent model studies suggesting that vitamin d is likely to exert activities directly on bone cells to improve or maintain bone mineral status ( 19 ) .
clinical studies since the early 1980 s have reported that an increased risk of hip fracture amongst the elderly occurs at mean levels of serum 25d of some 40 nmol / l ( 20 ) , a level now considered as adequate for maintaining plasma calcium homeostasis .
furthermore population studies from the us have demonstrated that bone mineral density increases with increasing serum 25d levels reaching a plateau at serum 25d levels of approximately 75 nmol / l ( 21 ) .
data from randomised clinical trials of vitamin d supplementation and fracture incidence indicate that anti - fracture efficacy at the hip or other non - vertebral sites is not achieved until serum 25d levels of 75
interesting confirmatory data for a direct anabolic action of vitamin d on bone have arisen from animal model studies .
mouse models in which vitamin d activity has been ablated either by knocking out the gene for the vdr or the cyp27b1 enzyme , as stated previously develop rickets when fed a normal calcium diet . when the dietary calcium and the phosphate levels are markedly increased sufficient to normalise plasma calcium and phosphate levels , the rickets phenotype is rescued and as mentioned above , many researchers have described the bone as
one research group has extended the feeding of the rescue diet to these genetically modified mice for a longer period ( 16 weeks instead of 10 weeks ) ( 23 ) .
however these mice had lower levels of normally mineralised bone than wild type mice ; that is they had osteoporosis .
as well the gene knockout mice had fewer osteogenic bone marrow cells although there was no difference in the number of osteoclasts in the bone of wild type and gene knockout mice .
thus these data support the concept that vitamin d activity is necessary for normal bone mineral levels at least in adult mice due to the production of an optimal number of osteoblast forming cells and osteoblast activity .
further independent data from other mouse models support this concept that vitamin d activity within mature osteoblast cells exerts an anabolic action on bone tissue .
the osvdr transgenic mouse line was prepared to over express the human gene for vdr only in mature osteoblasts and some osteocytes by utilising the promoter region of the human osteocalcin gene to regulate vdr transgene expression ( 24 ) .
adult osvdr mice demonstrate a strong bone phenotype compared with wild type mice with an increase of both cortical and trabecular bone volumes of some 15% at a number of sites in the skeleton . the increased bone volume is a result of both an increase in bone formation and decreased bone resorption ( 24 ) .
a similar but distinct transgenic mouse line ( osc mice ) has recently been prepared in which the human cyp27b1 gene is expressed under the control of the human osteocalcin promoter .
these osc mice demonstrate increased synthesis of 1,25d from 25d only in mature osteoblasts and some osteocytes .
these mice demonstrate increased trabecular bone volume in females at 20 weeks of age ( 25 ) .
these latest results have only been published as abstracts and are only preliminary at this time .
another line of inquiry has utilised dietary studies in rodent models to demonstrate that at levels of serum 25d between 20 and 80
nmol / l , trabecular bone volume is reduced over 3 months as a result of increased bone resorption from increased osteoclastogenesis due to increased bone expression of the key osteoclastogenic cytokine , rankl ( figure 2 ) , ( 26 ) . at 25d levels below 20 nmol / l and dietary calcium of 4% , osteomalacia is observed . at values above 20 nmol / l the mineralisation lag time is normal , which therefore excludes osteomalacia . in these animals it was only the serum 25d level and
not serum 1,25d or pth which significantly correlated with trabecular bone volume , osteoclast surface or bone rankl mrna levels .
all of these data are consistent with a model that local metabolism of vitamin d by bone cells to form 1,25d and its local action within bone cells can increase bone mineral status ( 27 ) .
, pth does not increase the synthesis of 1,25d in bone ( 28 ) and there is direct evidence that pth treatment of bone cells does not activate the promoter region of the human gene for cyp27b1(29 ) .
in fact high pth levels are associated with decreased synthesis expression of the cyp27b1 gene in bone tissue ( 28 ) .
perhaps the most important finding in this area is that increased dietary calcium intake increases the expression of the cyp27b1 gene in bone by three - fold and reduces the expression of this gene in the kidney ( 30 ) .
certainly rodent studies have found that optimal bone status is dependent on adequate vitamin d and dietary calcium status rather than either one alone ( 21 ) .
when rats are fed either an adequate vitamin d level but low dietary calcium or vice versa they develop osteoporosis and when fed both a low vitamin d and calcium diet they develop osteomalacia ( figure 3 ) .
the data reviewed here provide a plausible model for both catabolic and anabolic actions of vitamin d on bone depending on the dietary calcium intake . with either an inadequate dietary calcium intake or low vitamin d status with a marginal dietary calcium intake , increased activity of the plasma calcium and phosphate homeostatic mechanisms , including endocrine vitamin d activity , are required to maintain plasma calcium and phosphate levels within physiological levels .
this involves increased activity of pth when serum 25 d levels fall below 60 nmol / l ( 32 ) presumably arising from stimulation of the parathyroid gland calcium - sensing receptor as a result of a slight fall in plasma ionized calcium level .
however it is also possible that the increased pth secretion arises as a direct result of the fall in serum 25d ( 33 ) .
the increased pth acts to elevate the level of cyp27b1 enzyme in the kidney with the increased enzyme activity allowing continued renal synthesis of 1,25d and maintenance of serum 1,25d levels while serum 25d levels fall into the depleted levels ( eg levels below 60
the maintenance of serum 1,25d optimises intestinal calcium and phosphate absorption and the interaction between pth and serum 1,25d stimulates osteoclastogenesis and bone resorption increasing the flow of calcium and phosphate into the plasma compartment .
this mechanism is able to maintain plasma calcium and phosphate levels until serum 25d levels fall below 20 nmol / l at which time the substrate levels are too low to maintain serum 1,25d levels .
perhaps most importantly it is apparent that both pth and serum 1,25d are required for adequate bone resorption because the development of hypocalcaemia indicates that even when pth levels are very high there is inadequate flow of calcium from the bone compartment to maintain normocalcemia in the plasma compartment ( 15 ) . under these conditions
when dietary calcium intakes are adequate to meet all the demands of the calcium economy the data reviewed above suggest that in concert with an adequate vitamin d status ( eg , serum 25d levels greater than 75
nmol / l ) , increased levels of bone cyp27b1 enzyme activity ensure greater synthesis of 1,25d by bone cells associated with reduced circulating pth levels .
there are data to suggest that bone formation variables are also increased but it is unclear whether such changes are direct effects of 1,25d on osteoblasts or indirect effects of suppressed osteoclast activity ( 26 ) .
these latest findings provide further light on the delicate relationship between plasma calcium homeostasis , bone mineral homeostasis and vitamin d activities involving either endocrine or bone autocrine /paracrine activities .
these findings also provide a plausible mechanism for at least two critical levels for serum 25d , one for calcium homeostasis and a second for bone mineral homeostasis . to maintain plasma calcium homeostasis
, a serum 25d level of 20 nmol / l or greater appears to be sufficient to provide adequate substrate for the renal cyp27b1 enzyme level .
this lower level is adequate because the renal cyp27b1 enzyme level is increased by increasing levels of pth when serum 25d levels fall below 60 nmol / l ( 32 ) .
in contrast bone cyp27b1 enzyme levels are not increased by pth and therefore higher levels of serum 25d as substrate ( ie serum 25d 75 nmol / l or greater ) are required for bone cells to produce sufficient bone tissue 1,25d to suppress osteoclastogenesis .
furthermore adequate dietary calcium intake is also required to increase bone cyp27b1 enzyme levels even at these higher levels of serum 25d . | currently there is an unprecedented level of interest regarding the purported wide - ranging beneficial effects of an adequate vitamin d status translating into marked increases in test requests for clinical laboratories .
the well characterised endocrine pathway of vitamin d metabolism and action is solely responsible for vitamin d regulating plasma calcium and phosphate homeostasis .
a large body of data confirm that vitamin d exerts activities within each of the major bone cells and that these same cells are capable of synthesising the active metabolite , 1,25-dihydroxyvitamin d from 25-hydroxyvitamin d. such data arising from in vitro studies , animal models and clinical sources are consistent with a paradigm that local metabolism of vitamin d by bone cells to form 1,25-dihydroxyvitamin d and its consequent local actions within bone cells exerts an anabolic effect to increase bone mineral status .
the data reviewed here provide plausible mechanisms for both catabolic and anabolic actions of vitamin d on bone depending on dietary calcium intake . | INTRODUCTION
The actions of vitamin D to regulate plasma calcium and phosphate homeostasis
The actions of vitamin D to regulate bone mineral homeostasis
Regulation of bone synthesis of 1,25D
DISCUSSION | numerous beneficial effects of an adequate vitamin d status on a wide range of clinical conditions have been proposed particularly since the turn of the 21st century . these claims have generated an unprecedented level of interest in the medical media and lay press alike translating into great interest in assessing vitamin d status particularly amongst patients presenting to family or general practitioners . this growth in requests for serum 25-hydroxyvitamin d levels and the consequent issues for the clinical laboratory have recently been well addressed in this journal ( 1 ) . a major factor generating this marked interest in vitamin d
has been the reports of simple associations between a particular disease state or condition and low vitamin d status . it is clearly apparent that an improved vitamin d status can be associated with many attributes of good health relating to mobility and activities promoting sunlight exposure with out the involvement of a direct biological action of vitamin d. however during the 21st century there has been a flowering of knowledge of metabolism of vitamin d within a range of tissues including synthesis of the active metabolite 1,25-dihydroxyvitamin d ( 1,25d ) and activation of the vitamin d receptor ( vdr ) within the tissue of synthesis . such a mechanism now provides plausible physiological and molecular mechanisms for a diverse array of activities and tissue responses as has been reviewed recently ( 2 ) . in this article
i will review the current knowledge for the action of vitamin d on plasma calcium and phosphate homeostasis and bone mineral homeostasis with particular focus on the knowledge gaps . data published in the 21st century are challenging the concept of a single paradigm for the actions of vitamin d. an alternate paradigm involving local bone tissue metabolism of vitamin d with actions different from those observed with the endocrine source of 1,25d provides a plausible mechanism for activities observed in population studies and from randomised controlled trials of vitamin d supplementation to reduce the risk of fracture in the elderly . the well characterised endocrine pathway of vitamin d metabolism and action is summarised in figure 1 and is considered to be solely responsible for the contribution of vitamin d to regulate plasma calcium and phosphate . the identification of the endocrine pathway for activation of vitamin d in the 1970 s was a major achievement . the biologically active metabolite , 1,25d in plasma arises from sequential hydroxylations of vitamin d , firstly by the liver to form 25d and then by the kidney to form 1,25d ( 4 ) . the catabolism of vitamin d metabolites through the action of the 25-hydroxyvitamin-24-hydroxylase ( cyp24 ) has also been demonstrated to be an essential regulatory pathway for vitamin d homeostasis . under conditions
when this enzyme activity is reduced or ablated the serum half - lives of both 25d and 1,25d are increased and contribute to the development of hypercalcaemia , particularly following vitamin d supplementation ( 5 ) . this mechanism has recently been identified to account for idiopathic infantile hypercalcemia and other toxic effects of vitamin d ( 6 ) the binding of 1,25d to its highly specific nuclear receptor , vdr , modulates transcriptional activity of vitamin d responsive genes through binding to vitamin d response elements ( vdre s ) located within the promoter regions of these genes . another study indicated that approximately 913 genes were responsive to vdr in a squamous cell cancer cell line with 80% of genes being up - regulated and that these effects take some 6 to 12 hours to reach measurable levels ( 8) . 1,25d in association with pth increases the number of osteoclasts by way of an indirect mechanism through increased expression of the osteoblast - derived cytokine , rankl , which promotes osteoclastic differentiation ( 11 ) . alternatively it is important to note that in vitamin d deficiency or when vitamin d activity is ablated through gene mutations , hypocalcaemia develops despite a high pth in humans or rodents ( 13 , 14 ) . these findings suggest that pth requires 1,25d in order to increase osteoclast number and stimulate bone resorption to normalise extracellular fluid ( ecf ) calcium . each of these activities operates to restore the low plasma ionised calcium to its homeostatic level . pth also acts on the kidney to stimulate transcription of the gene coding for the enzyme which converts 25d to 1,25d , 25-hydroxyvitamin d-1-hydroxylase ( cyp27b1 ) increasing the levels in frank hypocalcaemia with secondary hyperparathyroidism by some 100 fold ( 15 ) . these data suggest that the critical level of serum 25d to maintain the endocrine actions of 1,25d to maintain plasma calcium homeostasis is 20 nmol / l or greater . the level of bone mineral at any one time is the result of at least two opposing cellular actions , bone formation by osteoblasts and bone resorption by osteoclasts . rickets in children ( or osteomalacia in adults when bone growth has ceased ) is the index disease for vitamin d deficiency . strong data have been generated from rodent models and human disease to indicate that dietary deficiency or genetic ablation of vitamin d activity results in hypocalcaemia , hypophosphataemia , hyperparathyroidism and rickets ( 17 ) . such a result
therefore it has been concluded that the action of vitamin d on bone mineral can be completely accounted for by its actions to maintain plasma calcium and phosphate homeostasis with no requirement for direct actions of vitamin d on bone cells to maintain or increase bone mineral status . the calcaemic action of 1,25d particularly in conjunction with pth to stimulate osteoclastogenesis and bone resorption as discussed above is a direct endocrine action of 1,25d on bone cells to reduce bone mineral in order to maintain plasma calcium levels . in contrast to these findings a large body of data have been and continue to be reported on the expression of vdr by each of the major types bone cells and on effects of 1,25d in vitro to inhibit proliferation of osteoblast - like cells and to stimulate osteoblast maturation and mineralisation ( 18 ) . osteoblasts as well as osteocytes and osteoclasts express the vdr and the cyp27b1 and cyp24 enzyme genes as well as a number of vitamin d responsive genes associated with mineralisation in osteoblasts including type i collagen , alkaline phosphatase and osteocalcin and other genes necessary for osteoblast and osteocyte maturation amongst a much larger number of proteins . furthermore there is evidence including clinical and rodent model studies suggesting that vitamin d is likely to exert activities directly on bone cells to improve or maintain bone mineral status ( 19 ) . furthermore population studies from the us have demonstrated that bone mineral density increases with increasing serum 25d levels reaching a plateau at serum 25d levels of approximately 75 nmol / l ( 21 ) . data from randomised clinical trials of vitamin d supplementation and fracture incidence indicate that anti - fracture efficacy at the hip or other non - vertebral sites is not achieved until serum 25d levels of 75 nmol / l are achieved ( 22 ) . interesting confirmatory data for a direct anabolic action of vitamin d on bone have arisen from animal model studies . when the dietary calcium and the phosphate levels are markedly increased sufficient to normalise plasma calcium and phosphate levels , the rickets phenotype is rescued and as mentioned above , many researchers have described the bone as
one research group has extended the feeding of the rescue diet to these genetically modified mice for a longer period ( 16 weeks instead of 10 weeks ) ( 23 ) . thus these data support the concept that vitamin d activity is necessary for normal bone mineral levels at least in adult mice due to the production of an optimal number of osteoblast forming cells and osteoblast activity . further independent data from other mouse models support this concept that vitamin d activity within mature osteoblast cells exerts an anabolic action on bone tissue . a similar but distinct transgenic mouse line ( osc mice )
has recently been prepared in which the human cyp27b1 gene is expressed under the control of the human osteocalcin promoter . another line of inquiry has utilised dietary studies in rodent models to demonstrate that at levels of serum 25d between 20 and 80 nmol / l , trabecular bone volume is reduced over 3 months as a result of increased bone resorption from increased osteoclastogenesis due to increased bone expression of the key osteoclastogenic cytokine , rankl ( figure 2 ) , ( 26 ) . all of these data are consistent with a model that local metabolism of vitamin d by bone cells to form 1,25d and its local action within bone cells can increase bone mineral status ( 27 ) . , pth does not increase the synthesis of 1,25d in bone ( 28 ) and there is direct evidence that pth treatment of bone cells does not activate the promoter region of the human gene for cyp27b1(29 ) . perhaps the most important finding in this area is that increased dietary calcium intake increases the expression of the cyp27b1 gene in bone by three - fold and reduces the expression of this gene in the kidney ( 30 ) . certainly rodent studies have found that optimal bone status is dependent on adequate vitamin d and dietary calcium status rather than either one alone ( 21 ) . when rats are fed either an adequate vitamin d level but low dietary calcium or vice versa they develop osteoporosis and when fed both a low vitamin d and calcium diet they develop osteomalacia ( figure 3 ) . the well characterised endocrine pathway of vitamin d metabolism and action is summarised in figure 1 and is considered to be solely responsible for the contribution of vitamin d to regulate plasma calcium and phosphate . new findings of the interaction between fgf23 and 1,25d and plasma phosphate levels are coming to light providing further insight in this area ( 3 ) . the identification of the endocrine pathway for activation of vitamin d in the 1970 s was a major achievement . the biologically active metabolite , 1,25d in plasma arises from sequential hydroxylations of vitamin d , firstly by the liver to form 25d and then by the kidney to form 1,25d ( 4 ) . the catabolism of vitamin d metabolites through the action of the 25-hydroxyvitamin-24-hydroxylase ( cyp24 ) has also been demonstrated to be an essential regulatory pathway for vitamin d homeostasis . under conditions
when this enzyme activity is reduced or ablated the serum half - lives of both 25d and 1,25d are increased and contribute to the development of hypercalcaemia , particularly following vitamin d supplementation ( 5 ) . this mechanism has recently been identified to account for idiopathic infantile hypercalcemia and other toxic effects of vitamin d ( 6 ) the binding of 1,25d to its highly specific nuclear receptor , vdr , modulates transcriptional activity of vitamin d responsive genes through binding to vitamin d response elements ( vdre s ) located within the promoter regions of these genes . another study indicated that approximately 913 genes were responsive to vdr in a squamous cell cancer cell line with 80% of genes being up - regulated and that these effects take some 6 to 12 hours to reach measurable levels ( 8) . the current controversial issue in this area is what is the critical level for serum 25d to maintain plasma calcium homeostasis ? 1,25d in association with pth increases the number of osteoclasts by way of an indirect mechanism through increased expression of the osteoblast - derived cytokine , rankl , which promotes osteoclastic differentiation ( 11 ) . pth also acts on the kidney to stimulate transcription of the gene coding for the enzyme which converts 25d to 1,25d , 25-hydroxyvitamin d-1-hydroxylase ( cyp27b1 ) increasing the levels in frank hypocalcaemia with secondary hyperparathyroidism by some 100 fold ( 15 ) . these data suggest that the critical level of serum 25d to maintain the endocrine actions of 1,25d to maintain plasma calcium homeostasis is 20 nmol / l or greater . the level of bone mineral at any one time is the result of at least two opposing cellular actions , bone formation by osteoblasts and bone resorption by osteoclasts . rickets in children ( or osteomalacia in adults when bone growth has ceased ) is the index disease for vitamin d deficiency . strong data have been generated from rodent models and human disease to indicate that dietary deficiency or genetic ablation of vitamin d activity results in hypocalcaemia , hypophosphataemia , hyperparathyroidism and rickets ( 17 ) . such a result
therefore it has been concluded that the action of vitamin d on bone mineral can be completely accounted for by its actions to maintain plasma calcium and phosphate homeostasis with no requirement for direct actions of vitamin d on bone cells to maintain or increase bone mineral status . the calcaemic action of 1,25d particularly in conjunction with pth to stimulate osteoclastogenesis and bone resorption as discussed above is a direct endocrine action of 1,25d on bone cells to reduce bone mineral in order to maintain plasma calcium levels . in contrast to these findings a large body of data have been and continue to be reported on the expression of vdr by each of the major types bone cells and on effects of 1,25d in vitro to inhibit proliferation of osteoblast - like cells and to stimulate osteoblast maturation and mineralisation ( 18 ) . osteoblasts as well as osteocytes and osteoclasts express the vdr and the cyp27b1 and cyp24 enzyme genes as well as a number of vitamin d responsive genes associated with mineralisation in osteoblasts including type i collagen , alkaline phosphatase and osteocalcin and other genes necessary for osteoblast and osteocyte maturation amongst a much larger number of proteins . furthermore there is evidence including clinical and rodent model studies suggesting that vitamin d is likely to exert activities directly on bone cells to improve or maintain bone mineral status ( 19 ) . furthermore population studies from the us have demonstrated that bone mineral density increases with increasing serum 25d levels reaching a plateau at serum 25d levels of approximately 75 nmol / l ( 21 ) . data from randomised clinical trials of vitamin d supplementation and fracture incidence indicate that anti - fracture efficacy at the hip or other non - vertebral sites is not achieved until serum 25d levels of 75
interesting confirmatory data for a direct anabolic action of vitamin d on bone have arisen from animal model studies . when the dietary calcium and the phosphate levels are markedly increased sufficient to normalise plasma calcium and phosphate levels , the rickets phenotype is rescued and as mentioned above , many researchers have described the bone as
one research group has extended the feeding of the rescue diet to these genetically modified mice for a longer period ( 16 weeks instead of 10 weeks ) ( 23 ) . thus these data support the concept that vitamin d activity is necessary for normal bone mineral levels at least in adult mice due to the production of an optimal number of osteoblast forming cells and osteoblast activity . further independent data from other mouse models support this concept that vitamin d activity within mature osteoblast cells exerts an anabolic action on bone tissue . another line of inquiry has utilised dietary studies in rodent models to demonstrate that at levels of serum 25d between 20 and 80
nmol / l , trabecular bone volume is reduced over 3 months as a result of increased bone resorption from increased osteoclastogenesis due to increased bone expression of the key osteoclastogenic cytokine , rankl ( figure 2 ) , ( 26 ) . all of these data are consistent with a model that local metabolism of vitamin d by bone cells to form 1,25d and its local action within bone cells can increase bone mineral status ( 27 ) . , pth does not increase the synthesis of 1,25d in bone ( 28 ) and there is direct evidence that pth treatment of bone cells does not activate the promoter region of the human gene for cyp27b1(29 ) . in fact high pth levels are associated with decreased synthesis expression of the cyp27b1 gene in bone tissue ( 28 ) . perhaps the most important finding in this area is that increased dietary calcium intake increases the expression of the cyp27b1 gene in bone by three - fold and reduces the expression of this gene in the kidney ( 30 ) . certainly rodent studies have found that optimal bone status is dependent on adequate vitamin d and dietary calcium status rather than either one alone ( 21 ) . when rats are fed either an adequate vitamin d level but low dietary calcium or vice versa they develop osteoporosis and when fed both a low vitamin d and calcium diet they develop osteomalacia ( figure 3 ) . the data reviewed here provide a plausible model for both catabolic and anabolic actions of vitamin d on bone depending on the dietary calcium intake . with either an inadequate dietary calcium intake or low vitamin d status with a marginal dietary calcium intake , increased activity of the plasma calcium and phosphate homeostatic mechanisms , including endocrine vitamin d activity , are required to maintain plasma calcium and phosphate levels within physiological levels . this involves increased activity of pth when serum 25 d levels fall below 60 nmol / l ( 32 ) presumably arising from stimulation of the parathyroid gland calcium - sensing receptor as a result of a slight fall in plasma ionized calcium level . however it is also possible that the increased pth secretion arises as a direct result of the fall in serum 25d ( 33 ) . the increased pth acts to elevate the level of cyp27b1 enzyme in the kidney with the increased enzyme activity allowing continued renal synthesis of 1,25d and maintenance of serum 1,25d levels while serum 25d levels fall into the depleted levels ( eg levels below 60
the maintenance of serum 1,25d optimises intestinal calcium and phosphate absorption and the interaction between pth and serum 1,25d stimulates osteoclastogenesis and bone resorption increasing the flow of calcium and phosphate into the plasma compartment . this mechanism is able to maintain plasma calcium and phosphate levels until serum 25d levels fall below 20 nmol / l at which time the substrate levels are too low to maintain serum 1,25d levels . perhaps most importantly it is apparent that both pth and serum 1,25d are required for adequate bone resorption because the development of hypocalcaemia indicates that even when pth levels are very high there is inadequate flow of calcium from the bone compartment to maintain normocalcemia in the plasma compartment ( 15 ) . under these conditions
when dietary calcium intakes are adequate to meet all the demands of the calcium economy the data reviewed above suggest that in concert with an adequate vitamin d status ( eg , serum 25d levels greater than 75
nmol / l ) , increased levels of bone cyp27b1 enzyme activity ensure greater synthesis of 1,25d by bone cells associated with reduced circulating pth levels . these latest findings provide further light on the delicate relationship between plasma calcium homeostasis , bone mineral homeostasis and vitamin d activities involving either endocrine or bone autocrine /paracrine activities . to maintain plasma calcium homeostasis
, a serum 25d level of 20 nmol / l or greater appears to be sufficient to provide adequate substrate for the renal cyp27b1 enzyme level . in contrast bone cyp27b1 enzyme levels are not increased by pth and therefore higher levels of serum 25d as substrate ( ie serum 25d 75 nmol / l or greater ) are required for bone cells to produce sufficient bone tissue 1,25d to suppress osteoclastogenesis . furthermore adequate dietary calcium intake is also required to increase bone cyp27b1 enzyme levels even at these higher levels of serum 25d . | [
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the fundamental mechanisms of modulating neural circuits in relation to specific physiological actions and behaviors in organisms are an area of current interest for basic research as well as clinical treatments .
neural circuits change in activity by modulators such as serotonin ( 5-hydroxytryptamine , 5-ht ) in altering sensitization in the gill - withdrawal reflex in aplysia .
biogenic amines can act as neurotransmitters and neuromodulators and are indispensable for the normal development of neural connections in organisms [ 3 , 4 ] .
the serotonergic ( 5-htergic ) system plays many roles in drosophila and other insects which are crucial for the animal survival and locomotion , feeding behaviors , learning and memory , aggression , circadian rhythms , sleep , heart rate , salivary secretion , insulin signaling , and synaptic transmission [ 817 ] . being able to manipulate subsets of specific neurons in drosophila melanogaster allows one to address the role of neurons associated with particular neuromodulators in altering specific behaviors [ 1820 ] .
in addition , the fruit fly nervous system is an attractive model to decode the neural circuits and interrogate the function of genes of interest in animal behavior [ 21 , 22 ] . also , drosophila have a relatively simple 5-htergic system that consists of 84 5-htergic neurons in the larval central nervous system ( cns ) [ 23 , 24 ] and 106 5-htergic neurons in adult cns .
serotonin is biosynthesized from an essential amino acid tryptophan , which is catalyzed by two enzymes , tryptophan hydroxylase ( trh ) and aromatic - l - amino acid decarboxylase .
after release of 5-ht into the synaptic cleft at nerve terminals by synaptic vesicles fusion , the action of 5-ht is terminated by its uptake into the nerve terminals by the serotonin transporter ( sert ) .
drosophila genome contains a gene that encodes dsert , which is homologous to the vertebrate human and rodent sert ( hsert and rsert ) .
it has been shown that dsert can be inhibited by a variety of chemicals , such as fluoxetine , which is a potent inhibitor of the sert and is used as an antidepressant drug [ 27 , 28 ] .
the drosophila genome harbors five different genes for the 5-ht receptors ( 5-ht1adro , 5-ht1bdro , 5-ht2adro , and 5-ht7dro ) and the recently cloned 5-ht2bdro receptor subtype .
5-ht receptors in drosophila are g - protein coupled receptors ( gpcr ) , which are expressed in various neurons and selective regions of the nervous system which correlate with the 5-ht localization [ 14 , 30 ] .
serotonin modulates locomotion activity in diverse groups of animals across the animal kingdom , for instance , in c. elegans [ 31 , 32 ] and mammals .
recently , it has been shown that various 5-ht receptors play a role in locomotive behavior in drosophila .
in addition , we demonstrated that mutations in 5-ht receptor subtypes significantly reduce larval locomotion behavior .
moreover , we show that overactivation of 5-ht neurons in freely moving larvae and adults significantly compromises locomotor activity . at the cellular level
, it was already shown that serotonin increases evoked motor unit activity which innervates body wall muscle fibers . in this current study
, we further investigated the 5-ht receptor subtypes that mediate the modulatory action of 5-ht in a sensory - cns - motor circuit .
5-ht modulates the feeding behavior in mammals as well as in insects . in honeybees ,
5-ht increases the motility of the gut ; however , it decreases feeding behavior drive in the cns [ 7 , 10 , 14 ] .
also , it has been shown that if 5-ht2a receptor subtype is blocked , there is a decrease in feeding behavior in d. melanogaster .
, we address whether 5-ht alters mouth hook movements ( mhms ) , which is an assay for feeding behavior .
this study demonstrates the role of 5-ht receptor subtypes and function of 5-ht releasing neurons in various behaviors within the ever increasingly invertebrate model d. melanogaster .
trh - gal4 ( ii ) ( stock # 38388 ) , trh - gal4 ( iii ) ( stock # 38389 ) , d42-gal4 ( iii ) ( stock # 8816 ) , 5-ht1a - gal4 ( stock # 49583 ) , 5-ht1b - gal4 ( stock # 46023 ) , 5-ht2a - gal4 ( stock # 49574 ) , 5-ht-7-gal4 ( stock # 49414 ) , uas-5-ht1b - rnai ( stock # 33418 ) , uas-5-ht2a - rnai ( stock # 31882 ) , uas-5-ht7-rnai ( stock # 27273 ) , uas - trpa1 ( stock # 26263 ) , uas - trh ( stock # 27638 ) , mi-5-ht1b ( w ; mi{et1}5-ht1b ) ( stock # 24240 ) , pbac-5-ht2a ( w ; p{frt(w)}2a p{neofrt}82b pbac{gal4d , eyfp}5-ht2a ) ( stock # 19367 ) , mi-5-ht2b ( line 2 ) ( w ; mi{et1}5-ht2b ) ( stock # 29257 ) , mi-5-ht2b ( line 1 ) ( y w ; mi{mic}5-ht2b ) ( stock # 40810 ) , pbac-5-ht7 ( w ;
pbac{wh}5-ht7 ) ( stock # 18848 ) , and pbac - trh ( w ; pbac{pb}trh ) ( stock # 10531 ) were obtained from the bloomington drosophila stock center ( bloomington , in , usa ) .
the flies were raised at room temperature ( 22 - 23c ) , unless otherwise stated in figure legends , in vials containing cornmeal - agar - dextrose - yeast medium .
the overexpressing and knockdown strains of the various proteins were not confirmed in this study with quantitative measures such as rt - pcr .
the agonists and antagonists used are defined for mammalian preparations and details of binding affinity and efficacy of the various drosophila 5-ht receptor subtypes are not established .
8-hydroxy - dpat hydrobromide ( 5-ht1a agonist ) , cp 93129 dihydrochloride ( 5-ht1b agonist ) , -methyl-5-hydroxytryptamine maleate ( 5-ht2 agonist ) , and as19 ( 5-ht7 agonist ) were purchased from tocris bioscience ( bristol , uk ) .
5-hydroxytryptamine ( 5-ht ) , 5-hydroxytryptophan ( 5-htp ) , fluoxetine hydrochloride , para - chlorophenylalanine ( pcpa ) , 5-carboxamidotryptamine maleate salt , 5-methoxytryptamine , bw 723c86 ( 5-ht2b agonist ) , r()-doi hydrochloride , ( )-doi hydrochloride , 1-(3-chlorophenyl)piperazine hydrochloride ( m - cpp ) , and lp-44 ( 5-ht7 agonist ) were purchased from sigma ( st . louis , mo , usa ) .
fresh solutions of specific concentrations were made from stock solutions each time before starting an experiment .
the dmso concentration in lp-44 10 m , which is the highest concentration employed , was 0.1% .
adult males and females were put inside a container with an apple - juice agar plate at the bottom .
specific drugs were dissolved in one milliliter ( ml ) of distilled water and mixed with 2 grams of cornmeal - agar - dextrose - yeast medium ( shown in millimolar ( mm ) in the figure legends ) .
approximately fifteen ( 15 ) embryos were transferred from the apple - juice container to the drug mixture .
the embryos were left to develop to the third - instar larval stage which were raised at room temperature ( 22 - 23c ) , unless otherwise stated in figure legends .
the same protocol was used for the control animals except that no drug was added to the food .
it is known how much endogenous 5-ht concentrations are altered by feeding some of these compounds ( 50 mm pcpa , ) ; however , it is not known for other compounds we used such as the receptor agonists and antagonist .
such measures for each dosage and each compound are beyond the scope of these initial studies .
our approach was to provide varying dosages as a means to assess the potential varied concentrations on the effect of whole animal behaviors .
individual third - instar larvae were placed on apple - juice agar ( 1% agar ) petri dishes ( about 8.59 cm diameter ) .
the larvae were left inside the dish for one minute to acclimate to the environment , unless otherwise stated in figure legends . the body wall contractions ( bwcs )
were counted for one minute in a lightly illuminated environment at room temperature ( 22 - 23c ) .
the flies were raised at 23c , 12 : 12 ld cycle . to obtain the locomotion behavior at room temperature ( 22 - 23c ) for control and shibire expressing larvae ,
the larvae were left for 1 min to acclimate to the new environment , and body wall contractions were counted for the following 1 min . to obtain bwcs at restrictive temperature ( 37c ) for control and shibire expressing larvae
, larvae were placed inside a mixture of 0.5 gm fly food plus 0.5 ml water in a tube : 9.4 cm height , 2.4 cm top diameter , and 2.25 cm bottom diameter .
the temperature range used was as previously used by song et al . for shibire .
individual larvae were placed on prewarmed apple - juice agar on a hot plate ( 3032c ) .
the larvae were left for 1 min to acclimate , and the bwcs were counted for the following 1 min .
the flies were raised at 23c , 12 : 12 ld cycle . to obtain bwcs at room temperature ( 22 - 23c ) ,
individual larvae were placed on apple - juice agar at room temperature and left for 2 min to acclimate to the new environment .
to obtain locomotion behavior at restrictive temperature ( 32c ) , individual larvae were placed on a prewarmed apple - juice agar plate on a hot plate ( 3032c ) .
individual larvae were placed inside a small petri dish ( 5.5 cm diameter ) that contained yeast solution ( a few dried yeast granules were mixed with water ) .
the larvae were left for one minute , and then the mouth hook movements ( mhms ) were counted for one minute .
the adult flies were anesthetized by exposing them to co2 and then males and females sorted out one day prior to performing the behavioral assay .
six to fourteen adult flies ( 26 days old ) , males and females , were tested separately .
they were placed inside a tube : about 9.4 height , 2.4 cm top diameter , and 2.25 cm bottom diameter . during the experiment ,
the cotton plug was removed and another similar tube was placed over the top of the first tube .
the tube was tapped until all the flies fell into the bottom of the tube .
a 10 sec period was set to observe how many flies would cross the 8 cm line in this period .
this procedure was repeated 10 times , and there was one - minute interval between each trial in order for the flies to recover from first tapping .
the climbing rate was calculated by combining all the fly cohorts that were tested at room temperature ( modified from ) .
the same fly cohorts , which were used at room temperature climbing assay , were transferred into the water bath ( 37c ) , and they were left for 10 min .
after 10 min incubation , the flies were returned back to room temperature ( 22 - 23c ) to repeat the climbing assay .
this procedure was repeated for 10 times and with a one - minute interval between each trial .
the adult flies , females or males , were anesthetized by exposing them to co2 .
then , the flies ( less than 1 to 6 days old ) were anesthetized by cold .
adult flies were transferred into empty vials , and they were placed inside an ice bucket for 2030 sec . the flies ( 4 - 5 ) were transferred into a plastic petri dish ( 9 cm diameter and 0.8 cm height ) , and a glass cover was put over it .
the flies were left for 1013 min to recover from cold anesthesia at room temperature .
the fly locomotion activities were recorded with a webcam ( webcam hd4110 , hewlett - packard company , palo alto , ca ) , which was connected to a computer , and the activity was recorded at 5 frames per second ( 5 fps ) for 10 min using virtualdub-1.10.4 software ( http://www.virtualdub.org/ ) .
the locomotion activity was analyzed for two time points , for 10 sec at the 5th min and for 10 sec at 10th min using manual tracking ( http://rsbweb.nih.gov/ij/plugins/track/track.html ) in imagej software ( 1.47 v ) ( http://rsbweb.nih.gov/ij/download.html ) .
we used a recently created channelrhodopsin-2 ( chr2 ) line which is very sensitive to light : y w ; pbac{uas - chr2.xxl}vk00018 ( bdsc stock # 58374 ) .
virgin females from uas - chr2-xxl were crossed with males of trh - gal4 ( bdsc stock # 38389 ) line to express chr2-xxl variant in serotonergic neurons .
we also used uas - h134r - chr2 ; trh - gal4 ( iii ) homozygous line , which is kindly provided by dr .
schoofs et al . , for the electrophysiological study since the chr2-xxl was too sensitive for the electrophysiology experiments .
louis , mo , usa ) was dissolved in 17.6 ml absolute ethanol to make a 100 mm stock solution .
100 l of 100 mm stock solution was transferred to small tubes and wrapped in aluminum foil , to prevent being exposed to light , and kept in 20c freezer .
in order to prepare fly food supplemented with 1 mm atr , 10 ml fly food was dissolved in microwave . after the food was cooled , 100 l of 100 mm atr was mixed well with fly food or 100 l of absolute ethanol ( vehicle ) was mixed with food as a control .
the flies were transferred to a vial containing atr and kept in a dark place ( to keep the atr from degradation ) at room temperature ( 22 - 23c ) .
locomotion behavior was assessed by placing a single larva on an apple - juice agar plate .
the body wall contractions were being counted for one minute ( bwcs / min ) while the larva was being exposed to a dim white light .
also , body wall contractions were counted while the larva was exposed to focused focal blue light ( a focused light through a microscope eyepiece with a mounted led ; see ) .
the males and females are to be sorted out and transferred into separate vials in cohorts of 1014 flies .
the flies were left to recover for 24 hours before running the experiments . a plastic vial ( drosophila culture cylindrical vial 1 - 1/4 diameter 4 tall )
was marked at 8 cm length , and the 1014 cohort flies were transferred to that empty marked vial .
another plastic vial was placed on top of the marked one ( modified from ) .
the vials were tapped to knock down the flies to the bottom of the tube .
then , the number of flies which climbed across the 8 cm mark in 10 sec was recorded .
third - instar larvae were placed in the dissecting dish and pinned out on anterior and posterior regions with the dorsal side up .
a modified hl3 saline ( nacl 70 mm , kcl 5 mm , mgcl26h2o 20 mm , nahco3 10 mm , trehalose 5 mm , sucrose 115 mm , trizma acid 25 mm or bes 25 mm , and cacl22h2o 1 mm , ph 7.1 ) was used as physiological saline .
the two last segmental nerves , which innervate last body segment , were cut close to the posterior end .
sharp microelectrodes ( 3 m kcl ) were used for monitoring muscle fiber 6 or 7 .
the segmental nerves were stimulated at 40 hz , 10 pulses ( s88 stimulator , astro - med , inc . ,
the excitatory postsynaptic potentials ( epsps ) were recorded with an axoclamp 2b ( axon instruments , usa ) , converted with a powerlab , 4sp ( adinstruments , usa ) , and analyzed with labchart 7.0 ( adinstruments , usa ) .
the traces were measured by averaging the responses in 8 stimulations trains made with normal saline and 8 stimulations trains after exchanging saline with various compounds , unless otherwise stated in figure legends .
the suction electrodes which were used to stimulate the segmental nerves in each preparation were slightly different ; therefore , a range of stimulation voltages were used .
depending on how tight the seal is with the suction electrode and the nerve , the voltage must be adjusted to evoked action potentials in the sensory nerves .
the current is not directly varied but is a reflection of the voltage and the seal resistance for each preparation .
bonferroni t - test was performed to obtain significant levels ( p values ) of various groups of flies ( sigmaplot version 12.0 ) .
paired t - test was employed to compare the number of epsps between the saline and agonist or saline - containing compound .
student 's t - test was used to compare the treatment and control groups . the level of significance ( p value ) less than or equal to 0.05
the rationale for feeding 5-ht as compared to injections is to reduce the handling stress associated with systemic injections .
5-ht was orally administered to the larvae at various concentrations : 1 mm , 10 mm , 50 mm , and 100 mm .
the embryos were collected and were placed inside a mixture of 0.5 gm fly food and 0.5 ml water ( control ) or 0.5 gm food and 0.5 ml water plus various 5-ht concentrations .
5-ht significantly reduced the body wall contractions ( bwcs ) and mouth hook movements ( mhms ) at 100 mm high dose ( figures 1(a ) and 1(b ) ) .
1 mm did not significantly alter mouth hook movements ( mhms ) ; however , 100 mm significantly reduced the mhms ( figure 1(b ) ) .
also , 5-ht altered the developmental size of larvae . a low level of 5-ht ( 1 mm , 10 mm ) resulted in significant increases in body length ; however , at high concentrations ( 50 mm and 100 mm ) , the body length
another approach to perturb the 5-ht level in neurons and systemically is by providing more of the precursor to synthesize 5-ht .
we predicted a similar result with feeding the precursor to feeding 5-ht itself to the larvae .
the embryos were collected and were placed inside a mixture of 0.5 gm standard fly food and 0.5 ml water ( control ) or 0.5 gm food and 0.5 ml water in addition to various concentrations of 5-htp .
5-htp at 5 mm and 25 mm reduced the bwcs in third - instar larvae ( figure 2(a ) ) . on the other hand
body length did not change for 5-htp at 5 mm , but the lengths did decrease at 25 mm as compared to controls and ones fed 5 mm ( figure 2(c ) ) .
the rate - limiting enzyme of 5-ht synthesis was blocked by para - chlorophenylalanine ( pcpa ) administration .
so , instead of increasing 5-ht , a decrease in levels in the cns as well as systemic levels in 5-ht occurs .
a similar feeding regime was used as in earlier studies which measured 5-ht with hplc and showed a significant reduction in level of 5-ht .
the embryos were collected and were placed inside a mixture of 0.5 gm standard fly food and 0.5 ml water ( control cs ) or 0.5 gm food and 0.5 ml water plus pcpa . feeding pcpa 50 mm significantly reduced the bwcs and mhms ( figures 3(a ) and 3(b ) , resp . ) .
thus , overexpression of trh was predicted to result in similar alterations to feeding 5-ht or the 5-htp .
the flies were raised at 23c or 29c to increase the expression level of gal4 transcriptional activator .
the proposed trh overexpression significantly reduced the bwcs and mhms in third - instar larvae ( figures 4(a ) and 4(b ) , resp . ) .
the 5-ht levels were not measured within the hemolymph for this strain ; however , we assume the strain works as well as it has in previous studies .
in addition , one of the parental lines ( trh - gal4 ( ii ) ) was used as a control . an additional approach to raise 5-ht at the synaptic cleft is to block the reuptake of 5-ht back into the presynaptic nerve terminal with selective 5-ht reuptake blockers .
fluoxetine was shown to block reuptake for the 5-ht transporter in drosophila [ 27 , 47 ] .
early second - instar larvae were collected and then 0.5 gm fly food was mixed with 0.5 ml water in case of control .
fluoxetine 10 mm was dissolved in 0.5 ml water , and then it was mixed with 0.5 gm food .
since fluoxetine is light sensitive , therefore the larvae were placed in an incubator without light .
fluoxetine 10 mm significantly reduced the bwcs and mhms in w larvae and trh insertional mutant larvae compared to control groups ( figures 5(a ) and 5(b ) ) .
moreover , fluoxetine 10 mm in trh insertional mutant larvae significantly reduced bwcs and mhms in comparison to fluoxetine - fed w larvae ( figures 5(a ) and 5(b ) ) .
chronic activation of the 5-ht neurons through activating trpa1 channels selectively expressed in 5-ht neurons significantly increased bwcs and mhms ( figures 6(a ) and 6(b ) ) .
5-ht1b knockdown in 5-ht1b expressing neurons during development significantly reduced the bwcs and mhms ( figures 6(a ) and 6(b ) ) .
however , 5-ht2a and 5-ht7 receptor knockdowns in 5-ht2a and 5-ht7 receptor expressing neurons did not markedly influence the bwcs but did decrease mhms ( figures 6(a ) and 6(b ) ) .
similar results presented later in which the 5-ht containing neurons were stimulated with light , through expression of channel rhodopsin , complement these findings .
in addition , the use of one of the parental strains ( gal4 ) as a control supports the differential findings for the f1 strains . in determining which receptor subtypes may play a larger role in altering the larval behaviors , strains carrying insertional mutations in 5-ht receptors were examined .
in addition , a pbac - trh strain was examined which would perturb the synthesis of 5-ht . the insertional mutation in 5-ht2a ( homozygotes mutant ) and 5-ht7 ( homozygotes mutant ) receptors significantly reduced the bwcs and mhms in comparison to w larvae ( figures 7(a ) and 7(b ) ) ; however , trh mutant larvae did not show a noticeable change in bwcs and mhms compared to w larvae ( figures 7(a ) and 7(b ) )
. figures 8(a ) and 8(b ) show that the fly lines were significantly reduced in bwcs and mhms in comparison to control cs larvae . furthermore
, 5-ht2b mutant ( line 2 ) larvae significantly decreased bwcs in comparison to w larvae , although there was not a marked change in mhms between w and 5-ht2b mutant ( line 2 ) larvae ( figures 8(a ) and 8(b ) ) . the 5-ht2b mutant ( line 2 ) had a larger decrease in bwc ( figure 8(b ) ) than 5-ht2b mutant ( line 2 ) .
shi is temperature sensitive which functions well at permissive temperature ( 22 - 23c ) ; however , it does not function at restrictive temperature ( 37c ) .
thus , the synaptic vesicles within the presynaptic nerve terminals are not able to recycle .
so there is an overall reduction in 5-ht being released in evoked and spontaneous vesicular events .
the flies were raised at room temperature , and the third - instar larvae were selected and incubated at 37c for 20 min .
it was shown that expressing shi in sensory neurons significantly reduces locomotion behavior when they are incubated at 37c for 20 min .
in this study , larvae which were expressing shi in 5-ht neurons did not markedly alter the rate in the body wall contractions compared to control ( uas - shi ) ( figure 9(a ) ) .
it has been demonstrated that trpa1 does not become activated at room temperature ( 22 - 23c ) but when it is exposed to > 28c , the trpa1 channels become activated .
the larvae that were expressing trpa1 channels were put on a prewarmed ( 32c ) agar plate while the body wall contractions were being counted .
the data indicate that acute activation of 5-ht neurons in third - instar larvae significantly decreased bwcs at 32c compared to control group ( uas - trpa1 ) ( figure 9(b ) ) .
one of the parental lines ( uas - trpa1 ) was used for comparisons . the locomotion activity in third - instar larvae
was also decreased when 5-ht neurons were activated by light , through expression of channelrhodopsin-2 ( chr2 ) , which complements these findings ( see the optogenetics results ) .
flies were raised at room temperature ( 22 - 23c ) , which were expressing a temperature sensitive dominant negative shi allele in 5-ht neurons .
the climbing assay ( i.e. , negative geotaxic assay ) was carried out to observe the effect of acute activity in 5-ht neurons on locomotion behavior in adult flies .
the shi flies were crossed with motor neuron specific gal4 ( d42-gal4 ) and this cross was used as a positive control .
when the flies were placed at 37c for 10 min , locomotion activity significantly decreased .
furthermore , shi flies were crossed with 5-ht neurons specific gal4 driver ( trh - gal4 ( iii ) ) .
when these flies were incubated at 37c for 10 min in the water bath , no significant changes were observed in locomotion behavior in both males and females in comparison to control flies ( figure 10(a ) ) .
the climbing assay , which was performed in shi fly groups , was not a suitable assay for the locomotion behavior of trpa1 expressing flies since when they were placed at 37c , the control flies were very active but they stayed at the bottom while the climbing assay was being performed ( i.e. , they did not cross the 8 cm line at the 10 sec time period ) ; therefore , we decided to use another assay to record locomotion activity as depicted in figure 10(b1 ) .
adult flies ( 4 - 5 flies in each group , males and females separately ) were placed in a petri dish which allowed the adults to walk around horizontally but not fly . both controls ( uas - trpa1 ) and trpa1 expressing flies
the locomotor activity was recorded for 10 min , and the activity was analyzed for 10 sec at 5th and 10 sec at 10th min .
acute activation of trpa1 channels in 5-ht neurons significantly reduced the locomotor activity in male and female trpa1 expressing flies at 5th and 10th min compared to male and female control groups ( uas - trpa1 ) .
note that the females had a decreased locomotor activity compared to male flies ( figure 10(b2 ) ) .
the locomotor activity in adult flies was also decreased when 5-ht neurons were activated by the channelrhodopsin chr2-xxl ( figure 17(c ) ) .
the other less sensitive chr2 variants were not used in this experiment since the adult flies have a thick cuticle which reduces the level of blue light penetrance ; therefore , blue light can not activate enough chr2 to observe a change in the behavior .
also , we showed that blue light exposure is effective in flies that were not fed atr .
it has been shown previously that chr2-xxl can be activated in the absence of atr diet supplementation .
sensory - motor circuit activity was used to examine the influence of 5-ht on the circuit activity in dissected third - instar larvae .
the rationale of conducting these acute studies in dissected preparations is to directly examine the action of 5-ht on a neural circuit in which one could control the electrical activity of sensory neurons and examine a defined motor output on a body wall muscle .
the body wall muscle ( m6 ) is used in the intact larvae for movements measured in bwm behavioral assay .
therefore , application of compounds which alter synaptic 5-ht in the excised preparation can be correlated to the approaches used to manipulate 5-ht concentration through diet , pharmacology , and genetics .
sensory neurons of the last segmental nerves in early 3rd instars were stimulated at 40 hz , 10 pulses ( figure 11(a ) ) .
the segmental nerves were stimulated for 8 stimulation trains in the saline and 8 stimulation trains or more after exchanging saline with saline containing 5-ht , 5-ht agonists , a selective serotonin reuptake inhibitor ( ssri , fluoxetine ) , or prewarmed saline , which was used in trpa1 expressing larvae .
when saline was exchanged with saline - containing 5-ht , the number of evoked excitatory postsynaptic potentials ( epsps ) significantly increased ( figure 11(b ) ) .
application of 5-ht at 100 nm to the exposed larval brains significantly increased the number of epsps . when higher concentrations of 5-ht ( 1 m , 10 m ) were applied , the number of epsps was also significantly increased ; however , the percentage change at 100 nm was higher than the percentage change for higher concentrations ( figure 12(a ) ) .
this demonstrates that 5-ht7 agonist , as19 , and 5-ht2 agonist ( )-doi hcl noticeably increased the number of epsps ; however , no significant changes were observed after application of 8-oh - dpat , cp9312 , and -methyl-5-ht ( figure 12(b ) ) .
there is one study which demonstrated that doi appears to function at 5-ht2 receptors in drosophila .
other 5-ht agonists were used for further confirmation of the 5-ht receptors subtypes that mediate modulatory role of 5-ht in sensory - motor circuit activity .
5-meoht ( 1 m and 10 m ) , 5-cat ( 100 nm and 10 m ) ,
m - cpp ( 100 nm and 10 m ) , and lp-44 ( 100 nm and 10 m ) were applied to dissected third - instar larvae .
there were no significant changes in epsps after application of these agonists ( figure 13(a ) ) .
5-ht2 agonists ( bw723c86 ) ( 100 nm and 10 m ) and r()-doi hcl ( 1 m ) significantly increased the number of epsps ( figure 13(b ) ) . as far as we know , only r()-doi hcl was examined for binding to drosophila 5-ht receptors ; however , to the best of our knowledge , binding studies of bw723c86 have yet to be performed on drosophila specific 5-ht receptors .
5-ht receptor subtype mutant lines mentioned previously were used for electrophysiological studies to corroborate the pharmacological results .
5-ht significantly increased the number of epsps in trh mutant larvae . also , the response of trh mutant larvae to 5-ht was higher than control flies ( cs ) ( figure 14 ) .
in addition , the exogenous application of 5-ht in dissected third - instar larvae significantly increased the sensorimotor activity in 5-ht2a receptor mutant flies , although the response of 5-ht2a receptor flies to 5-ht was lower than the control flies ( cs ) .
in 5-ht7 mutant larvae , 5-ht application slightly increased the sensory - motor activity but at p > 0.1 for group comparisons from controls due to the high variation in this line ( figure 14 ) .
to examine overactivity of the 5-htergic neurons , the same sensory - cns - motor paradigm was used in third - instar larvae ( uas - trpa1 ) at room temperature ( 22 - 23c ) ( figure 15(a1 ) ) and when switched with prewarmed saline ( 37c ) .
the numbers of epsps of 7 - 8 stimulation trains bathed in saline and 7 - 8 stimulation trains inside prewarmed saline were counted ( figures 15(a1 ) and 15(a2 ) ) .
5-htergic neurons were activated while the motor output was being recorded from muscle fiber 6 or 7 in trpa1 expressing larvae in saline ( figure 15(b1 ) ) and warmed saline ( figure 15(b2 ) ) .
the results indicate that acute activation of 5-htergic neurons by trpa1 cation channels ( trh - gal4 ( iii)>uas - trpa1 ) showed a decrease in activity when warmed at 92.4% significance ( p = 0.076 ) and in comparing this line to the changes in the parental line ( uas - trpa1 ) only a change occurred at 91% significance ( p < 0.09 ) ( figure 15(c ) ) . we choose to use another approach which has recently become popular due to the specific activation of known neurons .
we used the optogenetic approach to selectively activate 5-htergic neurons while the sensorimotor neuron activity was being recorded . in dissected third - instar larvae ,
blue light exposure did not suppress sensorimotor activity in uaschr2h134r - mcherry ; trh - gal4 ( homozygous for both constructs ) larvae which were not fed atr ( figure 16(b1 ) ) .
but blue light exposure suppressed sensorimotor activity in uas - chr2h134r - mcherry ; trh - gal4 ( homozygous for both constructs ) larvae which were fed food supplemented with atr ( 1 mm ) ( figure 16(b2 ) ) .
the sensory - motor circuit was stimulated at 40 hz , 10 stimuli , and the epsps were recorded in saline ( control ) ( figure 17(a ) ) .
afterwards , fluoxetine 10 m or 100 m was applied to observe how it influenced the sensorimotor circuit physiology .
fluoxetine is a selective serotonin reuptake inhibitor ( ssri ) which would potentially cause a rise in the 5-ht within the synaptic cleft .
application of fluoxetine 100 m significantly reduced the number of epsps ( figure 17(b ) ) .
however , no significant reduction was observed when fluoxetine 10 m was applied to dissected third - instar larvae ( figure 17(c ) ) .
in the current study , we investigated the role of the 5-htergic system in locomotion and feeding behaviors as well as activity in a sensorimotor circuit . 5-ht alone can enhance the evoked activity ; however , 5-ht likely interacts with other known neuromodulators such as dopamine and octopamine , to fulfill specific physiological or behavioral functions .
it has been demonstrated that dysregulation in the 5-htergic system is related to various psychological conditions such as depression , anxiety , learning and memory impairment , schizophrenia , and autism in humans .
the disruption of physiological serotonin level in critical periods during mammalian development is postulated to abnormal development of neural circuitry that consequently results in the long - lasting psychological disorders .
many studies have already shown that 5-ht plays miscellaneous physiological and behavioral roles in drosophila .
also , it is known that 5-ht level plays a role in development of the 5-htergic neurons in the central nervous system ( cns ) of drosophila .
a previous study has demonstrated that 5-ht2 receptors are expressed in drosophila during the embryonic stage , and its physiological level of expression is essential for the normal development and survival .
however , the physiological role of 5-ht receptor subtypes in various neural circuit activities had yet to be further investigated .
neuromodulators , such as dopamine and serotonin , play a role in changing the activity of locomotor neural networks .
many studies have shown that 5-ht is implicated in locomotion behavior in diverse animals and relatively simple organisms such as c. elegans [ 32 , 55 ] .
it has been shown that the sensory input is indispensable for the locomotion activity of drosophila larvae .
the rapid central drive in locomotion is rapidly altered when an organism confronts eminent danger , which may well be influenced by neuromodulators .
neuromodulators are found in drosophila which function to modulate specific behaviors and thus maybe even survival in the outdoor environment under predatory stress .
it was also shown recently that various 5-ht receptors have a role in drosophila larvae locomotion and that 5-ht is involved in larval turning behavior . in the study herein ,
the 5-ht system was manipulated by using both pharmacological and genetic approaches and complements some aspects of the results reported by silva et al . .
we showed that feeding larvae high levels of 5-ht or 5-htp significantly reduced body wall contractions and reduced the body length .
it is likely that 5-htp also led to an increase in 5-ht synthesis but this does not necessarily mean more 5-ht is released with synaptic transmission . it was shown that administration of 5-htp increases the level of 5-ht in adult flies .
5-ht could alter drosophila development by acting on insulin producing cells ( ipcs ) in the brain [ 10 , 5860 ] .
this could prepare the organism for increased metabolic activity which can also be induced by promoting locomotive activity .
the mechanism for the retarded growth with very high levels of 5-ht is only speculative at this time .
additionally , the trh blocker , pcpa , significantly reduced locomotion behavior and it was demonstrated that pcpa administration results in the reduction of 5-ht in larval cns .
these results demonstrate that dysregulation of 5-ht with either too much or too little 5-ht influences locomotion behavior .
we further investigated the effect of 5-ht biosynthesis disturbance on locomotive behavior by overexpression of the trh gene specifically in 5-htergic neurons which also slowed larval locomotion . in order to determine whether 5-ht was increased in the synaptic cleft of 5-ht releasing neurons , we used an ssri ( fluoxetine ) . such potential increase in synaptic 5-ht also decreased locomotion behavior .
it has been confirmed that , in trh mutant larvae , the 5-ht level is reduced in 5-ht neurons .
possible residual 5-ht in neurons might be due to the 5-ht uptake by dsert from the hemolymph , which is made by the peripheral dthpu .
this may explain why administration of fluoxetine ( 5 mg / ml ) further reduces 5-ht level in trh mutant 5-ht neurons .
the affinity of fluoxetine for drosophila sert was previously determined to be lower ( ki = 72 nm ) than for mammals .
high concentrations of fluoxetine are toxic to larval and adult drosophila so we stuck to a 10 mm concentration for this study . increasing
the activity of 5-ht neurons through activation of the trpa1 channels selectively targeted these neurons and increased the locomotion behavior in third - instar larvae .
this is in contrast to the other manipulations in the levels of 5-ht which all decreased locomotion .
potentially over a long period of heightened neural activity there might be some compensation by possibly downregulating the expression of 5-ht receptors or possibly desensitization of the receptors . since we do not know if these neurons may have fatigued in the atp production required for vesicular fusion and recycling or possibly depleted the release of 5-ht with the electrical activity , more definitive studies are needed to answer these questions .
various 5-ht receptor subtypes ( 5-ht1a , 5-ht1b , 5-ht2a , 5-ht7 , and 5-ht2b ) are expressed in drosophila ; thus , our approach with rnai in 5-ht1b knockdown , in 5-ht1b expressing neurons , could specifically examine this receptor role in locomotion .
it is interesting to note that the 5-ht1b knockdown significantly reduced the locomotion but 5-ht2a and 5-ht7 receptor knockdown did not have an effect .
it is known that 5-ht acts on serotonergic neurons through activation of 5-ht autoreceptors to orchestrate the serotonergic neuron branch density [ 52 , 63 ] .
thus , the knockdown of 5-ht1b during early stages of development might increase the 5-ht neurons branching pattern which might result in increasing 5-ht concentration thereby changing the development of the neural circuitry .
this is another interesting avenue to follow up in the possible anatomical restructuring . in a recent study
, it has been shown that 5-ht receptor subtypes ( 5-ht1a , 5-ht1b , 5-ht2 , or 5-ht7 ) knockdown in the nervous
system increases locomotion activity when the flies were raised at 19c until the day before examining the behavior .
however , in our study , we raised the flies at 30c during early development .
the paradoxical results might be due to the effect of 5-ht1b on larval development since the gal4 activity would be high at 30c compared to 19c .
the efficiency of the 5-ht2 and 5-ht7 rnai lines to knock down 5-ht2 and 5-ht7 could be low since we did not observe a significant change in locomotion behavior .
5-ht receptors insertional mutant lines were also used and the results indicate that 5-ht2a and 5-ht7 insertional mutations significantly decreased locomotion behavior .
however , trh mutant larvae did not show a significant change in body wall contractions .
our results are consistent with silva et al . which demonstrated that 5-ht7 mutation leads to the reduction in locomotion activity ; however , they were contradictory to silva et al . where the trh mutation resulted in an increase in locomotive activity . in support of our findings for the trh mutant ,
the results are consistent with neckameyer et al . , where they demonstrated no significant difference between w and pbac - trh larvae body wall contractions .
the contradictory results might be due to using different approaches to measure the locomotion activity in various studies .
we wanted to address the effect of trh overexpression on larval locomotion and feeding behaviors as well since previous studies have shown that trh overexpression , which increases the 5-ht level , would change various fly behaviors such as sleep and aggression .
we observed that trh overexpression significantly reduced locomotion and feeding behaviors ( figures 4(a ) and 4(b ) ) , which corroborates the pharmacological results ( figure 2 ) . in the study herein , the 5-ht2b receptor mutation , also , decreased body wall contractions in third - instar larvae ( figure 8(a ) ) .
further support of 5-ht receptors being altered in their expression levels and the notion that the pbac - trh larvae decreased 5-ht synthesis which could lead to upregulation of the receptors is that these larvae show an enhanced response to exogenous application of 5-ht on the exposed sensory - cns - motor circuit .
together , these results demonstrate that dysfunction of 5-ht receptors during development negatively influences locomotion behavior .
additionally , we selectively activated 5-ht neurons with the use of optogenetics which demonstrated an effect on the sensory - cns - motor circuit . since an alteration in the 5-htergic system during development might have effect on neural circuit development , it is difficult to pinpoint specific mechanisms to account for the behavioral changes
. therefore , we also used genetic tools to acutely manipulate 5-ht neurons in freely moving animals .
the synaptic transmission of 5-ht neurons was blocked by expressing shi allele in 5-htergic neurons .
however , dumping of 5-ht by increasing the activity of the 5-htergic neurons , through trpa1 channel activation , significantly reduced the body wall contractions in larvae , which corroborates the recently published findings of pooryasin and fiala .
this could suggest that 5-ht spillover from 5-ht neurons in freely moving larvae has a negative effect on locomotion behavior . in a recent study
, it was stated that activation of trpa1 channels negatively impacts locomotion activity in drosophila larvae .
furthermore , our results also show that suppression of synaptic transmission in 5-ht neurons did not cause a significant change in climbing behavior ( figure 10(a ) ) ; however , acute activation of 5-ht by trpa1 significantly reduced the locomotion activities in adult male and female flies ( figures 10(b1 ) and 10(b2 ) ) .
it was previously shown that adult drosophila expressing shi allele in 5-ht neurons and control group would increase the locomotion activity when they are exposed to high temperature ( 30c ) compared to low temperature ( 19c ) .
this effect is related to the temperature change and not blocking the transmitter release by the neurons .
since the temperature likely has an effect on drosophila behavior per se , we used an optogenetic approach to activate 5-htergic neurons in freely moving larvae and adults .
our results show that activation of 5-htergic neurons reduces locomotion behavior in third - instar larvae and adult flies ( figure 16 ) .
the 5-htergic system , also , plays a role in feeding behavior in invertebrates [ 7 , 44 ] and vertebrates . for most parts , increasing or decreasing
5-ht altered the feeding behavior . in a previous study , it was demonstrated that 5-htp administration decreases feeding behavior in larvae ; however , reduction of 5-ht level through rnai - mediated trh knockdown increases feeding behavior . on the other hand , increasing trh level decreases feeding .
also , the mutation in trh gene increases the branching pattern of serotonergic neurons in proventriculus of the digestive system . in our study , chronic activation of 5-ht neurons by trpa1 channels increased feeding behavior .
however , 5-ht1b and 5-ht2a receptor knockdowns significantly decreased mouth hook movements , and 5-ht7 receptor knockdown did not produce significant change in feeding behavior .
it was demonstrated previously that blocking 5-ht2a receptors results in a reduction of feeding behavior which concurs with our studies .
5-ht1b receptor knockdown might increase the branching pattern of the 5-ht neurons to locate a target which can provide feedback regulation thereby increasing 5-ht release as volume transmission and suppressing feeding behavior . 5-ht2a and 5-ht7 mutant larvae significantly decreased mouth hook movements .
the reduction in 5-ht7 mutant feeding behavior might be due to the weak musculature structure because the larvae also had a significant reduction in body wall contractions .
the reduction of feeding behavior in 5-ht2a mutant larvae confirms the results of 5-ht2a knockdown larvae feeding behavior .
5-ht2b mutant larvae did not show a significant change in feeding behavior compared to w larvae .
taken together , our data demonstrate that 5-ht receptors play a role in feeding behavior of drosophila larvae .
it is already known that 5-ht application to the dissected third - instar larvae increases the evoked sensorimotor circuit activity [ 34 , 38 ] , even though the 5-ht receptor subtypes that mediate modulatory action of 5-ht had yet to be investigated . in the study herein , the results show that 5-ht increased sensorimotor circuit activity at low ( 100 nm ) concentrations but decreased the effect at higher concentrations
. this might be due to the desensitization of 5-ht receptors . in attempt to understand which 5-ht
for dopamine , it was recently shown that administration of a high concentration itself is toxic to drosophila .
thus , caution should be considered when exposing the drosophila larvae or adults to high concentration of any biogenic amines . since some of the 5-ht agonists responded in a similar fashion to 5-ht
5-ht2a mutant larvae ( homozygotes ) were less responsive at 100 nm concentration compared to control group ; however , 5-ht7 mutant larvae ( heterozygote mutants + / ) responded well compared to control group .
5-ht7 mutant larvae ( heterozygote mutant + / ) might have enough 5-ht7 receptors to respond well to 5-ht or 5-ht7 receptors might work cooperatively with 5-ht2 receptors to increase the circuit activity .
since trh mutant 5-ht neurons have less 5-ht , they might compensate for that by increasing sensitivity of the 5-ht receptor expressing neurons to 5-ht .
in future studies , it would be good to try some additional agonists and antagonists , such as clozapine , which are known to have a function in other species of insects .
we have shown that acute activation of 5-ht neurons by trpa1 channels decreased locomotion behavior in larvae . however , we do not know the underlying cellular mechanism behind this phenomenon .
therefore , 5-ht neurons were activated by trpa1 channels while the evoked sensorimotor activity was being recorded .
we noted that reduction in locomotion activity is due to the reduction in motor output .
activation of trpa1 channels in motor neurons , also , resulted in the reduction in locomotive activity but this reduction is due to the spastic contraction of the animals .
we have demonstrated that fluoxetine administration reduced locomotion behavior ; therefore , we applied fluoxetine to dissected third - instar larvae while evoked sensorimotor activities were being recorded .
fluoxetine at 100 m significantly reduced sensorimotor activity ; however , at a lower concentration , it did not have an effect .
a recent study has demonstrated that application of 100 m of fluoxetine while stimulating channelrhodopsin-2 expressing 5-ht neurons results in blockage of 5-ht reuptake in drosophila larval ventral nerve cord .
it has been shown that fluoxetine can block ion channels [ 68 , 69 ] ; therefore , the interpretation of these results is difficult since we have to ensure that the observed effect of fluoxetine is due to the dysregulation of 5-ht level and no other possible nonselective side effects ( see ) .
this opens new avenues for the future studies on the mechanism of action of fluoxetine in altering the development in neural circuitry . in order to confirm the effect of 5-htergic neurons activation by trpa1 channels
the results show that 5-htergic neuron activation , in chr2-expressing larvae and adults , by blue light exposure significantly decreased locomotion activity in both third - instar larvae and adult flies ( figure 16 ) .
the reason why canton - s and w controls flies showed differences in body wall contractions is not known but has been reported previously . however , it is known that the white gene product is part of tryptophan transporter , and tryptophan is the precursor of serotonin .
it has been shown that the white gene mutation results in the reduction of 5-ht , dopamine , and histamine content of synaptic vesicles ; therefore , the reduction in these biogenic amine levels in w larvae might account for the slow locomotion activity compared to cs larvae . the paradoxical action of exogenous 5-ht application in dissected third - instar larvae and acute activation of 5-htergic neurons on locomotion activity might be due to the activation of different 5-ht receptors and the degree of 5-htergic neuron activity . in mammalian system , the action of 5-ht also has shown various responses on spinal motor neuron activity .
low activity of 5-htergic neurons activates 5-ht2 receptors at the 5-ht - motor neuron synapse which in turn increases the excitability of motor neurons .
this results in a higher frequency of motor neuron activity compared to the absence of activity in the 5-htergic neurons .
however , when the 5-htergic neurons release a large amount of 5-ht , the 5-ht can reach 5-ht1a receptors outside the synapse , the activation of which reduces the activity of sodium channels , and in turn the frequency of action potentials decreases in motor neurons . as previously mentioned , the drosophila genome has five known 5-ht receptor genes .
activation of 5-ht1a or 5-ht1b receptor subtypes leads to the reduction of cytosolic camp level due to the suppression of adenylyl cyclase ( ac ) enzyme activity .
when 5-ht7 is activated , it will activate ac that leads to the increasing of cytosolic camp .
in mammalian systems , 5-ht2 is coupled with the gq protein and has also been shown for the fly calliphora vicina and other insects ( e.g. , honeybees ) .
when 5-ht binds to 5-ht2 receptors , it will activate phospholipase c ( plc ) enzyme .
these receptors are expressed in various neurons in multiple regions of the nervous system in drosophila .
it has been demonstrated that the sensory neurons of the gill - withdrawal reflex in aplysia can be sensitized by the action of 5-ht , which is released from interneurons .
active gs protein would synthesize camp from atp which in turn activates pka . in some cells , pka can lead to blockage of k channels by phosphorylation .
however , active gq can activate plc which in turn activates pkc , leading to phosphorylation of synaptic vesicle proteins .
we present a hypothetical model to explain the molecular mechanism of modulatory action of 5-ht in sensorimotor physiology ( figure 18 ) .
here we hypothesized that 5-ht binds with 5-ht2 receptor on presynaptic terminals activating gq and consequently phospholipase c ( plc ) , thereby enhancing protein kinase c ( pkc ) .
active pkc can then lead to the established phosphorylation of synaptic vesicle proteins to increase docking of synaptic vesicles .
5-ht might also activate 5-ht7 receptor which activates gs protein , which activates protein kinase a ( pka ) .
active pka can also phosphorylate various synaptic vesicle proteins also enhancing the number of docked vesicles and probability of evoked release .
local messenger rna ( mrna ) translation to make proteins , which is important for modulation of synaptic strength , might be another possible mechanism for the modulatory action by 5-ht .
it was demonstrated that , during long - term facilitation in an aplysia sensorimotor synapse , mrnas were being translated at the synaptic site . | serotonin modulates various physiological processes and behaviors .
this study investigates the role of 5-ht in locomotion and feeding behaviors as well as in modulation of sensory - motor circuits .
the 5-ht biosynthesis was dysregulated by feeding drosophila larvae 5-ht , a 5-ht precursor , or an inhibitor of tryptophan hydroxylase during early stages of development .
the effects of feeding fluoxetine , a selective serotonin reuptake inhibitor , during early second instars were also examined .
5-ht receptor subtypes were manipulated using rna interference mediated knockdown and 5-ht receptor insertional mutations . moreover ,
synaptic transmission at 5-ht neurons was blocked or enhanced in both larvae and adult flies .
the results demonstrate that disruption of components within the 5-ht system significantly impairs locomotion and feeding behaviors in larvae .
acute activation of 5-ht neurons disrupts normal locomotion activity in adult flies . to determine which 5-ht receptor subtype modulates the evoked sensory - motor activity , pharmacological agents were used .
in addition , the activity of 5-ht neurons was enhanced by expressing and activating trpa1 channels or channelrhodopsin-2 while recording the evoked excitatory postsynaptic potentials ( epsps ) in muscle fibers .
5-ht2 receptor activation mediates a modulatory role in a sensory - motor circuit , and the activation of 5-ht neurons can suppress the neural circuit activity , while fluoxetine can significantly decrease the sensory - motor activity . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion | the fundamental mechanisms of modulating neural circuits in relation to specific physiological actions and behaviors in organisms are an area of current interest for basic research as well as clinical treatments . the serotonergic ( 5-htergic ) system plays many roles in drosophila and other insects which are crucial for the animal survival and locomotion , feeding behaviors , learning and memory , aggression , circadian rhythms , sleep , heart rate , salivary secretion , insulin signaling , and synaptic transmission [ 817 ] . in addition , the fruit fly nervous system is an attractive model to decode the neural circuits and interrogate the function of genes of interest in animal behavior [ 21 , 22 ] . the drosophila genome harbors five different genes for the 5-ht receptors ( 5-ht1adro , 5-ht1bdro , 5-ht2adro , and 5-ht7dro ) and the recently cloned 5-ht2bdro receptor subtype . serotonin modulates locomotion activity in diverse groups of animals across the animal kingdom , for instance , in c. elegans [ 31 , 32 ] and mammals . in addition , we demonstrated that mutations in 5-ht receptor subtypes significantly reduce larval locomotion behavior . moreover , we show that overactivation of 5-ht neurons in freely moving larvae and adults significantly compromises locomotor activity . in this current study
, we further investigated the 5-ht receptor subtypes that mediate the modulatory action of 5-ht in a sensory - cns - motor circuit . 5-ht modulates the feeding behavior in mammals as well as in insects . this study demonstrates the role of 5-ht receptor subtypes and function of 5-ht releasing neurons in various behaviors within the ever increasingly invertebrate model d. melanogaster . the agonists and antagonists used are defined for mammalian preparations and details of binding affinity and efficacy of the various drosophila 5-ht receptor subtypes are not established . adult flies were transferred into empty vials , and they were placed inside an ice bucket for 2030 sec . the fly locomotion activities were recorded with a webcam ( webcam hd4110 , hewlett - packard company , palo alto , ca ) , which was connected to a computer , and the activity was recorded at 5 frames per second ( 5 fps ) for 10 min using virtualdub-1.10.4 software ( http://www.virtualdub.org/ ) . the locomotion activity was analyzed for two time points , for 10 sec at the 5th min and for 10 sec at 10th min using manual tracking ( http://rsbweb.nih.gov/ij/plugins/track/track.html ) in imagej software ( 1.47 v ) ( http://rsbweb.nih.gov/ij/download.html ) . a plastic vial ( drosophila culture cylindrical vial 1 - 1/4 diameter 4 tall )
was marked at 8 cm length , and the 1014 cohort flies were transferred to that empty marked vial . ,
the excitatory postsynaptic potentials ( epsps ) were recorded with an axoclamp 2b ( axon instruments , usa ) , converted with a powerlab , 4sp ( adinstruments , usa ) , and analyzed with labchart 7.0 ( adinstruments , usa ) . the suction electrodes which were used to stimulate the segmental nerves in each preparation were slightly different ; therefore , a range of stimulation voltages were used . depending on how tight the seal is with the suction electrode and the nerve , the voltage must be adjusted to evoked action potentials in the sensory nerves . a low level of 5-ht ( 1 mm , 10 mm ) resulted in significant increases in body length ; however , at high concentrations ( 50 mm and 100 mm ) , the body length
another approach to perturb the 5-ht level in neurons and systemically is by providing more of the precursor to synthesize 5-ht . so , instead of increasing 5-ht , a decrease in levels in the cns as well as systemic levels in 5-ht occurs . the 5-ht levels were not measured within the hemolymph for this strain ; however , we assume the strain works as well as it has in previous studies . in addition , one of the parental lines ( trh - gal4 ( ii ) ) was used as a control . chronic activation of the 5-ht neurons through activating trpa1 channels selectively expressed in 5-ht neurons significantly increased bwcs and mhms ( figures 6(a ) and 6(b ) ) . in addition , the use of one of the parental strains ( gal4 ) as a control supports the differential findings for the f1 strains . in determining which receptor subtypes may play a larger role in altering the larval behaviors , strains carrying insertional mutations in 5-ht receptors were examined . in addition , a pbac - trh strain was examined which would perturb the synthesis of 5-ht . thus , the synaptic vesicles within the presynaptic nerve terminals are not able to recycle . in this study , larvae which were expressing shi in 5-ht neurons did not markedly alter the rate in the body wall contractions compared to control ( uas - shi ) ( figure 9(a ) ) . it has been demonstrated that trpa1 does not become activated at room temperature ( 22 - 23c ) but when it is exposed to > 28c , the trpa1 channels become activated . the data indicate that acute activation of 5-ht neurons in third - instar larvae significantly decreased bwcs at 32c compared to control group ( uas - trpa1 ) ( figure 9(b ) ) . the locomotion activity in third - instar larvae
was also decreased when 5-ht neurons were activated by light , through expression of channelrhodopsin-2 ( chr2 ) , which complements these findings ( see the optogenetics results ) . , negative geotaxic assay ) was carried out to observe the effect of acute activity in 5-ht neurons on locomotion behavior in adult flies . when these flies were incubated at 37c for 10 min in the water bath , no significant changes were observed in locomotion behavior in both males and females in comparison to control flies ( figure 10(a ) ) . adult flies ( 4 - 5 flies in each group , males and females separately ) were placed in a petri dish which allowed the adults to walk around horizontally but not fly . both controls ( uas - trpa1 ) and trpa1 expressing flies
the locomotor activity was recorded for 10 min , and the activity was analyzed for 10 sec at 5th and 10 sec at 10th min . acute activation of trpa1 channels in 5-ht neurons significantly reduced the locomotor activity in male and female trpa1 expressing flies at 5th and 10th min compared to male and female control groups ( uas - trpa1 ) . the locomotor activity in adult flies was also decreased when 5-ht neurons were activated by the channelrhodopsin chr2-xxl ( figure 17(c ) ) . sensory - motor circuit activity was used to examine the influence of 5-ht on the circuit activity in dissected third - instar larvae . the rationale of conducting these acute studies in dissected preparations is to directly examine the action of 5-ht on a neural circuit in which one could control the electrical activity of sensory neurons and examine a defined motor output on a body wall muscle . the segmental nerves were stimulated for 8 stimulation trains in the saline and 8 stimulation trains or more after exchanging saline with saline containing 5-ht , 5-ht agonists , a selective serotonin reuptake inhibitor ( ssri , fluoxetine ) , or prewarmed saline , which was used in trpa1 expressing larvae . when saline was exchanged with saline - containing 5-ht , the number of evoked excitatory postsynaptic potentials ( epsps ) significantly increased ( figure 11(b ) ) . other 5-ht agonists were used for further confirmation of the 5-ht receptors subtypes that mediate modulatory role of 5-ht in sensory - motor circuit activity . 5-ht receptor subtype mutant lines mentioned previously were used for electrophysiological studies to corroborate the pharmacological results . in addition , the exogenous application of 5-ht in dissected third - instar larvae significantly increased the sensorimotor activity in 5-ht2a receptor mutant flies , although the response of 5-ht2a receptor flies to 5-ht was lower than the control flies ( cs ) . in 5-ht7 mutant larvae , 5-ht application slightly increased the sensory - motor activity but at p > 0.1 for group comparisons from controls due to the high variation in this line ( figure 14 ) . to examine overactivity of the 5-htergic neurons , the same sensory - cns - motor paradigm was used in third - instar larvae ( uas - trpa1 ) at room temperature ( 22 - 23c ) ( figure 15(a1 ) ) and when switched with prewarmed saline ( 37c ) . the results indicate that acute activation of 5-htergic neurons by trpa1 cation channels ( trh - gal4 ( iii)>uas - trpa1 ) showed a decrease in activity when warmed at 92.4% significance ( p = 0.076 ) and in comparing this line to the changes in the parental line ( uas - trpa1 ) only a change occurred at 91% significance ( p < 0.09 ) ( figure 15(c ) ) . the sensory - motor circuit was stimulated at 40 hz , 10 stimuli , and the epsps were recorded in saline ( control ) ( figure 17(a ) ) . fluoxetine is a selective serotonin reuptake inhibitor ( ssri ) which would potentially cause a rise in the 5-ht within the synaptic cleft . in the current study , we investigated the role of the 5-htergic system in locomotion and feeding behaviors as well as activity in a sensorimotor circuit . however , the physiological role of 5-ht receptor subtypes in various neural circuit activities had yet to be further investigated . neuromodulators , such as dopamine and serotonin , play a role in changing the activity of locomotor neural networks . it has been shown that the sensory input is indispensable for the locomotion activity of drosophila larvae . it was also shown recently that various 5-ht receptors have a role in drosophila larvae locomotion and that 5-ht is involved in larval turning behavior . in the study herein ,
the 5-ht system was manipulated by using both pharmacological and genetic approaches and complements some aspects of the results reported by silva et al . it was shown that administration of 5-htp increases the level of 5-ht in adult flies . additionally , the trh blocker , pcpa , significantly reduced locomotion behavior and it was demonstrated that pcpa administration results in the reduction of 5-ht in larval cns . these results demonstrate that dysregulation of 5-ht with either too much or too little 5-ht influences locomotion behavior . we further investigated the effect of 5-ht biosynthesis disturbance on locomotive behavior by overexpression of the trh gene specifically in 5-htergic neurons which also slowed larval locomotion . in order to determine whether 5-ht was increased in the synaptic cleft of 5-ht releasing neurons , we used an ssri ( fluoxetine ) . it has been confirmed that , in trh mutant larvae , the 5-ht level is reduced in 5-ht neurons . increasing
the activity of 5-ht neurons through activation of the trpa1 channels selectively targeted these neurons and increased the locomotion behavior in third - instar larvae . since we do not know if these neurons may have fatigued in the atp production required for vesicular fusion and recycling or possibly depleted the release of 5-ht with the electrical activity , more definitive studies are needed to answer these questions . various 5-ht receptor subtypes ( 5-ht1a , 5-ht1b , 5-ht2a , 5-ht7 , and 5-ht2b ) are expressed in drosophila ; thus , our approach with rnai in 5-ht1b knockdown , in 5-ht1b expressing neurons , could specifically examine this receptor role in locomotion . it is known that 5-ht acts on serotonergic neurons through activation of 5-ht autoreceptors to orchestrate the serotonergic neuron branch density [ 52 , 63 ] . thus , the knockdown of 5-ht1b during early stages of development might increase the 5-ht neurons branching pattern which might result in increasing 5-ht concentration thereby changing the development of the neural circuitry . in a recent study
, it has been shown that 5-ht receptor subtypes ( 5-ht1a , 5-ht1b , 5-ht2 , or 5-ht7 ) knockdown in the nervous
system increases locomotion activity when the flies were raised at 19c until the day before examining the behavior . 5-ht receptors insertional mutant lines were also used and the results indicate that 5-ht2a and 5-ht7 insertional mutations significantly decreased locomotion behavior . in support of our findings for the trh mutant ,
the results are consistent with neckameyer et al . we wanted to address the effect of trh overexpression on larval locomotion and feeding behaviors as well since previous studies have shown that trh overexpression , which increases the 5-ht level , would change various fly behaviors such as sleep and aggression . we observed that trh overexpression significantly reduced locomotion and feeding behaviors ( figures 4(a ) and 4(b ) ) , which corroborates the pharmacological results ( figure 2 ) . further support of 5-ht receptors being altered in their expression levels and the notion that the pbac - trh larvae decreased 5-ht synthesis which could lead to upregulation of the receptors is that these larvae show an enhanced response to exogenous application of 5-ht on the exposed sensory - cns - motor circuit . together , these results demonstrate that dysfunction of 5-ht receptors during development negatively influences locomotion behavior . additionally , we selectively activated 5-ht neurons with the use of optogenetics which demonstrated an effect on the sensory - cns - motor circuit . the synaptic transmission of 5-ht neurons was blocked by expressing shi allele in 5-htergic neurons . however , dumping of 5-ht by increasing the activity of the 5-htergic neurons , through trpa1 channel activation , significantly reduced the body wall contractions in larvae , which corroborates the recently published findings of pooryasin and fiala . in a recent study
, it was stated that activation of trpa1 channels negatively impacts locomotion activity in drosophila larvae . furthermore , our results also show that suppression of synaptic transmission in 5-ht neurons did not cause a significant change in climbing behavior ( figure 10(a ) ) ; however , acute activation of 5-ht by trpa1 significantly reduced the locomotion activities in adult male and female flies ( figures 10(b1 ) and 10(b2 ) ) . our results show that activation of 5-htergic neurons reduces locomotion behavior in third - instar larvae and adult flies ( figure 16 ) . in a previous study , it was demonstrated that 5-htp administration decreases feeding behavior in larvae ; however , reduction of 5-ht level through rnai - mediated trh knockdown increases feeding behavior . in our study , chronic activation of 5-ht neurons by trpa1 channels increased feeding behavior . 5-ht1b receptor knockdown might increase the branching pattern of the 5-ht neurons to locate a target which can provide feedback regulation thereby increasing 5-ht release as volume transmission and suppressing feeding behavior . taken together , our data demonstrate that 5-ht receptors play a role in feeding behavior of drosophila larvae . it is already known that 5-ht application to the dissected third - instar larvae increases the evoked sensorimotor circuit activity [ 34 , 38 ] , even though the 5-ht receptor subtypes that mediate modulatory action of 5-ht had yet to be investigated . in the study herein , the results show that 5-ht increased sensorimotor circuit activity at low ( 100 nm ) concentrations but decreased the effect at higher concentrations
. since some of the 5-ht agonists responded in a similar fashion to 5-ht
5-ht2a mutant larvae ( homozygotes ) were less responsive at 100 nm concentration compared to control group ; however , 5-ht7 mutant larvae ( heterozygote mutants + / ) responded well compared to control group . since trh mutant 5-ht neurons have less 5-ht , they might compensate for that by increasing sensitivity of the 5-ht receptor expressing neurons to 5-ht . we have shown that acute activation of 5-ht neurons by trpa1 channels decreased locomotion behavior in larvae . therefore , 5-ht neurons were activated by trpa1 channels while the evoked sensorimotor activity was being recorded . a recent study has demonstrated that application of 100 m of fluoxetine while stimulating channelrhodopsin-2 expressing 5-ht neurons results in blockage of 5-ht reuptake in drosophila larval ventral nerve cord . it has been shown that fluoxetine can block ion channels [ 68 , 69 ] ; therefore , the interpretation of these results is difficult since we have to ensure that the observed effect of fluoxetine is due to the dysregulation of 5-ht level and no other possible nonselective side effects ( see ) . in order to confirm the effect of 5-htergic neurons activation by trpa1 channels
the results show that 5-htergic neuron activation , in chr2-expressing larvae and adults , by blue light exposure significantly decreased locomotion activity in both third - instar larvae and adult flies ( figure 16 ) . however , it is known that the white gene product is part of tryptophan transporter , and tryptophan is the precursor of serotonin . it has been shown that the white gene mutation results in the reduction of 5-ht , dopamine , and histamine content of synaptic vesicles ; therefore , the reduction in these biogenic amine levels in w larvae might account for the slow locomotion activity compared to cs larvae . the paradoxical action of exogenous 5-ht application in dissected third - instar larvae and acute activation of 5-htergic neurons on locomotion activity might be due to the activation of different 5-ht receptors and the degree of 5-htergic neuron activity . low activity of 5-htergic neurons activates 5-ht2 receptors at the 5-ht - motor neuron synapse which in turn increases the excitability of motor neurons . this results in a higher frequency of motor neuron activity compared to the absence of activity in the 5-htergic neurons . however , when the 5-htergic neurons release a large amount of 5-ht , the 5-ht can reach 5-ht1a receptors outside the synapse , the activation of which reduces the activity of sodium channels , and in turn the frequency of action potentials decreases in motor neurons . it has been demonstrated that the sensory neurons of the gill - withdrawal reflex in aplysia can be sensitized by the action of 5-ht , which is released from interneurons . we present a hypothetical model to explain the molecular mechanism of modulatory action of 5-ht in sensorimotor physiology ( figure 18 ) . | [
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since the early 1970s there have been dramatic improvements in the availability and quality of treatment for persons with hemophilia ( mannucci and tuddenham 2001 ) . as a result of this progress , barring the consequences of the human immunodeficiency virus ( hiv ) epidemics in the 1980s , the life span of hemophiliacs has progressively become similar to that of males in the general population , at least in more developed countries ( mejia - carvajal et al 2006 ) .
accordingly , a considerable number of hemophiliacs now reach old age : in italy , for instance , nearly 8% of persons with severe hemophilia a or b are 65 years old or older ( tagliaferri et al 2006 ) .
however , with age , persons with hemophilia develop medical and surgical diseases ( eg , cardiovascular diseases , prostatic hypertrophy , cancers , renal disease ) not previously seen in this group .
this aspect , together with the management of these patients , is discussed in the first part of this review .
the second part addresses the main features and clinical management of acquired hemophilia due to anti - factor viii antibodies , an acquired disease that may occur in elderly people .
however , the vast majority of them have evaluated only subgroups of patients , not the whole population .
the mortality of patients infected by the hepatitis c virus ( hcv ) or hiv has been specially analyzed .
for example , darby et al ( 1997 ) analyzed mortality in hemophiliacs in the united kingdom infused with blood products contaminated with hcv .
other studies evaluated the mortality rate among hiv - infected patients in spain ( del amo et al 2006 ) , the united kingdom ( darby et al 2004a ) and canada ( arnold et al 2006 ) . finally , another study investigated the effect of factor viii and ix inhibitors on mortality in a hemophilia population ( darby 2004b ) .
although only a small number of studies have so far evaluated mortality in the whole population of hemophiliacs ( street et al 2006 ) , these are interesting because they cover the whole history of the clinical - therapeutic approach to hemophilia , from the dramatic increase in life expectancy between the 1940s and 1980s with the advent of replacement therapy with plasma - derived clotting factor to the catastrophic contamination of the plasma pool by hiv and hcv in the 1980s ( street et al 2006 ) . in parallel with the therapeutic progresses , the causes of death have changed in hemophilia , hemorrhage being replaced by the acquired immunodeficiency syndrome ( aids ) as the leading cause of death ( mejia - carvajal et al 2006 ) .
indeed , the mortality curve in hemophiliacs over the years has a biphasic form with two peaks : one before the 1960s ( before the widespread availability of factor replacement therapy ) and the other in the 1980s1990s ( after the occurrence of aids and of the clinical manifestations of earlier hcv infections ) .
this trend has been confirmed by a recent survey that analyzed the mortality rate and causes of death in 967 dutch hemophiliacs ( plug et al 2006 ) . during the period of the study ( 19922001 ) the mortality rate was 2.3 times higher in patients with hemophilia than in the general male population .
moreover , comparing these data with those previously reported by the same group ( rosendaal et al 1989 ; triemstra et al 1995 ) , the life expectancy of patients with severe hemophilia decreased from 63 years in the period 19731986 to 61 and 59 years in the period 19861992 and 19922001 , respectively .
however , the exclusion of virus - related deaths resulted in a life expectancy at birth of 74 years , similar to that of the unaffected dutch male population .
similar trends were observed in studies involving scottish ( ludlam 2000 ) and canadian ( walker 1998 ) hemophiliacs . from 1900 to 1942 the life expectancy of severe hemophiliacs in sweden was 16.5 years ; this increased to 23.2 years between 1943 and 1957 ( ramgren 1962 ) and to 50 years between 1957 and 1980 ( larsson and wiechel 1983 ) . for comparison , the life expectancy of unaffected swedish men during the latter period was 75 years
another study analyzed 163 patients with severe hemophilia a living in finland in the period between 193079 ( ikkala 1982 ) . during 50 years of observation
, the mean age at death increased from 7.8 years in 193039 to 25.5 years in 197079 .
the effect of hiv infection on age and causes of death among persons with hemophilia a was analyzed in a study conducted in the united states ( chorba et al 2001 ) . the median age at death decreased from 55 years in 19791982 to 40.5 years in 19871990 and increased to 46 years in 19951998 , although the median age at death of patients with hemophilia a and hiv - related disease in the latter period was 33 years , compared to 72 years for those without hiv - related disease . during the same period ,
hemophilia a - associated deaths decreased by 41% , with a 78% decrease among those who had hiv - related disease .
this decline in hiv mortality was consistent with that observed in the general population of hiv - infected persons and reflected improvements in anti - retroviral therapy .
on the other hand , another study that analyzed the causes of death among hemophiliacs not infected by hiv found that the leading cause of death was hemorrhage , followed by liver disease ( hepatitis c and b ) , stroke and cancer .
currently , children with hemophilia look forward to a normal life expectancy and excellent health - related quality of life ( gringeri et al 2006 ) .
the most important factors contributing to the improved quality of life , reduced morbidity and increased life expectancy are the availability for replacement therapy of high - quality factor concentrates ( ie , virus inactivated plasma - derived clotting factor concentrates and recombinant products ) , insights into the management of hemophilic arthropathy and liver disease ( ie , surveillance of patients with chronic hepatitis , especially with respect to cancer and liver failure , and newer treatment options such as antiviral treatment against hiv and hcv and liver transplantation ) and the improved medical management provided by specialized hemophilia treatment centers .
high - quality factor concentrates are important not only for reducing the likelihood of death from hemorrhage but also for improving the quality of life , especially through prophylactic regimens and home treatment .
secondary prophylaxis , in particular , is becoming frequently prescribed among hemophiliacs , although only few data are available in this setting ( tagliaferri et al 2006a ) .
the impact of comprehensive care has been demonstrated by soucie et al ( 2006 ) who found that hemophiliacs who had received care in a specialized hemophilia treatment center had a significantly decreased risk of death .
the impact of comprehensive care is further exemplified by the analysis of 164 patients treated at the international haemophilia training centre at bangkok with blood and blood components from 1971 to 2000 .
the estimated probability of survival beyond 13 years of age of patients with severe hemophilia increased from 0.85 during the first decade of observation to 0.94 and 1 in the second and third decades ( chuansumrit et al 2004 ) .
the mortality rate fell from 30% to 14% and 5% over the three decades of observation . despite the lack of treatment with high quality clotting factor
concentrates in this center , the patients outcome improved with the development of a comprehensive approach to management . on behalf of the italian association of hemophilia centers ( aice )
, we are conducting a multicenter study to evaluate the health status and health - related quality of life ( hr - qol ) of severely affected italian hemophiliacs of 65 years or older , recruited from italian hemophilia centers ( tagliaferri et al 2006b ) .
another primary goal of the study is to provide information on whether or not there are differences in the global health status and hr - qol between healthy elderly individuals and persons with hemophilia , who started their lives in the first part of the last century when the availability and quality of treatment were poor .
this study will also allow us to establish whether or not the burden of diseases other than hemophilia ( including cognitive impairment measured using instruments of geriatric medicine ) is similar or different in cases and controls , and to understand which factors help people with severe hemophilia to cope with their disease and reach old age .
hemophilia is associated with bleeding episodes , which often occur into the joints or muscles . over time , recurrent bleeding into joints and muscles can cause permanent problems such as arthritis , chronic pain and joint damage resulting in poor control of balance and falls which ultimately worsen quality of life .
thus joint disease remains the leading cause of morbidity in those elderly hemophiliacs who had little or no access to prophylactic regimens and home treatment when they were younger .
an ongoing australian study is evaluating the degree of joint - related dysfunction in persons with hemophilia older than 30 years and the feasibility of home exercise programs targeting balance training ( street 2006 ) .
we have recently reported the case of a 72-year - old hemophiliac with severe joint bleeding in whom treatment was converted from on - demand to a secondary prophylaxis regimen ( tagliaferri et al 2006a ) .
the latter significantly reduced the number of bleeding episodes in target joints , slowing the progression of the arthropathy and improving the patient s perceived well - being .
however , further studies , dealing with the approach to replacement therapy and the development of preventive physical therapy programs for elderly patients , are warranted .
attempts have been made to determine whether or not hemophilia protects from the development of atherosclerosis and cardiovascular events , but the results are contradictory .
studies of carotid and femoral artery intimalmedial thickness as a surrogate of atherosclerosis burden showed that hemophilia does not protect from atherosclerosis ( sramek et al 2001 ) . on the other hand ,
hemophiliacs have a decreased mortality rate due to ischemic heart disease , supporting the views that they are protected from arterial thrombosis ( sramek et al 2003 ) .
recently , girolami et al ( 2006 ) reviewed reported cardiovascular events in patients with hemophilia a. among the 42 cases published so far , nine occurred in individuals aged 65 or over . analyzing the literature data
, the authors concluded that the bleeding diathesis did not help to prevent the occurrence of arterial occlusions .
thus factors which are known to predispose to atherothrombosis ( eg , hypercholesterolemia , smoking , diabetes , hypertension , aging ) appear to overcome the coagulation defect .
concomitant replacement therapy , especially with bypassing agents ( ie , activated or non - activated prothrombin complex concentrates and recombinant activated factor vii ) , was identified as having a predominant role in thrombotic manifestations in the majority of the cases ( aledort 2004 ) .
a number of successful cardiovascular interventions in elderly hemophiliacs have been carried out ( bovenzi et al 2003 ; de bels et al 2004 ) .
however , these cases raise the question of how to conduct anticoagulant therapy in such naturally anticoagulated patients .
the management of cardiovascular events in elderly hemophiliacs necessarily differs from that of non - hemophilic patients .
thus , while the treatment of acute coronary syndromes in hemophiliacs requires a combination of antithrombotic and replacement therapies , thrombolytic therapy for myocardial infarctions is unjustified in these patients due to the unacceptable risk of hemorrhage and should be substituted by alternative techniques such as percutaneous coronary angioplasty ( mafrici et al 2003 ) .
similarly , long - term anticoagulation should be avoided whenever possible by using , for example , a bioprosthetic valve in valve surgery .
elderly people with hemophilia are also at increased risk of chronic renal disease ( kulkarni et al 2003 ) .
this is probably due to chronic renal bleeding associated with the inherited coagulopathy which leads to structural renal abnormalities .
moreover , the concomitant presence of viral infections ( eg , hiv and hcv ) or exposure to nephrotoxic antiviral therapy may increase the incidence of renal dysfunction over time .
chronic renal disease is responsible for hypertension ( scully et al 2005 ) , which is observed more frequently in elderly hemophiliacs than in the general population and is a risk factor not only for cardiac disease but also for cerebral hemorrhage .
although most inhibitors occur at a young age in patients with severe hemophilia , patients with mild hemophilia may develop inhibitors at an advanced age when they receive intensive factor viii replacement therapy on the occasion of a surgical or invasive procedure for age - related problems such as prostatic hypertrophy , cardiovascular disease or cancer ( franchini et al 2006a ) .
indeed , there are some concerns on the use of bypassing agents in those patients considered at higher risk of developing thrombotic complications .
however , leebek et al ( 2006 ) have recently reported the safe and effective use of recombinant activated factor vii in three elderly patients with mild hemophilia a and high - titer inhibitors .
however , the vast majority of them have evaluated only subgroups of patients , not the whole population .
the mortality of patients infected by the hepatitis c virus ( hcv ) or hiv has been specially analyzed .
for example , darby et al ( 1997 ) analyzed mortality in hemophiliacs in the united kingdom infused with blood products contaminated with hcv .
other studies evaluated the mortality rate among hiv - infected patients in spain ( del amo et al 2006 ) , the united kingdom ( darby et al 2004a ) and canada ( arnold et al 2006 ) . finally , another study investigated the effect of factor viii and ix inhibitors on mortality in a hemophilia population ( darby 2004b ) .
although only a small number of studies have so far evaluated mortality in the whole population of hemophiliacs ( street et al 2006 ) , these are interesting because they cover the whole history of the clinical - therapeutic approach to hemophilia , from the dramatic increase in life expectancy between the 1940s and 1980s with the advent of replacement therapy with plasma - derived clotting factor to the catastrophic contamination of the plasma pool by hiv and hcv in the 1980s ( street et al 2006 ) . in parallel with the therapeutic progresses , the causes of death have changed in hemophilia , hemorrhage being replaced by the acquired immunodeficiency syndrome ( aids ) as the leading cause of death ( mejia - carvajal et al 2006 ) .
indeed , the mortality curve in hemophiliacs over the years has a biphasic form with two peaks : one before the 1960s ( before the widespread availability of factor replacement therapy ) and the other in the 1980s1990s ( after the occurrence of aids and of the clinical manifestations of earlier hcv infections ) .
this trend has been confirmed by a recent survey that analyzed the mortality rate and causes of death in 967 dutch hemophiliacs ( plug et al 2006 ) . during the period of the study ( 19922001 ) the mortality rate was 2.3 times higher in patients with hemophilia than in the general male population .
moreover , comparing these data with those previously reported by the same group ( rosendaal et al 1989 ; triemstra et al 1995 ) , the life expectancy of patients with severe hemophilia decreased from 63 years in the period 19731986 to 61 and 59 years in the period 19861992 and 19922001 , respectively .
however , the exclusion of virus - related deaths resulted in a life expectancy at birth of 74 years , similar to that of the unaffected dutch male population .
similar trends were observed in studies involving scottish ( ludlam 2000 ) and canadian ( walker 1998 ) hemophiliacs . from 1900 to 1942 the life expectancy of severe hemophiliacs in sweden was 16.5 years ; this increased to 23.2 years between 1943 and 1957 ( ramgren 1962 ) and to 50 years between 1957 and 1980 ( larsson and wiechel 1983 ) . for comparison , the life expectancy of unaffected swedish men during the latter period was 75 years
another study analyzed 163 patients with severe hemophilia a living in finland in the period between 193079 ( ikkala 1982 ) . during 50 years of observation
, the mean age at death increased from 7.8 years in 193039 to 25.5 years in 197079 .
the effect of hiv infection on age and causes of death among persons with hemophilia a was analyzed in a study conducted in the united states ( chorba et al 2001 ) . the median age at death decreased from 55 years in 19791982 to 40.5 years in 19871990 and increased to 46 years in 19951998 , although the median age at death of patients with hemophilia a and hiv - related disease in the latter period was 33 years , compared to 72 years for those without hiv - related disease . during the same period ,
hemophilia a - associated deaths decreased by 41% , with a 78% decrease among those who had hiv - related disease .
this decline in hiv mortality was consistent with that observed in the general population of hiv - infected persons and reflected improvements in anti - retroviral therapy .
on the other hand , another study that analyzed the causes of death among hemophiliacs not infected by hiv found that the leading cause of death was hemorrhage , followed by liver disease ( hepatitis c and b ) , stroke and cancer .
currently , children with hemophilia look forward to a normal life expectancy and excellent health - related quality of life ( gringeri et al 2006 ) .
the most important factors contributing to the improved quality of life , reduced morbidity and increased life expectancy are the availability for replacement therapy of high - quality factor concentrates ( ie , virus inactivated plasma - derived clotting factor concentrates and recombinant products ) , insights into the management of hemophilic arthropathy and liver disease ( ie , surveillance of patients with chronic hepatitis , especially with respect to cancer and liver failure , and newer treatment options such as antiviral treatment against hiv and hcv and liver transplantation ) and the improved medical management provided by specialized hemophilia treatment centers .
high - quality factor concentrates are important not only for reducing the likelihood of death from hemorrhage but also for improving the quality of life , especially through prophylactic regimens and home treatment .
secondary prophylaxis , in particular , is becoming frequently prescribed among hemophiliacs , although only few data are available in this setting ( tagliaferri et al 2006a ) .
the impact of comprehensive care has been demonstrated by soucie et al ( 2006 ) who found that hemophiliacs who had received care in a specialized hemophilia treatment center had a significantly decreased risk of death .
the impact of comprehensive care is further exemplified by the analysis of 164 patients treated at the international haemophilia training centre at bangkok with blood and blood components from 1971 to 2000 .
the estimated probability of survival beyond 13 years of age of patients with severe hemophilia increased from 0.85 during the first decade of observation to 0.94 and 1 in the second and third decades ( chuansumrit et al 2004 ) .
the mortality rate fell from 30% to 14% and 5% over the three decades of observation . despite the lack of treatment with high quality clotting factor
concentrates in this center , the patients outcome improved with the development of a comprehensive approach to management . on behalf of the italian association of hemophilia centers ( aice )
, we are conducting a multicenter study to evaluate the health status and health - related quality of life ( hr - qol ) of severely affected italian hemophiliacs of 65 years or older , recruited from italian hemophilia centers ( tagliaferri et al 2006b ) .
another primary goal of the study is to provide information on whether or not there are differences in the global health status and hr - qol between healthy elderly individuals and persons with hemophilia , who started their lives in the first part of the last century when the availability and quality of treatment were poor .
this study will also allow us to establish whether or not the burden of diseases other than hemophilia ( including cognitive impairment measured using instruments of geriatric medicine ) is similar or different in cases and controls , and to understand which factors help people with severe hemophilia to cope with their disease and reach old age .
hemophilia is associated with bleeding episodes , which often occur into the joints or muscles . over time , recurrent bleeding into joints and muscles can cause permanent problems such as arthritis , chronic pain and joint damage resulting in poor control of balance and falls which ultimately worsen quality of life .
thus joint disease remains the leading cause of morbidity in those elderly hemophiliacs who had little or no access to prophylactic regimens and home treatment when they were younger .
an ongoing australian study is evaluating the degree of joint - related dysfunction in persons with hemophilia older than 30 years and the feasibility of home exercise programs targeting balance training ( street 2006 ) .
we have recently reported the case of a 72-year - old hemophiliac with severe joint bleeding in whom treatment was converted from on - demand to a secondary prophylaxis regimen ( tagliaferri et al 2006a ) .
the latter significantly reduced the number of bleeding episodes in target joints , slowing the progression of the arthropathy and improving the patient s perceived well - being .
however , further studies , dealing with the approach to replacement therapy and the development of preventive physical therapy programs for elderly patients , are warranted .
attempts have been made to determine whether or not hemophilia protects from the development of atherosclerosis and cardiovascular events , but the results are contradictory .
studies of carotid and femoral artery intimalmedial thickness as a surrogate of atherosclerosis burden showed that hemophilia does not protect from atherosclerosis ( sramek et al 2001 ) . on the other hand ,
hemophiliacs have a decreased mortality rate due to ischemic heart disease , supporting the views that they are protected from arterial thrombosis ( sramek et al 2003 ) .
recently , girolami et al ( 2006 ) reviewed reported cardiovascular events in patients with hemophilia a. among the 42 cases published so far , nine occurred in individuals aged 65 or over .
analyzing the literature data , the authors concluded that the bleeding diathesis did not help to prevent the occurrence of arterial occlusions .
thus factors which are known to predispose to atherothrombosis ( eg , hypercholesterolemia , smoking , diabetes , hypertension , aging ) appear to overcome the coagulation defect . concomitant replacement therapy , especially with bypassing agents ( ie , activated or non - activated prothrombin complex concentrates and recombinant activated factor vii ) , was identified as having a predominant role in thrombotic manifestations in the majority of the cases ( aledort 2004 ) .
a number of successful cardiovascular interventions in elderly hemophiliacs have been carried out ( bovenzi et al 2003 ; de bels et al 2004 ) .
however , these cases raise the question of how to conduct anticoagulant therapy in such naturally anticoagulated patients .
the management of cardiovascular events in elderly hemophiliacs necessarily differs from that of non - hemophilic patients .
thus , while the treatment of acute coronary syndromes in hemophiliacs requires a combination of antithrombotic and replacement therapies , thrombolytic therapy for myocardial infarctions is unjustified in these patients due to the unacceptable risk of hemorrhage and should be substituted by alternative techniques such as percutaneous coronary angioplasty ( mafrici et al 2003 ) .
similarly , long - term anticoagulation should be avoided whenever possible by using , for example , a bioprosthetic valve in valve surgery .
elderly people with hemophilia are also at increased risk of chronic renal disease ( kulkarni et al 2003 ) .
this is probably due to chronic renal bleeding associated with the inherited coagulopathy which leads to structural renal abnormalities .
moreover , the concomitant presence of viral infections ( eg , hiv and hcv ) or exposure to nephrotoxic antiviral therapy may increase the incidence of renal dysfunction over time .
chronic renal disease is responsible for hypertension ( scully et al 2005 ) , which is observed more frequently in elderly hemophiliacs than in the general population and is a risk factor not only for cardiac disease but also for cerebral hemorrhage .
although most inhibitors occur at a young age in patients with severe hemophilia , patients with mild hemophilia may develop inhibitors at an advanced age when they receive intensive factor viii replacement therapy on the occasion of a surgical or invasive procedure for age - related problems such as prostatic hypertrophy , cardiovascular disease or cancer ( franchini et al 2006a ) .
indeed , there are some concerns on the use of bypassing agents in those patients considered at higher risk of developing thrombotic complications .
however , leebek et al ( 2006 ) have recently reported the safe and effective use of recombinant activated factor vii in three elderly patients with mild hemophilia a and high - titer inhibitors .
acquired hemophilia is an uncommon but potentially life - threatening clinical syndrome characterized by the sudden onset of bleeding in patients with a negative family and personal history .
acquired hemophilia is caused by auto - antibodies that , in the vast majority of cases , are directed against functional epitopes of factor viii causing its neutralization and/or accelerated clearance from the plasma ( delgado et al 2003 ) .
the incidence of acquired hemophilia has been estimated to be 0.21.0 cases per 1 million persons per year , with a mortality rate estimated to be in the range of 8 to 22% ( franchini et al 2005a ) .
the age distribution of autoantibodies is typically biphasic , with a small peak between 20 and 30 years ( mainly postpartum inhibitors ) and a larger peak in patients aged 6880 years .
factor viii inhibitors are distributed equally between sexes , although females predominate in the younger age group because of the association with pregnancy while males constitute the majority of patients with inhibitors over the age of 60 ( boggio and green 2001 ) . in approximately 50% of cases
factor viii autoantibodies occur in patients without concomitant diseases ( spontaneous antibodies ) , and in nearly 10% of cases autoantibodies appear during the postpartum period , usually in primiparous women within 3 months of delivery ( green and lechner 1981 ; solymoss 1998 ) .
however , several other conditions and diseases ( eg , autoimmune disorders , malignancies and drugs ) have been associated with the development of factor viii inhibitors ( bossi et al 1998 ) .
the clinical picture of acquired hemophilia differs from that of classical hereditary hemophilia a. more than 80% of patients with factor viii autoantibodies bleed into the skin , muscles or soft tissues ( including retroperitoneal hematoma ) and mucous membranes ( eg , epistaxis , gastrointestinal and urological bleeds ) , whereas hemarthroses , a typical manifestation of congenital factor viii deficiency , are unusual . not rarely the hemorrhages in acquired hemophilia are serious or life - threatening , as in the case of cerebral hemorrhage or rapidly progressive retroperitoneal hematomas ( franchini et al 2005a ) . the diagnosis of acquired hemophilia is based on the demonstration of an isolated prolongation of the activated partial thromboplastin time , not corrected by incubating the patient s plasma with equal volumes of normal plasma ( mixing study ) , associated with low factor viii levels and evidence of a factor viii inhibitor ( which can be titrated in bethesda units ( bu)/ml ) , in a patient with no previous personal or family history of bleeding ( cohen and kessler 1996 ) .
figure 1 reports the laboratory algorithm for the diagnosis of acquired hemophilia . the treatment of acquired hemophilia is classically divided into two phases : acute and chronic .
acute phase treatment is focused on the bleeding symptoms ( ludlam et al 1994 ) .
efficient hemostasis can be achieved with a variety of methods such as correction of fviii deficiency ( plasma - derived or recombinant fviii concentrates , desmopressin ) or bypassing the inhibitor ( activated prothrombin complex concentrate [ apcc ] , recombinant activated fvii [ rfviia ] ) ( roberts 1999 ) .
the choice of antihemorrhagic therapy depends mainly on the site and the entity of the bleeding . in patients with a high titer of inhibitor and severe hemorrhage , extracorporeal removal of the autoantibody by therapeutic plasmapheresis or immunoadsorption of immunoglobulins to staphylococcal protein
the aim of the chronic phase of treatment is to eradicate the inhibitor , which is achieved by using immunosuppressive agents including corticosteroids and such drugs as cyclophosphamide , azathioprine , 6-mercaptopurine and vincristine ( green et al 1993 ) .
high - dose immunoglobulins and cyclosporin have also been shown to be effective in acquired hemophilia and can be considered as second - line therapy for those patients who do not respond to standard immunosuppressive regimens ( schwartz et al 1995 ; petrovic et al 2000 ) .
more recently , serotherapy with anti - cd20 monoclonal antibody ( rituximab ) has been successfully used in immune disorders resulting from autoantibody formation , including acquired hemophilia ( wiestner et al 2002 ) .
finally , immune tolerance induction protocols have been proposed for the eradication of autoantibodies against coagulation factors ( nemes and pitlik 2000 ) . as reported above , with the exception of post - partum cases ,
although acquired hemophilia is considered a very rare occurrence , its true incidence may be underestimated .
indeed , patients may be seen by several specialists and undergo dangerous invasive investigations and interventions before the correct diagnosis is made .
the high mortality rate reported previously is in part related to a delay in the diagnosis . besides misdiagnoses
, acquired hemophilia may be underdiagnosed as many cases of clinically silent low titer inhibitors may be unrecognized unless patients undergo surgery or trauma . in approximately 50% of the elderly patients with acquired hemophilia ,
no underlying primary disorders are identified ( idiopathic forms ) , while most of the remaining cases are associated with autoimmune diseases or cancer , which increase with age ( hauser et al 1999 ; yee et al 2000 ) .
the prognosis of acquired hemophilia in elderly patients is strongly linked to the associated primary disorder , which should be always searched for and , if possible , cured .
idiopathic cases with a low inhibitor titer and mild bleeding may be managed with desmopressin alone and may not require eradicating therapy but only a watch and wait strategy .
similarly , drug - induced inhibitors may require nothing else than close observation , since these inhibitors tend to disappear spontaneously within a few months after drug discontinuation .
on the other hand , it is unlikely that cases associated with autoimmune disorders will remit spontaneously and thus they require more aggressive eradication therapy with steroids , whether or not combined with cytotoxic agents such as cyclophosphamide ( franchini et al 2005b ) . however , particular care must be taken when using these agents in the elderly and the choice of drugs and dosages should be tailored to the individual patient s age .
an analysis of the data from the european acquired hemophilia registry ( baudo et al 2004 ) showed that infections related to immunosuppressive therapy are the first cause of death in patients with acquired hemophilia .
thus , less toxic agents should be preferred for inhibitor eradication in elderly patients . in this context
we have recently reviewed the literature data on the use of rituximab in acquired hemophilia and have collected 50 cases , including many elderly patients ( franchini et al 2006b ) .
although most of the reports were isolated cases , there was a high%age of complete or partial responses ( 86% ) when rituximab was used alone or in association with immunosuppressive therapy .
thus , if confirmed by further controlled studies , this agent could be very useful in the management of such patients .
the presence of an underlying malignancy was associated with a poorer prognosis in a recent meta - analysis by delgado et al ( 2003 ) .
however , sallah et al ( 2001 ) , in a previous review of literature data on 41 cases of patients with cancer - associated factor viii inhibitors , including patients with solid and hematologic malignancies , found a 70% rate of complete responses to treatment of the inhibitor .
low titer inhibitors associated with early stage tumors were more likely to disappear after treatment than high titer inhibitors and the eradication of autoantibody was associated with a higher overall survival .
the same authors also observed that 22% of responders achieved complete remission after treatment of the cancer . on the basis of this high response rate
, it appears that the treatment of the primary malignancy in patients with cancer - associated factors viii inhibitors is of great importance , because this facilitates eradication of the antibody .
the presence of an underlying cancer is not a contraindication to the use of immunosuppressive therapy aimed at eradicating the autoantibody , so that these patients should be treated in the same manner as other patients with acquired hemophilia .
although only a few thrombotic adverse events have been recorded in patients with acquired hemophilia following the use of bypassing agents ( guillet et al 2002 ; roberts et al 2004 ) , particular care should be exercised when these drugs are used in elderly patients who should be monitored not only for clinical evidence of hemorrhage but also for the possible onset of thrombotic complications .
concomitant cardiovascular diseases should be ruled out before using desmopressin in elderly patients with congenital or acquired hemophilia ( mannucci 1997 ) .
acquired hemophilia is an uncommon but potentially life - threatening clinical syndrome characterized by the sudden onset of bleeding in patients with a negative family and personal history .
acquired hemophilia is caused by auto - antibodies that , in the vast majority of cases , are directed against functional epitopes of factor viii causing its neutralization and/or accelerated clearance from the plasma ( delgado et al 2003 ) .
the incidence of acquired hemophilia has been estimated to be 0.21.0 cases per 1 million persons per year , with a mortality rate estimated to be in the range of 8 to 22% ( franchini et al 2005a ) .
the age distribution of autoantibodies is typically biphasic , with a small peak between 20 and 30 years ( mainly postpartum inhibitors ) and a larger peak in patients aged 6880 years .
factor viii inhibitors are distributed equally between sexes , although females predominate in the younger age group because of the association with pregnancy while males constitute the majority of patients with inhibitors over the age of 60 ( boggio and green 2001 ) . in approximately 50% of cases
factor viii autoantibodies occur in patients without concomitant diseases ( spontaneous antibodies ) , and in nearly 10% of cases autoantibodies appear during the postpartum period , usually in primiparous women within 3 months of delivery ( green and lechner 1981 ; solymoss 1998 ) .
however , several other conditions and diseases ( eg , autoimmune disorders , malignancies and drugs ) have been associated with the development of factor viii inhibitors ( bossi et al 1998 ) .
the clinical picture of acquired hemophilia differs from that of classical hereditary hemophilia a. more than 80% of patients with factor viii autoantibodies bleed into the skin , muscles or soft tissues ( including retroperitoneal hematoma ) and mucous membranes ( eg , epistaxis , gastrointestinal and urological bleeds ) , whereas hemarthroses , a typical manifestation of congenital factor viii deficiency , are unusual . not rarely the hemorrhages in acquired hemophilia are serious or life - threatening , as in the case of cerebral hemorrhage or rapidly progressive retroperitoneal hematomas ( franchini et al 2005a ) . the diagnosis of acquired hemophilia is based on the demonstration of an isolated prolongation of the activated partial thromboplastin time , not corrected by incubating the patient s plasma with equal volumes of normal plasma ( mixing study ) , associated with low factor viii levels and evidence of a factor viii inhibitor ( which can be titrated in bethesda units ( bu)/ml ) , in a patient with no previous personal or family history of bleeding ( cohen and kessler 1996 ) .
figure 1 reports the laboratory algorithm for the diagnosis of acquired hemophilia . the treatment of acquired hemophilia is classically divided into two phases : acute and chronic .
acute phase treatment is focused on the bleeding symptoms ( ludlam et al 1994 ) .
efficient hemostasis can be achieved with a variety of methods such as correction of fviii deficiency ( plasma - derived or recombinant fviii concentrates , desmopressin ) or bypassing the inhibitor ( activated prothrombin complex concentrate [ apcc ] , recombinant activated fvii [ rfviia ] ) ( roberts 1999 ) .
the choice of antihemorrhagic therapy depends mainly on the site and the entity of the bleeding . in patients with a high titer of inhibitor and severe hemorrhage , extracorporeal removal of the autoantibody by therapeutic plasmapheresis or immunoadsorption of immunoglobulins to staphylococcal protein
the aim of the chronic phase of treatment is to eradicate the inhibitor , which is achieved by using immunosuppressive agents including corticosteroids and such drugs as cyclophosphamide , azathioprine , 6-mercaptopurine and vincristine ( green et al 1993 ) .
high - dose immunoglobulins and cyclosporin have also been shown to be effective in acquired hemophilia and can be considered as second - line therapy for those patients who do not respond to standard immunosuppressive regimens ( schwartz et al 1995 ; petrovic et al 2000 ) .
more recently , serotherapy with anti - cd20 monoclonal antibody ( rituximab ) has been successfully used in immune disorders resulting from autoantibody formation , including acquired hemophilia ( wiestner et al 2002 ) .
finally , immune tolerance induction protocols have been proposed for the eradication of autoantibodies against coagulation factors ( nemes and pitlik 2000 ) .
as reported above , with the exception of post - partum cases , a large number of patients with acquired hemophilia are elderly .
although acquired hemophilia is considered a very rare occurrence , its true incidence may be underestimated .
indeed , patients may be seen by several specialists and undergo dangerous invasive investigations and interventions before the correct diagnosis is made .
the high mortality rate reported previously is in part related to a delay in the diagnosis . besides misdiagnoses
, acquired hemophilia may be underdiagnosed as many cases of clinically silent low titer inhibitors may be unrecognized unless patients undergo surgery or trauma . in approximately 50% of the elderly patients with acquired hemophilia ,
no underlying primary disorders are identified ( idiopathic forms ) , while most of the remaining cases are associated with autoimmune diseases or cancer , which increase with age ( hauser et al 1999 ; yee et al 2000 ) .
the prognosis of acquired hemophilia in elderly patients is strongly linked to the associated primary disorder , which should be always searched for and , if possible , cured .
idiopathic cases with a low inhibitor titer and mild bleeding may be managed with desmopressin alone and may not require eradicating therapy but only a watch and wait strategy .
similarly , drug - induced inhibitors may require nothing else than close observation , since these inhibitors tend to disappear spontaneously within a few months after drug discontinuation .
on the other hand , it is unlikely that cases associated with autoimmune disorders will remit spontaneously and thus they require more aggressive eradication therapy with steroids , whether or not combined with cytotoxic agents such as cyclophosphamide ( franchini et al 2005b ) . however , particular care must be taken when using these agents in the elderly and the choice of drugs and dosages should be tailored to the individual patient s age .
an analysis of the data from the european acquired hemophilia registry ( baudo et al 2004 ) showed that infections related to immunosuppressive therapy are the first cause of death in patients with acquired hemophilia .
thus , less toxic agents should be preferred for inhibitor eradication in elderly patients . in this context
we have recently reviewed the literature data on the use of rituximab in acquired hemophilia and have collected 50 cases , including many elderly patients ( franchini et al 2006b ) .
although most of the reports were isolated cases , there was a high%age of complete or partial responses ( 86% ) when rituximab was used alone or in association with immunosuppressive therapy .
thus , if confirmed by further controlled studies , this agent could be very useful in the management of such patients .
the presence of an underlying malignancy was associated with a poorer prognosis in a recent meta - analysis by delgado et al ( 2003 ) .
however , sallah et al ( 2001 ) , in a previous review of literature data on 41 cases of patients with cancer - associated factor viii inhibitors , including patients with solid and hematologic malignancies , found a 70% rate of complete responses to treatment of the inhibitor .
low titer inhibitors associated with early stage tumors were more likely to disappear after treatment than high titer inhibitors and the eradication of autoantibody was associated with a higher overall survival .
the same authors also observed that 22% of responders achieved complete remission after treatment of the cancer . on the basis of this high response rate
, it appears that the treatment of the primary malignancy in patients with cancer - associated factors viii inhibitors is of great importance , because this facilitates eradication of the antibody .
the presence of an underlying cancer is not a contraindication to the use of immunosuppressive therapy aimed at eradicating the autoantibody , so that these patients should be treated in the same manner as other patients with acquired hemophilia .
although only a few thrombotic adverse events have been recorded in patients with acquired hemophilia following the use of bypassing agents ( guillet et al 2002 ; roberts et al 2004 ) , particular care should be exercised when these drugs are used in elderly patients who should be monitored not only for clinical evidence of hemorrhage but also for the possible onset of thrombotic complications .
concomitant cardiovascular diseases should be ruled out before using desmopressin in elderly patients with congenital or acquired hemophilia ( mannucci 1997 ) .
with their greatly prolonged life expectancy , persons with congenital hemophilia are now developing medical and surgical conditions previously not seen in this group .
the new challenge for physicians in hemophilia centers is to provide optimal care for this aging population of patients .
their optimal management requires a hemophilia team with close cooperation among physicians from different specialties such as hematology , oncology , cardiology , and nephrology .
particular care must be taken when choosing the inhibitor eradication regimen , which should be tailored to the patient s age . | after the increasing rate of deaths observed during the 1980s due to human immunodeficiency virus ( hiv ) infection , the health - related quality of life and life expectancy of persons with hemophilia have improved , mainly due to the progresses of replacement therapy and antiviral drugs and to the improvement of the global comprehensive care provided by specialized centers . as a consequence
, an increasing number of hemophiliacs have reached an older age and nowadays physicians in hemophilia centers find that they must handle age - related clinical problems never previously observed in this population .
the management of elderly persons with congenital hemophilia is discussed in the first part of this review .
the second part describes the general aspects of acquired hemophilia due to anti - factor viii autoantibodies , focusing on the clinical management of elderly patients , one of the groups most frequently affected by this acquired bleeding disorder . | Introduction
Management of congenital hemophilia in the elderly
Life-expectancy, causes of death and quality of life
Clinical management of congenital hemophilia in elderly patients
Management of acquired hemophilia in the elderly
General aspects of acquired hemophilia
Clinical management of acquired hemophilia in elderly patients
Conclusions | since the early 1970s there have been dramatic improvements in the availability and quality of treatment for persons with hemophilia ( mannucci and tuddenham 2001 ) . as a result of this progress , barring the consequences of the human immunodeficiency virus ( hiv ) epidemics in the 1980s , the life span of hemophiliacs has progressively become similar to that of males in the general population , at least in more developed countries ( mejia - carvajal et al 2006 ) . accordingly , a considerable number of hemophiliacs now reach old age : in italy , for instance , nearly 8% of persons with severe hemophilia a or b are 65 years old or older ( tagliaferri et al 2006 ) . this aspect , together with the management of these patients , is discussed in the first part of this review . the second part addresses the main features and clinical management of acquired hemophilia due to anti - factor viii antibodies , an acquired disease that may occur in elderly people . although only a small number of studies have so far evaluated mortality in the whole population of hemophiliacs ( street et al 2006 ) , these are interesting because they cover the whole history of the clinical - therapeutic approach to hemophilia , from the dramatic increase in life expectancy between the 1940s and 1980s with the advent of replacement therapy with plasma - derived clotting factor to the catastrophic contamination of the plasma pool by hiv and hcv in the 1980s ( street et al 2006 ) . indeed , the mortality curve in hemophiliacs over the years has a biphasic form with two peaks : one before the 1960s ( before the widespread availability of factor replacement therapy ) and the other in the 1980s1990s ( after the occurrence of aids and of the clinical manifestations of earlier hcv infections ) . during the period of the study ( 19922001 ) the mortality rate was 2.3 times higher in patients with hemophilia than in the general male population . moreover , comparing these data with those previously reported by the same group ( rosendaal et al 1989 ; triemstra et al 1995 ) , the life expectancy of patients with severe hemophilia decreased from 63 years in the period 19731986 to 61 and 59 years in the period 19861992 and 19922001 , respectively . however , the exclusion of virus - related deaths resulted in a life expectancy at birth of 74 years , similar to that of the unaffected dutch male population . from 1900 to 1942 the life expectancy of severe hemophiliacs in sweden was 16.5 years ; this increased to 23.2 years between 1943 and 1957 ( ramgren 1962 ) and to 50 years between 1957 and 1980 ( larsson and wiechel 1983 ) . for comparison , the life expectancy of unaffected swedish men during the latter period was 75 years
another study analyzed 163 patients with severe hemophilia a living in finland in the period between 193079 ( ikkala 1982 ) . the effect of hiv infection on age and causes of death among persons with hemophilia a was analyzed in a study conducted in the united states ( chorba et al 2001 ) . the median age at death decreased from 55 years in 19791982 to 40.5 years in 19871990 and increased to 46 years in 19951998 , although the median age at death of patients with hemophilia a and hiv - related disease in the latter period was 33 years , compared to 72 years for those without hiv - related disease . this decline in hiv mortality was consistent with that observed in the general population of hiv - infected persons and reflected improvements in anti - retroviral therapy . currently , children with hemophilia look forward to a normal life expectancy and excellent health - related quality of life ( gringeri et al 2006 ) . the most important factors contributing to the improved quality of life , reduced morbidity and increased life expectancy are the availability for replacement therapy of high - quality factor concentrates ( ie , virus inactivated plasma - derived clotting factor concentrates and recombinant products ) , insights into the management of hemophilic arthropathy and liver disease ( ie , surveillance of patients with chronic hepatitis , especially with respect to cancer and liver failure , and newer treatment options such as antiviral treatment against hiv and hcv and liver transplantation ) and the improved medical management provided by specialized hemophilia treatment centers . the estimated probability of survival beyond 13 years of age of patients with severe hemophilia increased from 0.85 during the first decade of observation to 0.94 and 1 in the second and third decades ( chuansumrit et al 2004 ) . on behalf of the italian association of hemophilia centers ( aice )
, we are conducting a multicenter study to evaluate the health status and health - related quality of life ( hr - qol ) of severely affected italian hemophiliacs of 65 years or older , recruited from italian hemophilia centers ( tagliaferri et al 2006b ) . another primary goal of the study is to provide information on whether or not there are differences in the global health status and hr - qol between healthy elderly individuals and persons with hemophilia , who started their lives in the first part of the last century when the availability and quality of treatment were poor . an ongoing australian study is evaluating the degree of joint - related dysfunction in persons with hemophilia older than 30 years and the feasibility of home exercise programs targeting balance training ( street 2006 ) . however , further studies , dealing with the approach to replacement therapy and the development of preventive physical therapy programs for elderly patients , are warranted . on the other hand ,
hemophiliacs have a decreased mortality rate due to ischemic heart disease , supporting the views that they are protected from arterial thrombosis ( sramek et al 2003 ) . concomitant replacement therapy , especially with bypassing agents ( ie , activated or non - activated prothrombin complex concentrates and recombinant activated factor vii ) , was identified as having a predominant role in thrombotic manifestations in the majority of the cases ( aledort 2004 ) . although most inhibitors occur at a young age in patients with severe hemophilia , patients with mild hemophilia may develop inhibitors at an advanced age when they receive intensive factor viii replacement therapy on the occasion of a surgical or invasive procedure for age - related problems such as prostatic hypertrophy , cardiovascular disease or cancer ( franchini et al 2006a ) . although only a small number of studies have so far evaluated mortality in the whole population of hemophiliacs ( street et al 2006 ) , these are interesting because they cover the whole history of the clinical - therapeutic approach to hemophilia , from the dramatic increase in life expectancy between the 1940s and 1980s with the advent of replacement therapy with plasma - derived clotting factor to the catastrophic contamination of the plasma pool by hiv and hcv in the 1980s ( street et al 2006 ) . indeed , the mortality curve in hemophiliacs over the years has a biphasic form with two peaks : one before the 1960s ( before the widespread availability of factor replacement therapy ) and the other in the 1980s1990s ( after the occurrence of aids and of the clinical manifestations of earlier hcv infections ) . during the period of the study ( 19922001 ) the mortality rate was 2.3 times higher in patients with hemophilia than in the general male population . moreover , comparing these data with those previously reported by the same group ( rosendaal et al 1989 ; triemstra et al 1995 ) , the life expectancy of patients with severe hemophilia decreased from 63 years in the period 19731986 to 61 and 59 years in the period 19861992 and 19922001 , respectively . however , the exclusion of virus - related deaths resulted in a life expectancy at birth of 74 years , similar to that of the unaffected dutch male population . from 1900 to 1942 the life expectancy of severe hemophiliacs in sweden was 16.5 years ; this increased to 23.2 years between 1943 and 1957 ( ramgren 1962 ) and to 50 years between 1957 and 1980 ( larsson and wiechel 1983 ) . for comparison , the life expectancy of unaffected swedish men during the latter period was 75 years
another study analyzed 163 patients with severe hemophilia a living in finland in the period between 193079 ( ikkala 1982 ) . the effect of hiv infection on age and causes of death among persons with hemophilia a was analyzed in a study conducted in the united states ( chorba et al 2001 ) . the median age at death decreased from 55 years in 19791982 to 40.5 years in 19871990 and increased to 46 years in 19951998 , although the median age at death of patients with hemophilia a and hiv - related disease in the latter period was 33 years , compared to 72 years for those without hiv - related disease . this decline in hiv mortality was consistent with that observed in the general population of hiv - infected persons and reflected improvements in anti - retroviral therapy . currently , children with hemophilia look forward to a normal life expectancy and excellent health - related quality of life ( gringeri et al 2006 ) . the most important factors contributing to the improved quality of life , reduced morbidity and increased life expectancy are the availability for replacement therapy of high - quality factor concentrates ( ie , virus inactivated plasma - derived clotting factor concentrates and recombinant products ) , insights into the management of hemophilic arthropathy and liver disease ( ie , surveillance of patients with chronic hepatitis , especially with respect to cancer and liver failure , and newer treatment options such as antiviral treatment against hiv and hcv and liver transplantation ) and the improved medical management provided by specialized hemophilia treatment centers . the estimated probability of survival beyond 13 years of age of patients with severe hemophilia increased from 0.85 during the first decade of observation to 0.94 and 1 in the second and third decades ( chuansumrit et al 2004 ) . on behalf of the italian association of hemophilia centers ( aice )
, we are conducting a multicenter study to evaluate the health status and health - related quality of life ( hr - qol ) of severely affected italian hemophiliacs of 65 years or older , recruited from italian hemophilia centers ( tagliaferri et al 2006b ) . another primary goal of the study is to provide information on whether or not there are differences in the global health status and hr - qol between healthy elderly individuals and persons with hemophilia , who started their lives in the first part of the last century when the availability and quality of treatment were poor . an ongoing australian study is evaluating the degree of joint - related dysfunction in persons with hemophilia older than 30 years and the feasibility of home exercise programs targeting balance training ( street 2006 ) . however , further studies , dealing with the approach to replacement therapy and the development of preventive physical therapy programs for elderly patients , are warranted . on the other hand ,
hemophiliacs have a decreased mortality rate due to ischemic heart disease , supporting the views that they are protected from arterial thrombosis ( sramek et al 2003 ) . although most inhibitors occur at a young age in patients with severe hemophilia , patients with mild hemophilia may develop inhibitors at an advanced age when they receive intensive factor viii replacement therapy on the occasion of a surgical or invasive procedure for age - related problems such as prostatic hypertrophy , cardiovascular disease or cancer ( franchini et al 2006a ) . acquired hemophilia is caused by auto - antibodies that , in the vast majority of cases , are directed against functional epitopes of factor viii causing its neutralization and/or accelerated clearance from the plasma ( delgado et al 2003 ) . the incidence of acquired hemophilia has been estimated to be 0.21.0 cases per 1 million persons per year , with a mortality rate estimated to be in the range of 8 to 22% ( franchini et al 2005a ) . factor viii inhibitors are distributed equally between sexes , although females predominate in the younger age group because of the association with pregnancy while males constitute the majority of patients with inhibitors over the age of 60 ( boggio and green 2001 ) . in approximately 50% of cases
factor viii autoantibodies occur in patients without concomitant diseases ( spontaneous antibodies ) , and in nearly 10% of cases autoantibodies appear during the postpartum period , usually in primiparous women within 3 months of delivery ( green and lechner 1981 ; solymoss 1998 ) . the clinical picture of acquired hemophilia differs from that of classical hereditary hemophilia a. more than 80% of patients with factor viii autoantibodies bleed into the skin , muscles or soft tissues ( including retroperitoneal hematoma ) and mucous membranes ( eg , epistaxis , gastrointestinal and urological bleeds ) , whereas hemarthroses , a typical manifestation of congenital factor viii deficiency , are unusual . the diagnosis of acquired hemophilia is based on the demonstration of an isolated prolongation of the activated partial thromboplastin time , not corrected by incubating the patient s plasma with equal volumes of normal plasma ( mixing study ) , associated with low factor viii levels and evidence of a factor viii inhibitor ( which can be titrated in bethesda units ( bu)/ml ) , in a patient with no previous personal or family history of bleeding ( cohen and kessler 1996 ) . the treatment of acquired hemophilia is classically divided into two phases : acute and chronic . in approximately 50% of the elderly patients with acquired hemophilia ,
no underlying primary disorders are identified ( idiopathic forms ) , while most of the remaining cases are associated with autoimmune diseases or cancer , which increase with age ( hauser et al 1999 ; yee et al 2000 ) . the prognosis of acquired hemophilia in elderly patients is strongly linked to the associated primary disorder , which should be always searched for and , if possible , cured . however , particular care must be taken when using these agents in the elderly and the choice of drugs and dosages should be tailored to the individual patient s age . an analysis of the data from the european acquired hemophilia registry ( baudo et al 2004 ) showed that infections related to immunosuppressive therapy are the first cause of death in patients with acquired hemophilia . in this context
we have recently reviewed the literature data on the use of rituximab in acquired hemophilia and have collected 50 cases , including many elderly patients ( franchini et al 2006b ) . however , sallah et al ( 2001 ) , in a previous review of literature data on 41 cases of patients with cancer - associated factor viii inhibitors , including patients with solid and hematologic malignancies , found a 70% rate of complete responses to treatment of the inhibitor . on the basis of this high response rate
, it appears that the treatment of the primary malignancy in patients with cancer - associated factors viii inhibitors is of great importance , because this facilitates eradication of the antibody . the presence of an underlying cancer is not a contraindication to the use of immunosuppressive therapy aimed at eradicating the autoantibody , so that these patients should be treated in the same manner as other patients with acquired hemophilia . concomitant cardiovascular diseases should be ruled out before using desmopressin in elderly patients with congenital or acquired hemophilia ( mannucci 1997 ) . acquired hemophilia is caused by auto - antibodies that , in the vast majority of cases , are directed against functional epitopes of factor viii causing its neutralization and/or accelerated clearance from the plasma ( delgado et al 2003 ) . the incidence of acquired hemophilia has been estimated to be 0.21.0 cases per 1 million persons per year , with a mortality rate estimated to be in the range of 8 to 22% ( franchini et al 2005a ) . factor viii inhibitors are distributed equally between sexes , although females predominate in the younger age group because of the association with pregnancy while males constitute the majority of patients with inhibitors over the age of 60 ( boggio and green 2001 ) . in approximately 50% of cases
factor viii autoantibodies occur in patients without concomitant diseases ( spontaneous antibodies ) , and in nearly 10% of cases autoantibodies appear during the postpartum period , usually in primiparous women within 3 months of delivery ( green and lechner 1981 ; solymoss 1998 ) . the clinical picture of acquired hemophilia differs from that of classical hereditary hemophilia a. more than 80% of patients with factor viii autoantibodies bleed into the skin , muscles or soft tissues ( including retroperitoneal hematoma ) and mucous membranes ( eg , epistaxis , gastrointestinal and urological bleeds ) , whereas hemarthroses , a typical manifestation of congenital factor viii deficiency , are unusual . the diagnosis of acquired hemophilia is based on the demonstration of an isolated prolongation of the activated partial thromboplastin time , not corrected by incubating the patient s plasma with equal volumes of normal plasma ( mixing study ) , associated with low factor viii levels and evidence of a factor viii inhibitor ( which can be titrated in bethesda units ( bu)/ml ) , in a patient with no previous personal or family history of bleeding ( cohen and kessler 1996 ) . in approximately 50% of the elderly patients with acquired hemophilia ,
no underlying primary disorders are identified ( idiopathic forms ) , while most of the remaining cases are associated with autoimmune diseases or cancer , which increase with age ( hauser et al 1999 ; yee et al 2000 ) . the prognosis of acquired hemophilia in elderly patients is strongly linked to the associated primary disorder , which should be always searched for and , if possible , cured . however , particular care must be taken when using these agents in the elderly and the choice of drugs and dosages should be tailored to the individual patient s age . an analysis of the data from the european acquired hemophilia registry ( baudo et al 2004 ) showed that infections related to immunosuppressive therapy are the first cause of death in patients with acquired hemophilia . in this context
we have recently reviewed the literature data on the use of rituximab in acquired hemophilia and have collected 50 cases , including many elderly patients ( franchini et al 2006b ) . however , sallah et al ( 2001 ) , in a previous review of literature data on 41 cases of patients with cancer - associated factor viii inhibitors , including patients with solid and hematologic malignancies , found a 70% rate of complete responses to treatment of the inhibitor . on the basis of this high response rate
, it appears that the treatment of the primary malignancy in patients with cancer - associated factors viii inhibitors is of great importance , because this facilitates eradication of the antibody . the presence of an underlying cancer is not a contraindication to the use of immunosuppressive therapy aimed at eradicating the autoantibody , so that these patients should be treated in the same manner as other patients with acquired hemophilia . concomitant cardiovascular diseases should be ruled out before using desmopressin in elderly patients with congenital or acquired hemophilia ( mannucci 1997 ) . with their greatly prolonged life expectancy , persons with congenital hemophilia are now developing medical and surgical conditions previously not seen in this group . the new challenge for physicians in hemophilia centers is to provide optimal care for this aging population of patients . | [
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] |
the hydration thermodynamics of analogs
of amino acid side chains
( or of amino acid side chains in small - model peptides ) has often been
used to understand protein folding and protein protein association .
a very small sampling of the many pioneering investigations where
this approach has been used includes work identifying hydrophobicity
as a dominant force in protein folding , investigations of protein denaturation , interpretation of calorimetric
data on protein unfolding , and , more recently , investigations
on the role of osmolytes in protein folding . drawing upon
insights attributed to cohn and edsall ,
,
the free energy of unfolding is given as a sum of the free energy
of transfer of the small component groups of the molecule ,
from the environment they have in the native form , to the environment
they have in the unfolded form .
accounting for subsequent refinements that included corrections
for the solvent - exposure of the small component groups
( cf . ref ( 9 ) ) , the
generic equation of the unfolding free energy ( gnu ) can be written
as1where gi is the free energy of transferring the group i from some reference phase to liquid water and i is a factor that corrects for the change
in solvent - exposure of the group i in protein unfolding .
in interpretation of calorimetric data , for example ,
a gas - phase reference is natural , as is also the case
in this article .
the same form of equation can also be used to describe
the effect of osmolytes on protein unfolding : identifying gi with the free energy for
transferring the group from water to the aqueous osmolyte solution
and i with the change in the solvent
exposure of the group upon unfolding in the osmolyte solution relative
to water , the previous equation directly gives the so - called m value for 1 m osmolyte . at a time when theory , simulations , and experimental techniques
were less developed than they are now , the group - additive approach
was a pragmatic first step to understand the hydration thermodynamics
of a complicated macromolecule .
but it is also important to assess
its limitations and probe if the physical conclusions based on this
approach are valid .
for example , the group - additive model appears
to capture the effect of osmolytes rather well , with the predicted and experimentally determined m values agreeing to within a couple kilocalories per mole
for proteins with about 100 residues . on this basis , it has been suggested
that some osmolytes largely act by tuning the solubility of the peptide
backbone , but it is unclear how to reconcile
this observation with simulations that suggest that urea - induced denaturation
is mediated by promiscuous urea protein interactions tuned
by typically nonspecific dispersion forces .
furthermore , our previous study on the solvation of a peptide group showed that in the water - to - osmolyte transfer
solvent - mediated correlations between peptide units ( in glyn ) cancel , and this allows one to identify a peptide - group
transfer free energy that is model - independent .
but in the vapor - to - liquid
transfer , a situation where solvent - mediated correlations are preserved ,
the identified group - additive contribution ( gi , eq 1 ) depends
on the model used to define the peptide group .
several recent
studies have explored the limitations of the additive
model . while our work emphasized solvent - mediated
correlations , knig et al .
have emphasized
the role of self - solvation in limiting additivity . in exploring context dependence of hydration ,
these authors also suggested how adding a methyl group to an amino
acid changes the solvent - exposure of the peptide backbone , thereby
influencing the excess free energy in a way that can not be captured
by side - chain analog data .
building on these efforts , here we study
how well a model of the side chain describes the hydration of the
side chain in the context of a protein .
in particular , our aim is
to better understand factors limiting additivity and the reasons why
scaling model - compound data to describe its properties in a protein
context may not always be satisfactory . to aid in parsing energetic
differences , we study the conditional solvation of an isoleucine residue ,
often regarded as very hydrophobic , in
the context of model - extended and helical peptides .
butane in the
gauche conformation , matching exactly the side - chain conformation
of isoleucine in extended peptides , is used as a small - molecule analog
of the side chain . in obtaining mechanistic insight from free - energy
calculations ,
any decomposition
into components that are not themselves state functions can lead to
path dependencies in the analysis .
however , with adequate care , such components can yield insights into the molecular scale features
underlying the free energy that are of first interest .
here we use
the quasichemical approach to separate the packing ( steric )
contributions from the hydrophilic contributions to both facilitate
the calculation and also provide insights into
the limitations of additivity . these components ( but not the net free
energy )
do depend on the specification of a hydration shell , but the
approach has nevertheless provided important insights into the physics
of hydration and in generating models of molecular solutions .
we complement the quasichemical analysis with the traditional
decomposition
of the excess free energy into its enthalpic and entropic contributions .
the quasichemical approach helps us better appreciate how the context
influences the hydration of the solute , while the enthalpy entropy
decomposition leads to the finding that nonobvious contributions from
the reference ( the context ) get folded into the conditional contribution
attributed to the side chain ( the solute ) that is of first interest
in additive models .
next , we outline the methods , following which we present the results and discussion .
it is convenient , after choice of a standard state and concentration
scale , to consider the chemical potential ( the partial molar gibbs
free energy ) as being composed of an ideal part with explicit dependence
on the concentration and an excess part depending on solute
( h ) and entropic ( s ) components are calculated in this work .
solvent interaction energy distribution p( ) and = 1/kbt , where t is the temperature and kb is the boltzmann constant .
we compute
using the quasichemical decomposition
of the potential distribution theorem . to this end
, we specify
an inner hydration shell around the solute by a length parameter
( figure 1 ) .
the inner outer demarcation
serves to separate strong , short - range solute solvent interactions
from their longer - range counterparts . with the definition of the inner
shell
, the excess chemical potential is given by2 schematic depicting the quasichemical organization of
. schematic reproduced with permission from ref ( 16 ) .
the constraint is a field of range
that serves to move the solvent away from the solute . typically , for
an inner shell extending up to the first hydration shell , p(| ) , the ( regularized )
probability density of the solute solvent binding energy is
gaussian .
the remaining components in
eq 2 have the
following physical meaning . the free energy to empty the inner shell
of solvent is kbt ln x0 , where x0 is the probability to find an empty inner shell . in terms
of equilibrium solute
solvent clustering within the inner shell ,
ln x0 = ln(1 + i1kiwi ) , where ki is the equilibrium constant to form
a solute plus an i - water cluster and w is the bulk density of water .
we can consider an analogous
process of solvent clustering within the same defined inner shell
but in the absence of the solute : the quantity kbt ln p0 is
the work done to create such a cavity in the liquid and is of fundamental
interest in understanding liquid structure and primitive hydrophobic
effects .
( the adjective primitive emphasizes that
this is a property solely of the neat liquid . ) in simulations ,
we grow the external field to calculate x0 and p0 . in practice
, the field has a
soft boundary at , unlike the
sharp - demarcation indicated in the
schematic .
it is straightforward to correct for this effect , but we do not pursue that here .
( please note
that the soft - cavity packing is a lower bound to the hard - cavity packing
contribution and vice versa for the chemistry term . )
we note in passing
that for solutes such as nonpolar molecules , water , and ions , including those with high charge density , the quasichemical approach leads to free energies
in good agreement with traditional free - energy perturbation or histogram
overlap approaches , with the added advantages of the physical insights
provided by the approach .
the excess entropy of hydration of
the solute is given approximately
by4where the average solute
solvent interaction
contribution to the excess enthalpy is esw and the average solvent reorganization contribution is ereorg .
the excess enthalpy h esw + ereorg , where we have neglected
small corrections due to pressure volume
effects and the finite isothermal compressibility of the pure liquid .
in the previous equation
we additionally ignore a small correction
due to the finite thermal expansion coefficient of the pure liquid .
the pentapeptides ggggg , ggigg , and igggg are modeled in the extended
configuration with the long axis aligned with the diagonal of the
simulation cell and the center of the peptide placed at the center
of the simulation cell .
helical deca - glycine ( g9 g ) and
a helical peptide with nine alanine and one glycine at position 6
( a9 g ) serve as references for the helical peptides , g9i and a9i , respectively .
all peptides had an acetylated
( ace ) n - terminus and an n - methyl - amide ( nme ) c - terminus .
butane in the gauche conformation was built using the isoleucine conformation
in the extended pentapeptide .
the solvent was modeled by the
tip3p model , and
the charmm force field with cmap correction
terms for dihedral angles was used for
the peptide .
a total of 2006 water molecules solvated the pentapeptide ;
3500 water molecules were used for the helical peptides .
the lennard - jones
parameters for the isoleucine side chain were used for g - butane . because c in isoleucine becomes a ch2 group in g - butane , the partial charges of
that center
in particular , the parameter we use for c is very slightly shifted from the parameter values for the corresponding
carbon atom in butane .
our parametrization was motivated by our desire
to be as close to isoleucine as possible , but this minor shift in
parameters is not expected to change any conclusions .
the g9 g and a9 g peptides were built from
helical deca - alanine .
these structures were energy - minimized
with weak restraints on heavy atoms to prevent large distortions of
the helix .
( after energy minimization , the solute atoms are held fixed
for the remainder of the simulation . )
the g9i and a9i helices were built by grafting the conformation of isoleucine
in the ggigg system onto position 6 .
thus the internal conformation
of the isoleucine is the same in g - butane , ggigg ,
and the helical peptides . with minor changes , the free - energy
calculations and error analysis
follow the procedure previously described . in brief , is applied such that
varies from 0 to 5 . for every unit angstrom ,
a five - point gauss - legendre
quadrature rule defines the points sampled .
the work to apply
the field is then obtained by quadratures . at each gauss - point , the
system was equilibrated for 0.5 ns , and the data were collected over
the subsequent 0.5 ns .
for the extended peptides , the long - range contribution
was obtained by performing particle - insertion calculations in the
appropriate molecular - shaped cavity ( figure 1 ) .
water with the appropriate cavity was simulated for 1 ns and 1250
frames from the last 0.625 ns used for analysis . confirming the gaussian
distribution of binding energies , we found that particle extraction
calculations ( eq 3 ) agree with the more robust particle - insertion procedure .
for the helical peptides ,
given their high dipole moment we made the conservative choice of
obtaining the electrostatic contribution to the long - range interaction
using a two - point gauss - legendre quadrature ; the van der waals ( vdw ) contribution was obtained using particle
insertion in the molecular cavity .
electrostatic self - interaction
corrections ( of 0.5 kcal / mol ) were applied .
( for the helices , the sum of the vdw and
quadrature - based electrostatic contribution deviates by 1
kcal / mol from the gaussian model , but this deviation is significant
at the statistical resolution of the chemistry and packing contributions . ) for the zwitterionic isoleucine and glycine , we obtained the free
energy in two stages .
first , the excess free energy of the completely
discharged amino acid was obtained using the quasichemical procedure .
then , the work required to turn - on the charges was obtained using
a three - point gauss - legendre quadrature .
the three - point rule gave
the same answer ( within 0.1 kcal / mol ) as the two - point rule , but these
estimates deviate by over 10 kcal / mol from the linear - response result .
the excess energy was obtained by adapting the shell - wise calculation
procedure previously used for studying the hydration of methane . for the peptides , water molecules in the first
two hydration shells
were considered , where the hydration shell is
defined by the union of shells of radius centered on the heavy
atoms : 5 defined the first , 5.0 < 8 defined the second ,
and 8.0 < 11.0 defined
the third hydration shell . within statistical uncertainties , the reorganization
contribution from the third shell is close to zero , justifying our
use of just the first two shells for calculating h. for calculating the excess energy , we equilibrated
the solvated peptide system an additional 1.5 ns ( beyond what was
used in the free - energy calculation ) and propagated the trajectory
for an additional 3 ns , collecting data every 500 ps for a total of
6000 frames .
entropies obtained using eq 4 agree
within statistical uncertainty with entropy calculated using /t ( manuscript
in preparation ) .
the conditional
hydration
free energy of the side - chain x in a peptide mx relative to the reference
mh ( the corresponding amino acid is glycine ) is defined by [x|mx ] = [mx][mh ] .
provided that additivity
holds , [x|mx ] will be proportional to [g - butane ] , the side - chain analog .
( the
same proportionality , by definition , must then also hold for h and s. ) to
better understand how the reference influences the hydration of the
side chain , we use the quasichemical approach to study the packing ,
chemistry , and long - range contributions of the side chain in the presence
of the reference .
( we term these the conditional quasichemical components . )
for the packing calculation , we open a cavity at the appropriate position
next to mh to accommodate the side chain , with mh fully coupled to
the solvent .
likewise , for the chemistry contribution , we expel solvent
only around the side chain x in mx . with solvent
thus excluded , the
long - range contribution was obtained using eq 3 . in additivity - based
approaches ,
a protein is partitioned down to single peptide units ( or even lower ) ,
and the thermodynamics of the macromolecule is obtained using equations
of the form of eq 1 .
to better understand the
limitations of additivity , here we partition the helical peptide s into just two groups , the isoleucine side chain ( sc ) and
the rest , generically termed the background ( back ) ; then5where sc - modelex refers to the conditional hydration
contribution of the side chain in a model compound .
typically ,
the conditional quantity based on the zwitterionic amino acid is combined with factors
based on the solvent exposure of the side - chain x in the gxg tripeptide .
here we consistently use the conditional quantity and the solvent
exposure from ggigg solely .
( our results make it apparent that using
conditional quantities from the zwitterionic model will only worsen
predictions of thermodynamic quantities . )
the factors here
are based on solvent - accessible surface area ( sasa ) obtained using
a standard code and atomic radii . as an example , let at be the sasa of g9i and asc be the contribution due to the isoleucine side chain .
furthermore ,
let asc0 be the sasa of the isoleucine side chain in ggigg and aback0 be the sasa of g9 g .
in evaluating the results later ,
it serves to first define the
error tolerance for an additive model to be satisfactory in predicting
energetics of protein folding .
dill has
suggested that the allowable error per group for a modest 100 amino - acid
protein is 10 cal / mol / group ( or 0.02 kbt / group at 298 k ) .
this estimate sets
a stringent precision level ; our results show that the additive approach
at best agrees at a couple of kilocalories per mole level .
table 1 summarizes
the results on the hydration thermodynamics
of the zwitterionic forms of isoleucine and glycine .
the difference
of these quantities ( denoted by in table 1 ) is typically identified as the group contribution of the
amino acid side chain .
table 1 shows
that whereas the conditional free energy of the isoleucine side chain
is in good agreement with the excess free energy of g - butane , comparing the discharged and charged analogs of the amino
acids shows that slightly over half of the 2.5 kcal / mol free energy
comes from differences in the free energy of charging the amino acids .
this is suggestive of screening of the zwitterion - dipole by the side
chain ( cf . ref ( 19 ) ) , but this physics associated with charging is not one envisioned
when considering the hydration of g - butane , an alkane .
furthermore , examining esw and ereorg reveals large deviations between g - butane and the side chain .
thus , these results suggest
physical differences in the conditional hydration of the side chain
and g - butane , its small molecule analog .
[x ] , where x = i or i is value
relative to g or g , respectively .
solvent
interaction contribution ( esw ) and solvent
reorganization contribution ( ereorg ) to h are also given .
the quantities ereorg , ts , and h have similar statistical uncertainties . the statistical
uncertainty in esw is less than 1/10th
that in h. standard error of the mean
is given at 1. all thermodynamic quantities are in units of
kilocalories per mole .
it is evident that hydration thermodynamics
of ggigg and igggg are different , suggesting that the conditional
free energy of isoleucine will depend on where it is placed along
the backbone .
the difference in is 1
kcal / mol , and it is all in the enthalpy of solution .
[x ] , where x = ggigg or igggg is the value
relative to ggggg .
sc = 0.619 is the ratio of the
solvent - accessible surface area
of the isoleucine side chain in ggigg to that for g - butane ; sc = 0.616 for igggg
note
that relative to g - butane the fractional
solvent exposure of isoleucine in ggigg and igggg is nearly the same ,
and hence the g - butane data corrected by fractional - solvent
exposure are only modestly successful in modeling the positional dependence
in this case . relative to g - butane ,
the conditional h is more positive ( less favorable ) , and this
effect primarily arises from changes in esw .
this result is intuitively reasonable given that the isoleucine
side chain in the pentapeptide is less solvent - exposed than g - butane , but the expected solvent exposure turns out to
be physically unrealistic .
one would need to scale the g - butane esw by 0.2 to match the corresponding
value for the side chain , but the observed fractional exposure is
0.6 .
furthermore , the scaling factors are different for , the subcomponents of h , and
hence also s. table 3 summarizes
the results on the conditional hydration
of isoleucine in the helical peptides g9i and a9i . once again , we find large deviations between the conditional contribution
and the value from the side - chain model appropriately scaled by the
solvent exposure .
the conditional esw contribution to h in a9i is positive , whereas esw for g - butane and the conditional esw in ggigg are both negative .
[x ] , where x = g9i or a9i , gives value relative
to g9 g or a9 g , respectively .
the fractional solvent exposure of isoleucine in g9i is
sc = 0.59 , and in a9i it is sc = 0.56 .
relative to isoleucine in ggigg , sc = 0.95 ( g91 ) and sc = 0.90 ( a9i ) .
subscript a denotes the predicted
properties obtained by combining [ggigg ] ( table 2 ) and [x ] ( table 3 , x = g9i or a9i ) using
eq 5 .
equation 5 , with a protein partitioned down
to a single peptide unit , for example , ref ( 9 ) ( or sometimes even lower , for example , ref ( 8) ) , is often used in group - additivity - based
interpretation of experimental data . following this precedent but
limiting the partitioning of the peptide into only two groups
the side chain and the rest we use eq 5 to reconstruct the free energy of the helical peptides using the
conditional hydration thermodynamics of isoleucine in ggigg together
with the appropriate reference for the helix ( table 4 ) .
it is evident that the error based on the additivity - based
approach ( eq 5 ) can be several kbt values ( and is itself comparable to
typical folding free energies ) .
the
results above show that at the precision level suggested by dill the conditional contribution of amino acid x
from a small - length - scale model or side - chain analog is of limited
utility in modeling the contribution of x in model helices ( and by
induction in more complicated proteins ) . to understand the reasons
for this , we look at two interrelated aspects of the problem : ( 1 )
hydration of the side chain in the presence of the background and
how this compares with the side - chain analog and ( 2 ) how the side
chain changes the hydration of the background and how this effect
is conflated with the conditional contribution attributed to the side
chain . table 5 gives the conditional
quasichemical components for isoleucine side chain in the context
of extended and helical peptides . except for isoleucine in g9i , the agreement in the conditional with values
obtained as [mx ]
( for g9i , the agreement is good only at
2. ) statistical uncertainties in chemistry
and packing are 0.1 kcal / mol , while that in the long - range
contribution is an order of magnitude lower .
table 5 shows that primitive hydrophobic
effects ( as assessed here by the soft - cavity packing contribution )
is context - sensitive . comparing a9i with g9i
and igggg with ggigg
, we find , as is expected , that opening a cavity near hydrophobic groups is more facile
than opening it next to a more polar surface .
this observation is
fundamentally related to how water density fluctuations vary depending
on the polarity of the surface .
furthermore ,
relative to g - butane , the primitive hydrophobic effect
is lower in the context of these peptide models , but the fractional
exposed area does not provide the correct proportionality . finally ,
the free energy to open a cavity , for cavity sizes of interest in
modeling amino acid side chains , is expected to scale with the volume
and not the area . mirroring the trend in the packing contribution ,
the chemistry
contribution is less negative when the side chain is near other hydrophobic
groups .
the long - range contribution also follows a trend similar to
the chemistry contribution .
the compensating variation in the packing
and hydrophilic contributions tends to balance , and ( excluding the
g9i case ) the net conditional free energy of the side chain
is not significantly different from that for g - butane .
like the packing contribution
, the short - range and long - range hydrophilic
contributions are not expected to have a simple dependency on the
solvent - exposed surface area . because different physical effects are
implicit in the net , no single scale factor can
be expected to map the g - butane data to the isoleucine
side chain in a given model , and , in general , properties of a small - scale
model to its value in a protein .
turning now to the effect of
the side chain on the background ,
we see that in grafting a g - butane onto a zwitterionic
glycine to construct the zwitterionic isoleucine the background ( here
the peptide backbone ) experiences an unfavorable change in esw of 6.3 (= 120.7114.4 ) kcal / mol and
the side chain contributes 8.1 kcal / mol for a net change of
1.8 kcal / mol .
esw[g ] , we ascribe 6.3 kcal / mol
of backbone contributions to the side chain .
the effects of this problem
are seen to varying extents in the other peptide systems studied here
( table 6 ) . for ggigg and igggg ,
the scaled - esw of g - butane ,
the side - chain
model ( sc - model ) , is given under scesw [ sc - model ] ; for the helices , the scaled - esw of the side chain in ggigg is given .
backesw[back ] is the scaled
value of the background reference ( back - model ) .
, we have chosen a specific , but illustrative ,
example of hydration of isoleucine in model peptide systems .
we have
also considered the hydration of the aromatic phenylalanine in the
ggfgg system and find that all qualitative conclusions of the isoleucine
study apply to this system as well .
( these results are collected in
the supporting information . ) a more extensive
investigation of sequence and structure space may be desirable to
better understand the success and limitation of the additivity approach ,
but within the context of the potential model chosen , the examples
considered here do serve to indicate the size of deviations from additivity
and , importantly , the compensating deviations in entropy and enthalpy
and the conflation of backbone - specific changes with effects attributed
to the side chain . in summary , the interdependence of background
hydration and side - chain
hydration previously indicated suggests that using y[gxg]y[ggg ] to model y[x ] ,
where y is some thermodynamic state function of interest ,
in proteins has important limitations .
acknowledging the changes in
the solvent exposure of both the background and the side chain in
the peptide under study is an intuitively reasonable step , but no
single scaling factor proves to be satisfactory in capturing both
the hydrophilic and hydrophobic aspects of hydration , especially if
one uses the stringent precision levels suggested by dill .
our results
show that there are nontrivial correlations between
the side chain , backbone , and water that do not allow a perfect pairwise
decomposition .
in essence , the protein context and its induced correlations
in water change the hydration of objects within that correlation range .
thus composing the enthalpy , entropy , and free energy
of a macromolecule using a group - additive approach will necessarily
include reference and group contributions in entirely nontransparent
ways .
constructing the free energy of a protein by scaling appropriate
model compound data by the fractional solvent - exposure of that group
in the protein is intuitively reasonable , but this approach attempts
to map simultaneously both packing and hydrophilic aspects of hydration ,
but each of these aspects addresses inherently different physical
features of solvent behavior and solute solvent interaction .
moreover , the derived conditional energetics of the side chain are
seen to contain nonobvious contributions from the changes in the hydration
of the background .
thus no single scale factor can be expected to
describe the conditional , h , and s of the side chain relative
to the property of the model compound .
furthermore , relative to a
model compound , the identification of greater or lesser hydrophobicity
or hydrophilicity will itself be context - dependent , raising questions
about the utility of small - molecule data to understand finely detailed
features of protein hydration
. this could be of concern in assessing
contributions of hydrophilic versus hydrophobic mutations in proteins .
if we take the stringent dill error criteria , our analysis shows that the hydration of g - butane is inadequate to model the conditional hydration of ggigg ,
and the conditional hydration of isoleucine in ggigg is inadequate
in modeling the hydration of isoleucine in model helices .
the successes
in using small - molecule data in modeling protein hydration should
thus be considered with respect to the errors required in a given
context .
if an additive description of free energy predicts the numerical
values with reasonable error expectations in accord with experiments ,
it is likely that this success arises due to some compensation in
the underlying components . | the
hydration thermodynamics of the amino acid x relative to the
reference g ( glycine ) or the hydration thermodynamics of a small - molecule
analog of the side chain of x is often used to model the contribution
of x to protein stability and solution thermodynamics .
we consider
the reasons for successes and limitations of this approach by calculating
and comparing the conditional excess free energy , enthalpy , and entropy
of hydration of the isoleucine side chain in zwitterionic isoleucine ,
in extended penta - peptides , and in helical deca - peptides .
butane in
gauche conformation serves as a small - molecule analog for the isoleucine
side chain .
parsing the hydrophobic and hydrophilic contributions
to hydration for the side chain shows that both of these aspects of
hydration are context - sensitive .
furthermore , analyzing the solute
solvent
interaction contribution to the conditional excess enthalpy of the
side chain shows that what is nominally considered a property of the
side chain includes entirely nonobvious contributions of the background .
the context - sensitivity of hydrophobic and hydrophilic hydration and
the conflation of background contributions with energetics attributed
to the side chain limit the ability of a single scaling factor , such
as the fractional solvent exposure of the group in the protein , to
map the component energetic contributions of the model - compound data
to their value in the protein . but
ignoring the origin of cancellations
in the underlying components the group - transfer model may appear to
provide a reasonable estimate of the free energy for a given error
tolerance . | Introduction
Theory
Methods
Results and Discussion
Concluding Discussion | the hydration thermodynamics of analogs
of amino acid side chains
( or of amino acid side chains in small - model peptides ) has often been
used to understand protein folding and protein protein association . a very small sampling of the many pioneering investigations where
this approach has been used includes work identifying hydrophobicity
as a dominant force in protein folding , investigations of protein denaturation , interpretation of calorimetric
data on protein unfolding , and , more recently , investigations
on the role of osmolytes in protein folding . drawing upon
insights attributed to cohn and edsall ,
,
the free energy of unfolding is given as a sum of the free energy
of transfer of the small component groups of the molecule ,
from the environment they have in the native form , to the environment
they have in the unfolded form . ref ( 9 ) ) , the
generic equation of the unfolding free energy ( gnu ) can be written
as1where gi is the free energy of transferring the group i from some reference phase to liquid water and i is a factor that corrects for the change
in solvent - exposure of the group i in protein unfolding . the same form of equation can also be used to describe
the effect of osmolytes on protein unfolding : identifying gi with the free energy for
transferring the group from water to the aqueous osmolyte solution
and i with the change in the solvent
exposure of the group upon unfolding in the osmolyte solution relative
to water , the previous equation directly gives the so - called m value for 1 m osmolyte . at a time when theory , simulations , and experimental techniques
were less developed than they are now , the group - additive approach
was a pragmatic first step to understand the hydration thermodynamics
of a complicated macromolecule . furthermore , our previous study on the solvation of a peptide group showed that in the water - to - osmolyte transfer
solvent - mediated correlations between peptide units ( in glyn ) cancel , and this allows one to identify a peptide - group
transfer free energy that is model - independent . but in the vapor - to - liquid
transfer , a situation where solvent - mediated correlations are preserved ,
the identified group - additive contribution ( gi , eq 1 ) depends
on the model used to define the peptide group . in exploring context dependence of hydration ,
these authors also suggested how adding a methyl group to an amino
acid changes the solvent - exposure of the peptide backbone , thereby
influencing the excess free energy in a way that can not be captured
by side - chain analog data . building on these efforts , here we study
how well a model of the side chain describes the hydration of the
side chain in the context of a protein . in particular , our aim is
to better understand factors limiting additivity and the reasons why
scaling model - compound data to describe its properties in a protein
context may not always be satisfactory . to aid in parsing energetic
differences , we study the conditional solvation of an isoleucine residue ,
often regarded as very hydrophobic , in
the context of model - extended and helical peptides . butane in the
gauche conformation , matching exactly the side - chain conformation
of isoleucine in extended peptides , is used as a small - molecule analog
of the side chain . these components ( but not the net free
energy )
do depend on the specification of a hydration shell , but the
approach has nevertheless provided important insights into the physics
of hydration and in generating models of molecular solutions . we complement the quasichemical analysis with the traditional
decomposition
of the excess free energy into its enthalpic and entropic contributions . the quasichemical approach helps us better appreciate how the context
influences the hydration of the solute , while the enthalpy entropy
decomposition leads to the finding that nonobvious contributions from
the reference ( the context ) get folded into the conditional contribution
attributed to the side chain ( the solute ) that is of first interest
in additive models . it is convenient , after choice of a standard state and concentration
scale , to consider the chemical potential ( the partial molar gibbs
free energy ) as being composed of an ideal part with explicit dependence
on the concentration and an excess part depending on solute
( h ) and entropic ( s ) components are calculated in this work . typically , for
an inner shell extending up to the first hydration shell , p(| ) , the ( regularized )
probability density of the solute solvent binding energy is
gaussian . the excess entropy of hydration of
the solute is given approximately
by4where the average solute
solvent interaction
contribution to the excess enthalpy is esw and the average solvent reorganization contribution is ereorg . in the previous equation
we additionally ignore a small correction
due to the finite thermal expansion coefficient of the pure liquid . the pentapeptides ggggg , ggigg , and igggg are modeled in the extended
configuration with the long axis aligned with the diagonal of the
simulation cell and the center of the peptide placed at the center
of the simulation cell . helical deca - glycine ( g9 g ) and
a helical peptide with nine alanine and one glycine at position 6
( a9 g ) serve as references for the helical peptides , g9i and a9i , respectively . butane in the gauche conformation was built using the isoleucine conformation
in the extended pentapeptide . the solvent was modeled by the
tip3p model , and
the charmm force field with cmap correction
terms for dihedral angles was used for
the peptide . the lennard - jones
parameters for the isoleucine side chain were used for g - butane . thus the internal conformation
of the isoleucine is the same in g - butane , ggigg ,
and the helical peptides . for the extended peptides , the long - range contribution
was obtained by performing particle - insertion calculations in the
appropriate molecular - shaped cavity ( figure 1 ) . for the helical peptides ,
given their high dipole moment we made the conservative choice of
obtaining the electrostatic contribution to the long - range interaction
using a two - point gauss - legendre quadrature ; the van der waals ( vdw ) contribution was obtained using particle
insertion in the molecular cavity . for the zwitterionic isoleucine and glycine , we obtained the free
energy in two stages . first , the excess free energy of the completely
discharged amino acid was obtained using the quasichemical procedure . for the peptides , water molecules in the first
two hydration shells
were considered , where the hydration shell is
defined by the union of shells of radius centered on the heavy
atoms : 5 defined the first , 5.0 < 8 defined the second ,
and 8.0 < 11.0 defined
the third hydration shell . within statistical uncertainties , the reorganization
contribution from the third shell is close to zero , justifying our
use of just the first two shells for calculating h. for calculating the excess energy , we equilibrated
the solvated peptide system an additional 1.5 ns ( beyond what was
used in the free - energy calculation ) and propagated the trajectory
for an additional 3 ns , collecting data every 500 ps for a total of
6000 frames . the conditional
hydration
free energy of the side - chain x in a peptide mx relative to the reference
mh ( the corresponding amino acid is glycine ) is defined by [x|mx ] = [mx][mh ] . ( the
same proportionality , by definition , must then also hold for h and s. ) to
better understand how the reference influences the hydration of the
side chain , we use the quasichemical approach to study the packing ,
chemistry , and long - range contributions of the side chain in the presence
of the reference . for the packing calculation , we open a cavity at the appropriate position
next to mh to accommodate the side chain , with mh fully coupled to
the solvent . in additivity - based
approaches ,
a protein is partitioned down to single peptide units ( or even lower ) ,
and the thermodynamics of the macromolecule is obtained using equations
of the form of eq 1 . to better understand the
limitations of additivity , here we partition the helical peptide s into just two groups , the isoleucine side chain ( sc ) and
the rest , generically termed the background ( back ) ; then5where sc - modelex refers to the conditional hydration
contribution of the side chain in a model compound . typically ,
the conditional quantity based on the zwitterionic amino acid is combined with factors
based on the solvent exposure of the side - chain x in the gxg tripeptide . as an example , let at be the sasa of g9i and asc be the contribution due to the isoleucine side chain . furthermore ,
let asc0 be the sasa of the isoleucine side chain in ggigg and aback0 be the sasa of g9 g . table 1 summarizes
the results on the hydration thermodynamics
of the zwitterionic forms of isoleucine and glycine . the difference
of these quantities ( denoted by in table 1 ) is typically identified as the group contribution of the
amino acid side chain . table 1 shows
that whereas the conditional free energy of the isoleucine side chain
is in good agreement with the excess free energy of g - butane , comparing the discharged and charged analogs of the amino
acids shows that slightly over half of the 2.5 kcal / mol free energy
comes from differences in the free energy of charging the amino acids . this is suggestive of screening of the zwitterion - dipole by the side
chain ( cf . furthermore , examining esw and ereorg reveals large deviations between g - butane and the side chain . thus , these results suggest
physical differences in the conditional hydration of the side chain
and g - butane , its small molecule analog . it is evident that hydration thermodynamics
of ggigg and igggg are different , suggesting that the conditional
free energy of isoleucine will depend on where it is placed along
the backbone . sc = 0.619 is the ratio of the
solvent - accessible surface area
of the isoleucine side chain in ggigg to that for g - butane ; sc = 0.616 for igggg
note
that relative to g - butane the fractional
solvent exposure of isoleucine in ggigg and igggg is nearly the same ,
and hence the g - butane data corrected by fractional - solvent
exposure are only modestly successful in modeling the positional dependence
in this case . relative to g - butane ,
the conditional h is more positive ( less favorable ) , and this
effect primarily arises from changes in esw . this result is intuitively reasonable given that the isoleucine
side chain in the pentapeptide is less solvent - exposed than g - butane , but the expected solvent exposure turns out to
be physically unrealistic . furthermore , the scaling factors are different for , the subcomponents of h , and
hence also s. table 3 summarizes
the results on the conditional hydration
of isoleucine in the helical peptides g9i and a9i . once again , we find large deviations between the conditional contribution
and the value from the side - chain model appropriately scaled by the
solvent exposure . the fractional solvent exposure of isoleucine in g9i is
sc = 0.59 , and in a9i it is sc = 0.56 . equation 5 , with a protein partitioned down
to a single peptide unit , for example , ref ( 9 ) ( or sometimes even lower , for example , ref ( 8) ) , is often used in group - additivity - based
interpretation of experimental data . following this precedent but
limiting the partitioning of the peptide into only two groups
the side chain and the rest we use eq 5 to reconstruct the free energy of the helical peptides using the
conditional hydration thermodynamics of isoleucine in ggigg together
with the appropriate reference for the helix ( table 4 ) . the
results above show that at the precision level suggested by dill the conditional contribution of amino acid x
from a small - length - scale model or side - chain analog is of limited
utility in modeling the contribution of x in model helices ( and by
induction in more complicated proteins ) . to understand the reasons
for this , we look at two interrelated aspects of the problem : ( 1 )
hydration of the side chain in the presence of the background and
how this compares with the side - chain analog and ( 2 ) how the side
chain changes the hydration of the background and how this effect
is conflated with the conditional contribution attributed to the side
chain . table 5 gives the conditional
quasichemical components for isoleucine side chain in the context
of extended and helical peptides . furthermore ,
relative to g - butane , the primitive hydrophobic effect
is lower in the context of these peptide models , but the fractional
exposed area does not provide the correct proportionality . the compensating variation in the packing
and hydrophilic contributions tends to balance , and ( excluding the
g9i case ) the net conditional free energy of the side chain
is not significantly different from that for g - butane . because different physical effects are
implicit in the net , no single scale factor can
be expected to map the g - butane data to the isoleucine
side chain in a given model , and , in general , properties of a small - scale
model to its value in a protein . turning now to the effect of
the side chain on the background ,
we see that in grafting a g - butane onto a zwitterionic
glycine to construct the zwitterionic isoleucine the background ( here
the peptide backbone ) experiences an unfavorable change in esw of 6.3 (= 120.7114.4 ) kcal / mol and
the side chain contributes 8.1 kcal / mol for a net change of
1.8 kcal / mol . esw[g ] , we ascribe 6.3 kcal / mol
of backbone contributions to the side chain . for ggigg and igggg ,
the scaled - esw of g - butane ,
the side - chain
model ( sc - model ) , is given under scesw [ sc - model ] ; for the helices , the scaled - esw of the side chain in ggigg is given . we have
also considered the hydration of the aromatic phenylalanine in the
ggfgg system and find that all qualitative conclusions of the isoleucine
study apply to this system as well . a more extensive
investigation of sequence and structure space may be desirable to
better understand the success and limitation of the additivity approach ,
but within the context of the potential model chosen , the examples
considered here do serve to indicate the size of deviations from additivity
and , importantly , the compensating deviations in entropy and enthalpy
and the conflation of backbone - specific changes with effects attributed
to the side chain . in summary , the interdependence of background
hydration and side - chain
hydration previously indicated suggests that using y[gxg]y[ggg ] to model y[x ] ,
where y is some thermodynamic state function of interest ,
in proteins has important limitations . acknowledging the changes in
the solvent exposure of both the background and the side chain in
the peptide under study is an intuitively reasonable step , but no
single scaling factor proves to be satisfactory in capturing both
the hydrophilic and hydrophobic aspects of hydration , especially if
one uses the stringent precision levels suggested by dill . in essence , the protein context and its induced correlations
in water change the hydration of objects within that correlation range . thus composing the enthalpy , entropy , and free energy
of a macromolecule using a group - additive approach will necessarily
include reference and group contributions in entirely nontransparent
ways . constructing the free energy of a protein by scaling appropriate
model compound data by the fractional solvent - exposure of that group
in the protein is intuitively reasonable , but this approach attempts
to map simultaneously both packing and hydrophilic aspects of hydration ,
but each of these aspects addresses inherently different physical
features of solvent behavior and solute solvent interaction . moreover , the derived conditional energetics of the side chain are
seen to contain nonobvious contributions from the changes in the hydration
of the background . thus no single scale factor can be expected to
describe the conditional , h , and s of the side chain relative
to the property of the model compound . furthermore , relative to a
model compound , the identification of greater or lesser hydrophobicity
or hydrophilicity will itself be context - dependent , raising questions
about the utility of small - molecule data to understand finely detailed
features of protein hydration
. if we take the stringent dill error criteria , our analysis shows that the hydration of g - butane is inadequate to model the conditional hydration of ggigg ,
and the conditional hydration of isoleucine in ggigg is inadequate
in modeling the hydration of isoleucine in model helices . the successes
in using small - molecule data in modeling protein hydration should
thus be considered with respect to the errors required in a given
context . if an additive description of free energy predicts the numerical
values with reasonable error expectations in accord with experiments ,
it is likely that this success arises due to some compensation in
the underlying components . | [
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macrophages are endowed with highly dynamic projections of their cell surface , allowing them to move and to survey the extracellular milieu with extensions such as filopodia , lamellipodia and dorsal ruffles .
cell migration is a well - studied process , and lamellipodia and filopodia are essential for migration and chemotaxis of immune cells . at the leading edge , cell motility
is driven by lamellipodia ( thin membrane sheets containing a branched actin substructure ) and filopodia ( elongated membrane fingers ( 100300 m ) comprising tight parallel bundles of actin filaments ) that both have the growing barbed ends of actin filaments oriented to push the leading edge forward .
these structures are abundant on macrophages and their distinctive and recognisable morphologies can be appreciated by viewing the macrophage surface using scanning electron microscopy ( figure 1 ) .
lamellipodia extend through actin - based polymerisation and branching of actin filaments using machinery including the arp2/3 and wasp - family verprolin - homologous protein ( wave ) regulatory complexes .
the same basic machinery is used to nucleate actin for the actin - based motility of bacterial pathogens , that invade host cells and induce the classical actin comet tails ' to move around in the cytoplasm .
the molecular composition of filopodia and their formation rely heavily on actin - binding and modifying proteins .
ena / vasodilator - stimulated phosphoprotein ( vasp ) proteins prevent capping of actin - barbed ends at the tips of filopodia , formins like dia2 are involved in producing unbranched actin filaments , fascin for actin bundling and unconventional myosin x motors that can move up and down filopodia , are all well - known elements of these structures .
filopodia can form anywhere on the cell surface , for instance , emerging from lamellipodia at the cell 's leading edge during migration ( figure 1a ) .
cell adhesion and cell cell contact can be driven by filopodia loaded with integrins or cadherins at their tips or displayed along their length .
macrophages extend many filopodia , commonly using them as probes ' to explore the surrounding milieu .
macrophages can also use filopodia as tentacles to draw particles towards the cell body and in an extreme form of this process , very long filopodia wrap around and entrap bacteria in the coiling phagocytosis ' described for lassoing legionella for uptake .
filopodia also have cholesterol - rich and highly ordered lipid - raft - enriched membranes that are poised to contribute to receptor signalling and membrane trafficking , which in turn are important for the probing functions of these structures in cells like macrophages .
dorsal ruffles ( or hereafter referred to as ruffles ) are highly distinctive , sometimes circular ( hence the alternative name
circular dorsal ruffles ' ) , veils of membrane that rise up from the dorsal surface of adherent cells or sometimes occur at peripheral edges of cells , noting however their distinction from the ruffling leading edges of migrating cells .
dorsal ruffles behave in a wave - like manner , rising up and then collapsing back onto the surface , and any fluid , particles or pathogens encircled by the collapsing ruffles are internalised into the cell along with the ruffle membrane .
the collapsing ruffles can form macropinosomes for fluid - phase uptake or phagosomes to engulf and internalise larger particulate matter or pathogens .
dorsal ruffles share some morphologically and molecular features with other f - actin - rich surface structures , notably , podosomes and the invadopodia of cancer cells .
epidermal growth factor ( egf ) and platelet - derived growth factor ( pdgf ) stimulate dorsal ruffle formation in fibroblasts and epithelial cells .
the respective receptor tyrosine kinases ( rtks ) are concentrated in the ruffles , which act as signalling centres and ruffles are enriched in signalling molecules including ras , rac , src , pi3k , akt , mapk and pak1 .
the formation of dorsal ruffles in fibroblasts also involves crosstalk and co - signalling from integrins and rtks .
the collapse of ruffles into macropinosomes provides for rapid internalisation and downregulation of these clustered rtks .
thus , dorsal ruffles have both positive and negative regulatory roles in growth factor signalling , highlighting their importance to key processes in development and disease .
dorsal ruffles are dependent on the transient production of f - actin , and inhibitors of f - actin polymerisation , such as cytochalasin d , abolish their formation .
classical f - actin assembly complexes are localised to sites of dorsal ruffle formation . through their proline - rich domains , neural wiskott
aldrich syndrome protein , wave1 and wave2 can bind to sh3-domain containing proteins , which include arp2/3 and membrane phospholipids to help concentrate relevant actin machinery in the dynamic ruffle environment .
rho gtpases are also a feature of the ruffle machinery , with both rac1 and rac2 implicated in specialised ruffle actin - associated complexes and signalling cascades , along with other rho family members including rhoa and cdc42 .
active rac is needed for the development of pre - ruffle protrusions and for ruffle formation in macrophages . in macrophages and immature dendritic cells ,
membrane ruffling occurs constitutively , and the perpetual cycles of f - actin polymerisation are promoted by membrane phospholipids at the ruffle sites .
phosphatidic acid in the pre - ruffle membrane helps to generate f - actin , via either recruiting and activating rac or by activating phosphatidylinositol 4,5-biphosphate ( ptdins(4,5)p2 ) , to support continual actin polymerisation at sites of ruffling .
overall , ruffles are highly enriched in phospholipids , which support the charge - based recruitment and attachment of proteins .
the inner leaflet of the plasma membrane has the highest pool of ptdins(4,5)p2 that supports protein and cytoskeletal interactions for a large suite of host processes including signalling , endocytosis , cell movement and enzyme activation . in ruffles ,
this ptdins(4,5)p2 is also a substrate for the local production of ptdins(3,4,5)p3 , which is important for recruiting signalling proteins and also potentially for closure of the dorsal ruffle .
the ptdins(3,4,5)p3 produced in ruffles then persists after closure and through the early stages of formation of macropinosomes and phagocytic cups .
these phosphoinositide transitions can be observed using probes such as the pleckstrin homology domain of protein kinase b ( akt ) .
the phosphoinositides also recruit effector proteins to the membrane such as transporter associated with antigen processing 1 and lowe oculocerebrorenal syndrome protein , which can influence the dynamics of the cytoskeleton and subsequent compartment maturation .
the dorsal ruffles are also replete with molecular machinery and families of regulators such as gtpases ( rhos , rabs , arfs and ras ) that participate in signalling and membrane trafficking , although in many cases , specific members of these families have not yet been identified specifically on ruffles in different cell types .
interestingly , the adp - ribosylation factor - like ( arl ) protein , arl13b , has recently been demonstrated in dorsal ruffles .
is better known as a key component of primary cilia , specialised organelles for mechanosensory regulation and signal transduction .
related to this , arl13b is a casual locus mutated in the congenital ciliopathy , joubert syndrome , associated with severely impaired development .
arl13b associated with f - actin in dorsal ruffles and loss of arl13b also reduces pdgf - induced dorsal ruffle formation . in this role , and in its other roles in endocytic recycling , the arl13b shares a non - muscle myosin iia as an effector .
it is of emerging interest that arl13b is necessary for formation of dorsal ruffles and cilia as distinct signal transduction membrane domains .
innate immunity relies on a series of host pattern recognition receptors that detect non - self , danger signals and pathogen - derived molecules .
toll - like receptors ( tlrs ) are one such receptor family and different tlrs are positioned on the cell surface or endosomes to detect and respond to selected extracellular or intracellular pathogens .
although macrophages ruffle constitutively , dorsal ruffling is enhanced by exposing the cells experimentally to lipopolysaccharide ( lps ) of gram - negative bacteria , indicating that ruffling is also a pathogen
lps is a ligand for tlr4 and activated tlr4 generates signals from the cell surface or from endosomes / macropinosomes using different sets of adaptor molecules to elicit cytokine release and other downstream responses . tlr4 and
its cell surface adaptors can be found clustered in dorsal ruffles where they are poised with the relevant machinery for pi3k - akt signalling .
as the ruffles collapse , tlr4 is delivered to macropinosomes , or to phagosomes during pathogen engulfment , where signalling from different adaptors varies the signalling and downstream responses .
thus , in addition to receptor ( tlr4 ) trafficking for degradation or recycling , the collapsing ruffle creates another signalling platform for biasing immune outputs .
in addition to tlr4-mediated transcriptional regulation and synthesis of cytokines , the dorsal ruffles and pre - phagosome membranes are also sites for release of cytokines such as tnf .
newly synthesised tnf is delivered to recycling endosomes that feed membrane to the base of the ruffles and pre - phagosomal membranes , simultaneously exporting tnf to the surface at this point for release from the cell .
innate immunity relies on a series of host pattern recognition receptors that detect non - self , danger signals and pathogen - derived molecules .
toll - like receptors ( tlrs ) are one such receptor family and different tlrs are positioned on the cell surface or endosomes to detect and respond to selected extracellular or intracellular pathogens .
although macrophages ruffle constitutively , dorsal ruffling is enhanced by exposing the cells experimentally to lipopolysaccharide ( lps ) of gram - negative bacteria , indicating that ruffling is also a pathogen
lps is a ligand for tlr4 and activated tlr4 generates signals from the cell surface or from endosomes / macropinosomes using different sets of adaptor molecules to elicit cytokine release and other downstream responses . tlr4 and
its cell surface adaptors can be found clustered in dorsal ruffles where they are poised with the relevant machinery for pi3k - akt signalling .
as the ruffles collapse , tlr4 is delivered to macropinosomes , or to phagosomes during pathogen engulfment , where signalling from different adaptors varies the signalling and downstream responses .
thus , in addition to receptor ( tlr4 ) trafficking for degradation or recycling , the collapsing ruffle creates another signalling platform for biasing immune outputs .
in addition to tlr4-mediated transcriptional regulation and synthesis of cytokines , the dorsal ruffles and pre - phagosome membranes are also sites for release of cytokines such as tnf .
newly synthesised tnf is delivered to recycling endosomes that feed membrane to the base of the ruffles and pre - phagosomal membranes , simultaneously exporting tnf to the surface at this point for release from the cell .
macropinocytosis is one of the most ancient , prevalent and yet perhaps least well - defined forms of endocytosis .
it is the process of fluid gulping practiced at some level by most cells of the body , but scaled up to a high volume highway in macrophages as they perform surveillance of the surrounding tissue .
the pathway results in the ingestion of fluid , plus any fluid - phase cargo and a large swathe of plasma membrane with its resident transmembrane proteins and any molecules , particles or pathogens attached to them on the external face of the plasma membrane . unlike receptor - mediated endocytosis and phagocytosis , macropinocytosis is not driven by receptor - ligand interactions .
macropinocytosis is therefore distinguished by being a large volume , non - selective , actin - dependent pathway for internalisation and this is reflected in the nature of the macropinosomes themselves , which are uncoated , nondescript vesicles varying in size from 0.2 to > 2 m in diameter .
as discussed below , the non - selective , large volume nature of macropinocytosis means that both inevitably , and by design , it serves for the uptake of pathogens .
, who show that once macrophages have been activated by contact with pathogens , they upregulate receptor - independent macropinocytosis to override the less efficient process of receptor - mediated phagocytosis to enhance their capacity to clear the pathogens .
this switch they found to be mediated by phosphorylation of the actin - binding protein coronin 1 , which is known as a prominent regulator of early actin assembly forming a crown - like ring around newly forming macropinosomes in dictyostelium and other organisms .
the fate of macropinosomes has classically been studied by tracking the uptake of fluid - phase cargo , such as fluorescent dextran , or fluorescently - tagged membrane proteins or lipids in live or fixed cells .
these studies routinely show that soon after internalisation , macropinosomes shrink and move towards the centre of the cell as they undergo maturation ( figure 1 ) .
evidence from tracking cargo mixing shows that macropinosomes have fused with other endosomes within 1 min of uptake .
the rab11-positive recycling endosomal network in macrophages is vast and forms an important repository for plasma membrane and proteins recycling back to the surface , as well as new exocytic traffic , in this high turnover environment .
alternatively , live imaging also reveals that macropinosomes can undergo rapid and extensive tubulation after internalisation , forming a transient network of elongated membranes or tubes that help to sort membrane proteins and fluid - phase cargo into various endosomes , many of which mature into lamp-1/rab7 late endosomes / lysosomes where degradation can occur .
the actin polymerisation in dorsal ruffles or at the leading edges of motile cells provides the platform of branched actin filaments needed to assemble the macropinosomes .
multiple small gtpases contribute to actin regulation during macropinocytosis including the rho , rac , cdc42 , rab , arf and ras subfamilies .
arf6 is involved in ruffling and macropinocytosis through roles that include recruiting rac1 for the production of pi(4,5)p2 or for the activation of phospholipase d ( reviewed in ) . with the expression and manipulation of photoactivatable rac1 , fujii et al .
were able to demonstrate the cycles of rac1 activation needed to initiate ruffling and macropinosome formation and the important deactivation of rac1 that accompanies closure and maturation .
rab5a is a signature gtpase on macropinosomes , recruited for roles in actin remodelling , signalling and in the closed , maturing macropinosome where pi(3)p has generated the activation of rab5a and recruitment of its effectors , which may help to stabilise these compartments .
other rabs , such as rab34 , rab21 and rab31 can be found on macropinosomes serving to recruit a variety of additional effectors .
the transition of pi(4,5)p2 to pi(3,4,5)p3 to pi(3,4)p2 and then to pi(3)p is conserved during the processes of macropinosome closure and maturation from c.elegans to mammalian cells .
pi3kinases are crucially involved in macropinocytosis and the broad spectrum pi3kinase inhibitors , ly294002 and wortmannin blocks macropinosome formation , and specifically macropinosome closure , without necessarily affecting the preceding ruffling .
the transient appearance of pi(3,4,5)p3 is accompanied by rac1 activation and diacylglycerol production , and this is followed by the appearance of pi(3,4)p2 during macropinosome closure .
lipid probes variably show accumulation of pi(3,4,5)p3 on the macropinosome membranes before or just after closure .
pi(3,4,5)p3 can then activate phospholipase c and diacylglycerol , as well as protein kinase c and ras during macropinosome maturation .
accordingly , inhibitors for these enzymes variably block macropinocytosis ( table 1 ) . the sorting nexins ( snxs ) with their signature
phox homology ( px ) lipid - binding domains are an important class of phosphoinositide effectors , and multiple snx family members are associated with , and functionally implicated in , macropinocytosis .
snx5 , for instance , is required for macropinocytosis . at the behest of egfr activation ,
snx5 is recruited to pi(3,4)p2-enriched membranes on early macropinosomes and it is present on tubulating macropinosomes , where in both cases , the snx5 bar domain may contribute to membrane curvature .
macrophages , but interestingly not immature dendritic cells , from snx5 knockout mice have significantly reduced macropinocytosis and dorsal ruffling .
a diagnostic feature of macropinocytosis , used to distinguish this pathway from clathrin - mediated endocytosis , is its sensitivity to inhibitors of na / h ion exchangers , such as amiloride .
although the mode of action of amiloride is controversial , it remains a useful experimental tool for blocking macopinocytic uptake of markers or pathogens . in the immune system , macropinocytosis is an important process for antigen presentation through the environmental sampling performed by antigen - presenting cells such as macrophages and immature dendritic cells .
activated and mature dendritic cells downregulate macropinocytosis in favour of migration to lymph nodes for presentation to t cells .
antigenic peptides derived from internalised material are loaded onto major histocompatibility complex ( mhc ) molecules for presentation to t cells to invoke adaptive immune responses .
antigens presented by either mhci , ( usually self or endogenous antigens ) or mhcii complexes ( foreign or extracellular antigens ) can come from macropinosomes .
fluid - phase markers ingested by dendritic cells can be delivered to the mhcii compartment , denoted by some late endosome / lysosome markers , for processing and loading .
macropinocytosis can also result in the cross - presentation of extracellular antigens on mhci after processing of these antigens to peptides in the endosomal system or in the cytoplasm .
finally , the non - selective nature of macropinocytosis lends itself to exploitation for the delivery of a variety of particles , soluble drugs , genetic material and biological modifiers into cells .
membrane - penetrating peptides such as trans - activating transcriptional activator ( tat ) are taken up into macropinosomes where they can cross the membrane into the cytoplasm providing an important portal for genetic or pharmacologic modifiers .
the macropinocytic and ligand - directed uptake of quantum dot nanoparticles can be demonstrated with the potential to target delivery to specific cell types or situations .
recently , the supercharging of macropinocytosis in tumour cells by activating epidermal growth factor receptor ( egfr ) and oncogenic ras provided efficient delivery of engineered cytotoxic exosomes into cells .
macropinocytosis will continue to be an important tool in fighting disease in this and many other ways .
phagocytosis is a process initiated in macrophages or other professional phagocytes , confronted with a pathogen , a dead cell or large ( > 0.5 m ) opsonised particle .
phagocytosis is a deliberate , receptor - driven process based on recognition of the pathogen or object and engagement with it by receptors on macrophage cell surface ruffles , filopodia or the phagocytic cups that surround and engulf the
the typical course of phagosome maturation is through fusion with successive endosome and lysosomes , acidification of the lumen and generation of reactive oxygen species , which lead to a destructive phagolysosome designed to kill pathogens and degrade them or any inert particles that have been phagocytosed .
phagocytosis is essential for innate immunity , organ development and maintaining homoeostasis throughout tissues of the body .
it contributes , either directly or inadvertently , to a wide range of diseases from bacterial pathogenesis and infection , to the accumulation of aberrant proteins in neurodegenerative diseases , the advancement of atherosclerotic lesions and to cancer and chronic diseases like chronic obstructive pulmonary disease ( copd ) and cystic fibrosis .
pattern recognition receptors ( receptors for polysaccharides on yeast , e.g. , dectin-1 ) , opsonic receptors ( immunoglobulin or complement receptors , e.g. , fcr or cr1 ) and apoptotic cell or corpse receptors ( phosphatidylserine receptors , e.g. , tim-1 ) are among those that can be co - engaged ( reviewed in flannagan et al . ) . through receptor crosstalk , signalling from co - activated tlrs , for instance , concomitantly drives cell death / survival and inflammatory responses appropriate for the nature of the pathogen or particle .
receptor signalling takes place in the phospholipid and actin - rich environment of the phagocytic cups where receptor trafficking is also supported by prolific membrane exchange between recycling endosomes or early endosomes and the early phagocytic membranes .
actin filaments are a feature of only the early stage ruffles , pseudopods and phagocytic cups , leading up to sealing and initial internalisation of the phagosomes whereupon f - actin is rapidly depolymerised and dispersed .
the precise timing of this association means that f - actin is a useful experimental marker for early phagosomes .
actin polymerisation is activated by different receptors ( through diverse pathways ) and engaged for the f - actin and receptor - mediated protrusion of the membranes over the surface of the prey during engulfment .
activated fcrs recruit arp2/3 , whereas cr3 and integrin - dependent phagocytosis recruits talin and other actin - binding proteins .
cholesterol is needed for phagocytic cup formation , and sphingolipids and phospholipids are also extensively engaged .
the phospholipid transitions that accompany ruffling and macropinocytosis have also been mapped during phagocytosis , beginning with conversion of the pi(4,5)p2 to pi(3,4,5)p3 in presealed phagocytic cups and pi(3)p in fully sealed , internalised phagosomes .
multiple ( > 20 ) rab gtpases , their accessory proteins and varied effectors are especially recruited for phagocytosis , warranted by the intense membrane and protein trafficking , endosome fusion and receptor signalling inherent in the remodelling stages of early phagocytosis , followed throughout the whole process by further trafficking and fusion events .
the sequential nature of rab recruitment also experimentally defines successive stages of phagocytosis . with its focus as the entrapment and destruction of pathogens ,
some bacterial virulence factors aim to block phagocytic uptake , whereas for intracellular pathogens , other virulence factors are geared to allow phagocytic entry but subvert its destructive capacity . on the other hand , some pathogenic bacteria have developed the ability to actively induce phagocytosis and/or macropinocytosis to gain entry into the host cell . during this type of entry ,
dorsal ruffles and macropinosomes are co - opted or induced by a wide range of human pathogens and infectious agents , from viruses , to bacteria , protozoa and prions , for entry into human cells .
can actively infect almost any cell type through the injection of bacterially - derived effector proteins that modulate a number of host processes such as f - actin polymerisation to induce macropinocytosis .
typically salmonella typhimumirum that causes gasteroenteritis or typhoid fever , invades epithelial cells or macrophages .
one of the salmonella effector proteins is sipa , which has been shown to promote the generation of plasma membrane protrusions and ruffles by promoting the polymerisation of f - actin filaments .
salmonella induce large localised patches of ruffling on the surface of epithelial cells , whereas on macrophages , large areas of exaggerated ruffling are induced ( figure 1 ) , serving to capture and internalise multiple bacteria near the cell surface .
additional bacterial effectors hijack cell signalling and the maturation of macropinosomes to avoid detection and destruction , creating protective vacuoles inside host cells for sequestration and proliferation .
many of the salmonella effectors have been characterised , serving to inform us about the roles of various host proteins in normal macropinocytic processes .
bacteria like legionella , which invade macrophages through modifying macropinocytic or phagocytic processes , inject hundreds of effectors , many of which remain to be characterised . pathogenic viruses commonly infect human cells via macropinocytosis .
the highly infectious and deadly ebola virus and mosquito - borne flaviviruses like dengue , west nile , yellow fever and the newly emergent zika viruses , all rely on macropinocytosis for entry into human cells , where they gain access to intracellular host membranes and machinery for replication .
ebola entry into macrophages is calcium dependent , relying on the class of endosomal two - pore channels , and disrupting the function of these channels either genetically or with pharmacological inhibitors such as tetrandrine , can prevent virus infection in human macrophages .
vaccinia virus also uses macropinocytosis as a main but possibly not exclusive pathway for entering dendritic cells .
in fact , vaccinia can also use an elegant apoptotic mimicry mechanism to enter human cells , enhancing macropinocytic uptake , but also cleverly encasing its infective virions inside a flipped ' host plasma membrane exposing phosphatidylserine on the outer surface to mimic apoptotic bodies .
virions budding from host cells with this false apoptotic coating are readily taken up by other host cells to spread the infection .
hiv infects a variety of cells through cell surface portals and different endocytic mechanisms , and virions can gain entry into macrophages via macropinosomes followed by evidence of capsid release into the cytoplasm .
hiv hijacks cell cell interactions between dendritic cells and cd4 t cells , by inducing long actin - dependent filopodia loaded at their tips with immature hiv virions , to spread the virus to their target t cells .
approaches and information emerging from the fields of cell and molecular biology , immunology and biochemistry are adding to our knowledge of the cell surface as morphological and functional
organelle ' , an entity that is customised for interacting with the extracellular milieu in many different physiological and patho - physiological situations .
many pathogenic species have already deciphered and learned to exploit aspects of mammalian cell surface behaviour and function to aid infection and their survival .
ever more powerful imaging and microscopy methods will be powerful tools for viewing cell surface behaviour and dysfunction ; gene editing techniques , genomics and proteomics are also approaches that are adding to our molecular inventory of cell surface regulators and machinery . moreover .
the knowledge acquired about cell surfaces is essential for devising new preventative measures , new vaccines and new drugs to combat the ever - present threat of common and emerging infectious diseases .
host cell targets for vaccines and drugs offer the potential to avoid or reduce the complications of acquired resistance or rapidly evolving pathogens that thwart our ability to treat and eradicate some of the most prevalent pathogens in our midst . | the surface of mammalian cells offers an interface between the cell interior and its surrounding milieu . as part of the innate immune system , macrophages have cell surface features optimised for probing and sampling as they patrol our tissues for pathogens , debris or dead cells .
their highly dynamic and constantly moving cell surface has extensions such as lamellipodia , filopodia and dorsal ruffles that help detect pathogens .
dorsal ruffles give rise to macropinosomes for rapid , high volume non - selective fluid sampling , receptor internalisation and plasma membrane turnover .
ruffles can also generate phagocytic cups for the receptor - mediated uptake of pathogens or particles .
the membrane lipids , actin cytoskeleton , receptors and signalling proteins that constitute these cell surface domains are discussed .
although the cell surface is designed to counteract pathogens , many bacteria , viruses and other pathogens have evolved to circumvent or hijack these cell structures and their underlying machinery for entry and survival .
nevertheless , these features offer important potential for developing vaccines , drugs and preventative measures to help fight infection . | Filopodia and lamellipodia
Dorsal ruffles
Ruffles for receptor signalling and pathogen detection
Macropinocytosis
Phagocytosis
Pathogen entry via ruffles and macropinosomes
Conclusion | macrophages are endowed with highly dynamic projections of their cell surface , allowing them to move and to survey the extracellular milieu with extensions such as filopodia , lamellipodia and dorsal ruffles . the same basic machinery is used to nucleate actin for the actin - based motility of bacterial pathogens , that invade host cells and induce the classical actin comet tails ' to move around in the cytoplasm . the molecular composition of filopodia and their formation rely heavily on actin - binding and modifying proteins . filopodia can form anywhere on the cell surface , for instance , emerging from lamellipodia at the cell 's leading edge during migration ( figure 1a ) . macrophages extend many filopodia , commonly using them as probes ' to explore the surrounding milieu . macrophages can also use filopodia as tentacles to draw particles towards the cell body and in an extreme form of this process , very long filopodia wrap around and entrap bacteria in the coiling phagocytosis ' described for lassoing legionella for uptake . filopodia also have cholesterol - rich and highly ordered lipid - raft - enriched membranes that are poised to contribute to receptor signalling and membrane trafficking , which in turn are important for the probing functions of these structures in cells like macrophages . dorsal ruffles ( or hereafter referred to as ruffles ) are highly distinctive , sometimes circular ( hence the alternative name
circular dorsal ruffles ' ) , veils of membrane that rise up from the dorsal surface of adherent cells or sometimes occur at peripheral edges of cells , noting however their distinction from the ruffling leading edges of migrating cells . dorsal ruffles behave in a wave - like manner , rising up and then collapsing back onto the surface , and any fluid , particles or pathogens encircled by the collapsing ruffles are internalised into the cell along with the ruffle membrane . the collapsing ruffles can form macropinosomes for fluid - phase uptake or phagosomes to engulf and internalise larger particulate matter or pathogens . the formation of dorsal ruffles in fibroblasts also involves crosstalk and co - signalling from integrins and rtks . the collapse of ruffles into macropinosomes provides for rapid internalisation and downregulation of these clustered rtks . thus , dorsal ruffles have both positive and negative regulatory roles in growth factor signalling , highlighting their importance to key processes in development and disease . dorsal ruffles are dependent on the transient production of f - actin , and inhibitors of f - actin polymerisation , such as cytochalasin d , abolish their formation . through their proline - rich domains , neural wiskott
aldrich syndrome protein , wave1 and wave2 can bind to sh3-domain containing proteins , which include arp2/3 and membrane phospholipids to help concentrate relevant actin machinery in the dynamic ruffle environment . rho gtpases are also a feature of the ruffle machinery , with both rac1 and rac2 implicated in specialised ruffle actin - associated complexes and signalling cascades , along with other rho family members including rhoa and cdc42 . active rac is needed for the development of pre - ruffle protrusions and for ruffle formation in macrophages . the inner leaflet of the plasma membrane has the highest pool of ptdins(4,5)p2 that supports protein and cytoskeletal interactions for a large suite of host processes including signalling , endocytosis , cell movement and enzyme activation . in ruffles ,
this ptdins(4,5)p2 is also a substrate for the local production of ptdins(3,4,5)p3 , which is important for recruiting signalling proteins and also potentially for closure of the dorsal ruffle . the ptdins(3,4,5)p3 produced in ruffles then persists after closure and through the early stages of formation of macropinosomes and phagocytic cups . these phosphoinositide transitions can be observed using probes such as the pleckstrin homology domain of protein kinase b ( akt ) . the phosphoinositides also recruit effector proteins to the membrane such as transporter associated with antigen processing 1 and lowe oculocerebrorenal syndrome protein , which can influence the dynamics of the cytoskeleton and subsequent compartment maturation . the dorsal ruffles are also replete with molecular machinery and families of regulators such as gtpases ( rhos , rabs , arfs and ras ) that participate in signalling and membrane trafficking , although in many cases , specific members of these families have not yet been identified specifically on ruffles in different cell types . interestingly , the adp - ribosylation factor - like ( arl ) protein , arl13b , has recently been demonstrated in dorsal ruffles . arl13b associated with f - actin in dorsal ruffles and loss of arl13b also reduces pdgf - induced dorsal ruffle formation . in this role , and in its other roles in endocytic recycling , the arl13b shares a non - muscle myosin iia as an effector . it is of emerging interest that arl13b is necessary for formation of dorsal ruffles and cilia as distinct signal transduction membrane domains . innate immunity relies on a series of host pattern recognition receptors that detect non - self , danger signals and pathogen - derived molecules . toll - like receptors ( tlrs ) are one such receptor family and different tlrs are positioned on the cell surface or endosomes to detect and respond to selected extracellular or intracellular pathogens . although macrophages ruffle constitutively , dorsal ruffling is enhanced by exposing the cells experimentally to lipopolysaccharide ( lps ) of gram - negative bacteria , indicating that ruffling is also a pathogen
lps is a ligand for tlr4 and activated tlr4 generates signals from the cell surface or from endosomes / macropinosomes using different sets of adaptor molecules to elicit cytokine release and other downstream responses . tlr4 and
its cell surface adaptors can be found clustered in dorsal ruffles where they are poised with the relevant machinery for pi3k - akt signalling . as the ruffles collapse , tlr4 is delivered to macropinosomes , or to phagosomes during pathogen engulfment , where signalling from different adaptors varies the signalling and downstream responses . in addition to tlr4-mediated transcriptional regulation and synthesis of cytokines , the dorsal ruffles and pre - phagosome membranes are also sites for release of cytokines such as tnf . newly synthesised tnf is delivered to recycling endosomes that feed membrane to the base of the ruffles and pre - phagosomal membranes , simultaneously exporting tnf to the surface at this point for release from the cell . toll - like receptors ( tlrs ) are one such receptor family and different tlrs are positioned on the cell surface or endosomes to detect and respond to selected extracellular or intracellular pathogens . although macrophages ruffle constitutively , dorsal ruffling is enhanced by exposing the cells experimentally to lipopolysaccharide ( lps ) of gram - negative bacteria , indicating that ruffling is also a pathogen
lps is a ligand for tlr4 and activated tlr4 generates signals from the cell surface or from endosomes / macropinosomes using different sets of adaptor molecules to elicit cytokine release and other downstream responses . tlr4 and
its cell surface adaptors can be found clustered in dorsal ruffles where they are poised with the relevant machinery for pi3k - akt signalling . as the ruffles collapse , tlr4 is delivered to macropinosomes , or to phagosomes during pathogen engulfment , where signalling from different adaptors varies the signalling and downstream responses . in addition to tlr4-mediated transcriptional regulation and synthesis of cytokines , the dorsal ruffles and pre - phagosome membranes are also sites for release of cytokines such as tnf . newly synthesised tnf is delivered to recycling endosomes that feed membrane to the base of the ruffles and pre - phagosomal membranes , simultaneously exporting tnf to the surface at this point for release from the cell . macropinocytosis is one of the most ancient , prevalent and yet perhaps least well - defined forms of endocytosis . it is the process of fluid gulping practiced at some level by most cells of the body , but scaled up to a high volume highway in macrophages as they perform surveillance of the surrounding tissue . the pathway results in the ingestion of fluid , plus any fluid - phase cargo and a large swathe of plasma membrane with its resident transmembrane proteins and any molecules , particles or pathogens attached to them on the external face of the plasma membrane . unlike receptor - mediated endocytosis and phagocytosis , macropinocytosis is not driven by receptor - ligand interactions . macropinocytosis is therefore distinguished by being a large volume , non - selective , actin - dependent pathway for internalisation and this is reflected in the nature of the macropinosomes themselves , which are uncoated , nondescript vesicles varying in size from 0.2 to > 2 m in diameter . as discussed below , the non - selective , large volume nature of macropinocytosis means that both inevitably , and by design , it serves for the uptake of pathogens . , who show that once macrophages have been activated by contact with pathogens , they upregulate receptor - independent macropinocytosis to override the less efficient process of receptor - mediated phagocytosis to enhance their capacity to clear the pathogens . this switch they found to be mediated by phosphorylation of the actin - binding protein coronin 1 , which is known as a prominent regulator of early actin assembly forming a crown - like ring around newly forming macropinosomes in dictyostelium and other organisms . the fate of macropinosomes has classically been studied by tracking the uptake of fluid - phase cargo , such as fluorescent dextran , or fluorescently - tagged membrane proteins or lipids in live or fixed cells . these studies routinely show that soon after internalisation , macropinosomes shrink and move towards the centre of the cell as they undergo maturation ( figure 1 ) . the rab11-positive recycling endosomal network in macrophages is vast and forms an important repository for plasma membrane and proteins recycling back to the surface , as well as new exocytic traffic , in this high turnover environment . alternatively , live imaging also reveals that macropinosomes can undergo rapid and extensive tubulation after internalisation , forming a transient network of elongated membranes or tubes that help to sort membrane proteins and fluid - phase cargo into various endosomes , many of which mature into lamp-1/rab7 late endosomes / lysosomes where degradation can occur . arf6 is involved in ruffling and macropinocytosis through roles that include recruiting rac1 for the production of pi(4,5)p2 or for the activation of phospholipase d ( reviewed in ) . other rabs , such as rab34 , rab21 and rab31 can be found on macropinosomes serving to recruit a variety of additional effectors . the transition of pi(4,5)p2 to pi(3,4,5)p3 to pi(3,4)p2 and then to pi(3)p is conserved during the processes of macropinosome closure and maturation from c.elegans to mammalian cells . the sorting nexins ( snxs ) with their signature
phox homology ( px ) lipid - binding domains are an important class of phosphoinositide effectors , and multiple snx family members are associated with , and functionally implicated in , macropinocytosis . macrophages , but interestingly not immature dendritic cells , from snx5 knockout mice have significantly reduced macropinocytosis and dorsal ruffling . a diagnostic feature of macropinocytosis , used to distinguish this pathway from clathrin - mediated endocytosis , is its sensitivity to inhibitors of na / h ion exchangers , such as amiloride . although the mode of action of amiloride is controversial , it remains a useful experimental tool for blocking macopinocytic uptake of markers or pathogens . in the immune system , macropinocytosis is an important process for antigen presentation through the environmental sampling performed by antigen - presenting cells such as macrophages and immature dendritic cells . macropinocytosis can also result in the cross - presentation of extracellular antigens on mhci after processing of these antigens to peptides in the endosomal system or in the cytoplasm . finally , the non - selective nature of macropinocytosis lends itself to exploitation for the delivery of a variety of particles , soluble drugs , genetic material and biological modifiers into cells . membrane - penetrating peptides such as trans - activating transcriptional activator ( tat ) are taken up into macropinosomes where they can cross the membrane into the cytoplasm providing an important portal for genetic or pharmacologic modifiers . the macropinocytic and ligand - directed uptake of quantum dot nanoparticles can be demonstrated with the potential to target delivery to specific cell types or situations . phagocytosis is a deliberate , receptor - driven process based on recognition of the pathogen or object and engagement with it by receptors on macrophage cell surface ruffles , filopodia or the phagocytic cups that surround and engulf the
the typical course of phagosome maturation is through fusion with successive endosome and lysosomes , acidification of the lumen and generation of reactive oxygen species , which lead to a destructive phagolysosome designed to kill pathogens and degrade them or any inert particles that have been phagocytosed . phagocytosis is essential for innate immunity , organ development and maintaining homoeostasis throughout tissues of the body . through receptor crosstalk , signalling from co - activated tlrs , for instance , concomitantly drives cell death / survival and inflammatory responses appropriate for the nature of the pathogen or particle . receptor signalling takes place in the phospholipid and actin - rich environment of the phagocytic cups where receptor trafficking is also supported by prolific membrane exchange between recycling endosomes or early endosomes and the early phagocytic membranes . actin filaments are a feature of only the early stage ruffles , pseudopods and phagocytic cups , leading up to sealing and initial internalisation of the phagosomes whereupon f - actin is rapidly depolymerised and dispersed . actin polymerisation is activated by different receptors ( through diverse pathways ) and engaged for the f - actin and receptor - mediated protrusion of the membranes over the surface of the prey during engulfment . activated fcrs recruit arp2/3 , whereas cr3 and integrin - dependent phagocytosis recruits talin and other actin - binding proteins . the phospholipid transitions that accompany ruffling and macropinocytosis have also been mapped during phagocytosis , beginning with conversion of the pi(4,5)p2 to pi(3,4,5)p3 in presealed phagocytic cups and pi(3)p in fully sealed , internalised phagosomes . with its focus as the entrapment and destruction of pathogens ,
some bacterial virulence factors aim to block phagocytic uptake , whereas for intracellular pathogens , other virulence factors are geared to allow phagocytic entry but subvert its destructive capacity . during this type of entry ,
dorsal ruffles and macropinosomes are co - opted or induced by a wide range of human pathogens and infectious agents , from viruses , to bacteria , protozoa and prions , for entry into human cells . can actively infect almost any cell type through the injection of bacterially - derived effector proteins that modulate a number of host processes such as f - actin polymerisation to induce macropinocytosis . one of the salmonella effector proteins is sipa , which has been shown to promote the generation of plasma membrane protrusions and ruffles by promoting the polymerisation of f - actin filaments . salmonella induce large localised patches of ruffling on the surface of epithelial cells , whereas on macrophages , large areas of exaggerated ruffling are induced ( figure 1 ) , serving to capture and internalise multiple bacteria near the cell surface . many of the salmonella effectors have been characterised , serving to inform us about the roles of various host proteins in normal macropinocytic processes . bacteria like legionella , which invade macrophages through modifying macropinocytic or phagocytic processes , inject hundreds of effectors , many of which remain to be characterised . the highly infectious and deadly ebola virus and mosquito - borne flaviviruses like dengue , west nile , yellow fever and the newly emergent zika viruses , all rely on macropinocytosis for entry into human cells , where they gain access to intracellular host membranes and machinery for replication . ebola entry into macrophages is calcium dependent , relying on the class of endosomal two - pore channels , and disrupting the function of these channels either genetically or with pharmacological inhibitors such as tetrandrine , can prevent virus infection in human macrophages . in fact , vaccinia can also use an elegant apoptotic mimicry mechanism to enter human cells , enhancing macropinocytic uptake , but also cleverly encasing its infective virions inside a flipped ' host plasma membrane exposing phosphatidylserine on the outer surface to mimic apoptotic bodies . hiv infects a variety of cells through cell surface portals and different endocytic mechanisms , and virions can gain entry into macrophages via macropinosomes followed by evidence of capsid release into the cytoplasm . approaches and information emerging from the fields of cell and molecular biology , immunology and biochemistry are adding to our knowledge of the cell surface as morphological and functional
organelle ' , an entity that is customised for interacting with the extracellular milieu in many different physiological and patho - physiological situations . many pathogenic species have already deciphered and learned to exploit aspects of mammalian cell surface behaviour and function to aid infection and their survival . ever more powerful imaging and microscopy methods will be powerful tools for viewing cell surface behaviour and dysfunction ; gene editing techniques , genomics and proteomics are also approaches that are adding to our molecular inventory of cell surface regulators and machinery . the knowledge acquired about cell surfaces is essential for devising new preventative measures , new vaccines and new drugs to combat the ever - present threat of common and emerging infectious diseases . host cell targets for vaccines and drugs offer the potential to avoid or reduce the complications of acquired resistance or rapidly evolving pathogens that thwart our ability to treat and eradicate some of the most prevalent pathogens in our midst . | [
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the
biological activity of many proteins is intimately linked with
their interactions with interfaces , most notably cellular membranes .
for instance , antimicrobial peptides ( amps ) , used by many organisms
to defend against infection , may function by permeabilizing bacterial
membranes .
a number of other peptides , such as voltage - gated
ion channels , possess specialized structures that allow them to span
the membrane .
moreover , some peptides
are thought to malfunction via membrane interactions ; for example ,
the membrane interactions of human islet amyloid polypeptide ( hiapp )
may be important to its aggregation , a process speculated to damage
the membrane .
membrane - active proteins often function through
a series of fast
structural changes . to study them , therefore , it is desirable to employ
an experimental technique that is selectively sensitive to the structure
of interfacial proteins and that operates on the fast time scale of
protein conformational change . here
one powerful technique for determining the structures
of interfacial proteins in realistic environments is nuclear magnetic
resonance , which has been used to probe many systems , including amps
and proton channels in micelles and lipid membranes .
electron paramagnetic resonance
with site - specific spin labeling , meanwhile , has allowed researchers
to examine particular elements of protein structure in detail and
to characterize their dynamics , and has provided detailed information
on protein conformational change in membranes . finally ,
infrared ( ir ) spectroscopic techniques provide a powerful means of
studying protein structure and dynamics due to the sensitivity of
vibrational line shapes to environmental influences and ( for time - domain
studies ) due to the sub - picosecond periods of ir pulses , which enable
these techniques to distinguish transient protein conformations . for
example
, linear and two - dimensional ( 2d ) ir methods have been used
to study the conformations of amps on a bilayer surface and the aggregation of hiapp in the presence
of lipid vesicles .
attenuated total reflection fourier
transform ir spectroscopy provides additional interface selectivity
due to the small penetration depth of the probe radiation , and it
has been used to determine the orientation of membrane proteins .
researchers using these techniques frequently employ detergent micelles
or vesicles ( instead of the native membrane ) in order to create a
soluble protein lipid system , or remove excess solvent from
a bilayer system .
vibrational sum frequency generation ( sfg )
spectroscopy offers
an attractive alternative to these techniques because of its intrinsic
surface selectivity ( described below ) , which obviates the need for
vesicles or desolvated systems , and its fast dynamical time scale .
thus , it has become much - used for the study of interfacial proteins and has enabled researchers to study these systems in real time
and in situ . in particular ,
many recent experiments have focused on the amide
i ( primarily co - stretch ) mode , which has also been extensively used
for linear and 2d ir studies .
this mode is particularly attractive for proteins because it exhibits
distinct spectral features for different secondary structures . in vibrational sfg spectroscopy ,
a resonant ir pulse and a non - resonant
visible pulse
are overlapped spatially and temporally on a sample
surface , and the signal is detected at the sum frequency of the incident
beams .
typically ,
the pulses are controlled to propagate in a single plane ( perpendicular
to the sample surface ) , with polarizations either parallel ( p ) or
perpendicular ( s ) to that plane , giving eight possible polarization
combinations : ppp , pps , psp , spp , pss , sps , ssp , and sss ( letters
listed in decreasing frequency order sum frequency , then visible ,
then ir ) . within the dipole approximation , second - order nonlinear
techniques such as sfg give no signal in centrosymmetric systems
and are thus sensitive to interfaces , where centrosymmetry breaks .
in systems of achiral molecules with random azimuthal orientation ,
the non - zero signals are ssp , sps , pss , and ppp ; these polarization combinations have been much - used in previous
work on proteins . in systems that additionally lack symmetry with respect to an arbitrary
mirror plane
, randomly oriented
chiral molecules the spp , psp , and pps signals can be non - zero
as well .
chiral sfg has been used to study
interfacial proteins . in particular , yan
and co - workers have found that different secondary structures can
be distinguished on the basis of the existence of detectable psp signals
for the protein amide i and nh - stretch modes .
for example , a parallel -sheet exhibited a distinct signal
only for the amide i mode , an -helix exhibited a signal only
for the nh - stretch mode , and an antiparallel -sheet exhibited
signals for both modes .
yan and co - workers have applied this method
to monitor in real time the aggregation of hiapp at a lipid membrane .
chiral sfg thus holds great promise as a means
for interfacial secondary structure determination .
commonly ,
vibrational sfg spectra are interpreted in terms of a
summation over independent contributions from amide vibrations in
fixed orientations relative to the surface .
that is , the amide i vibrations
are described in terms of a limited number of local or normal modes ;
the local modes are assigned a fixed molecular - frame transition dipole
and transition polarizability ; and the protein is assumed to be fixed
in a single conformation relative to the surface ( i.e. , the orientational
distribution of each amide group is assumed to be a delta function ) .
first , vibrational
coupling may cause the difference between the sfg signals of the local
and normal modes to become significant .
second , peptide dynamics can
alter the spectrum through effects such as motional narrowing ( a decrease
in line widths due to frequency self - averaging ) . finally , even if
coupling and dynamical effects can be ignored , the orientational distribution
of the local amide modes is never truly a delta function . in
order to determine the relative importance of each of these
factors ,
it is necessary to develop a theoretical framework within
which experimental results may be interpreted .
several researchers
have made strides in this direction for peptides , applying theory
to static structures or to relatively short ( 2 ns
or less ) simulations of fairly large proteins , whose sfg spectra are
difficult to interpret in detail .
( it should be noted ,
however , that such short simulation times may actually be more justified
for large proteins as opposed to smaller peptides , since large proteins
tend to have stable structures that give rise to quickly converging
spectra . ) in this work , we take a different approach , using long ( 200
ns1.25 s ) molecular dynamics ( md ) simulations to examine
in detail both chiral and non - chiral sfg spectra for relatively small
model systems . in previous works , we have developed a theoretical
strategy for
the calculation of peptide amide i spectra from md simulations and
have applied this strategy to study , e.g. , protein thermal unfolding
and amyloid aggregation . here
, we extend this method to the calculation of sfg spectra , providing
a means to bridge sfg experiments and md simulation , and to interpret
experimental spectra in a detailed manner .
we then apply this method
to study the ssp and psp spectra of three model systems , which are
displayed pictorially in figure 1 .
first , we
examine a single - chromophore achiral model amide , n - methylacetamide ( nma ) , in order to validate our spectroscopic model
and to demonstrate a zero psp signal for an achiral system .
next ,
we study a single - chromophore chiral molecule , vg dipeptide ( in its
zwitterionic form ) , and illustrate how the presence of a single chiral
center gives rise to a significant psp signal .
we then calculate spectra
for gramicidin s ( gs10 ) , a cyclic decapeptide with the sequence ( vklyp)2 , which possesses overall c2 symmetry
and a highly stable anti - parallel -sheet structure .
analysis of the results provides insight into the influence of secondary
structure , vibrational coupling , and dynamics on sfg spectra .
we conclude
by suggesting a simple method for using ratios of experimental heterodyne - detected
signals for different polarizations to determine a chromophore s
orientation , and testing this method via our simulations .
carbon atoms are shown in
tan , oxygen in red , nitrogen in blue , and hydrogen in silver .
note
that aliphatic ch , ch2 , and ch3 groups are treated as united atoms in the force field , so
these hydrogens are not shown .
in vibrational sfg , the sample interacts
with a tunable ir beam
with frequency and polarization k ,
and with a non - resonant visible beam with frequency vis and polarization .
the signal is emitted
with frequency s = + vis and polarization , and is enhanced when
the ir beam is resonant with a system vibration . in figure 2 , we diagram
the sfg setup . typically , the scattering plane is perpendicular to
the surface ; we therefore define the z - axis as the
surface normal , and the x z plane as the scattering
plane .
the beams are polarized either in the x z plane ( p polarization ) or in the y - direction ( s
polarization ) .
the angles of the input and sum - frequency wavevectors
with respect to the surface normal are , vis , and s ; by convention , we always consider the
angle with respect to vectors pointing away from the surface , such
that is between 0 and 90 for all three beams .
all these
definitions apply to the laboratory frame , symbolized here using capital
letters ( x / y / z and i / j / k ) .
, , and k indicate
the polarization directions for the sum frequency , visible , and ir
beams , respectively ( here drawn for ppp polarization ) .
indicates sum frequency
and vis indicates visible ; no subscript indicates
ir .
we now focus our derivation on
the lab - frame ssp and psp signals ,
which consist of three types of factors : elements of the ( second - rank )
fresnel tensor l , which describe effects caused by the
different refractive indices of the media under study ; factors arising
from the angles of the input and sum - frequency beams ; and elements
of the complex third - rank tensor ( individual
elements are symbolized as ijk ) ,
which describe the intrinsic response of the sample .
we symbolize the full lab - frame response
( including all three effects ) as , the effective
response .
is related to by the equation1to write the final expressions for
the effective
ssp and psp signals , sspeff and pspeff , note that s - polarization corresponds
to - polarization in the lab frame , whereas
p - polarization corresponds to polarization in the direction cos x + sin for
the ir beam , cos visx + sin vis for the
visible beam , or cos
sx + sin s for the
sum - frequency beam . substituting these polarization vectors into eq 1
gives the results:23 in this work , we will assume = vis = 45 ;
because vis
and s sin s = vis sin vis + sin , we can also set s = 45.
within this limit , the angular terms merely contribute constant factors
to both sspeff and pspeff , and we will ignore these factors .
equations 2 and 3 can
be simplified for systems with azimuthal symmetry ( with respect to
an arbitrary rotation around the z - axis ) , such as
those considered here .
frame , symbolized using lower - case letters ( x / y / z and i / j / k ) , for which = , while the x- and y - axes have been rotated by an angle such that x = cos x + sin and = sin x + cos .
azimuthal
symmetry is imposed by averaging over all angles . note that
this causes all terms with an even number of z - components
to vanish , since these terms have an odd number of sin
or cos factors when converted into the surface - fixed
frame .
thus , azimuthal averaging gives
the following results:45where the equality yxz = xyz , though not true
in general , is proven for our model in the next section .
because the
fresnel factors only contribute constant factors to both sspeff and pspeff , we now drop
them as well . defining the resulting quantities as simply ssp and psp , we have67 it is worth considering the
effect of replacing with = sin x
cos in the above analysis .
because sspeff has two y - polarized components , this reflection does not affect
the ssp signal ; pspeff , however , has only one such component , so
this reflection will reverse the sign of the psp signal . ( the fresnel
and angular factors are not affected by this procedure . )
thus , for
systems with reflection symmetry ( i.e. , achiral systems ) , pspeff = pspeff = 0 .
note also that , due to the small time and length scales employed
in simulated systems , azimuthal symmetry is not fully realized in
our simulations .
the experimental systems that we are attempting to
model are azimuthally symmetric , however , and this ought to be reflected
in the spectra .
accordingly , we identify the coordinate frame of the
simulation not with the lab frame ( x / y / z ) but with the surface - fixed frame ( x / y / z ) , for which we impose azimuthal
symmetry .
( hereafter , we will refer to this frame as the simulation
frame . )
practically , this means that we calculate spectra using eqs 6 and 7 , rather than eqs 2 and 3 . for gs10
the derivation for ppp is analogous to those for ssp and psp and is presented in appendix 1 . again ignoring the fresnel and angular factors , the result is8it
should
be noted that the justification
for ignoring the fresnel coefficients is not as strong here as for
ssp and ppp ; see appendix 1 . in order to calculate sfg signals using eqs 68 , we require a model for the
second - order nonlinear susceptibility elements ijk in the simulation frame .
ijk consists of both resonant ( r ) terms , which
depend mostly on , and non - resonant
these are represented , respectively , as
ijkr( ) and ijknr(vis ) . in this work ,
we focus exclusively on the heterodyne - detected
signal , which is given by the imaginary part of ijkr( ) and is the most physically meaningful part of the response .
ijkr( ) can be expressed as the following
quantum time - correlation function ( tcf):9here , the trace is over
all nuclear quantum
states , is the equilibrium density operator for the nuclear
hamiltonian , ij(t ) is an element of the ground electronic state 10 transition
polarizability tensor , and k(0 )
is an element of the 10 transition dipole .
this equation represents
ijkr( ) as a quantum equilibrium statistical
mechanical average , which is extremely difficult to evaluate for proteins
in the condensed phase . to simplify the analysis
, we divide
the system into a quantum subspace consisting of the amide i vibrations
and a classical bath consisting of low - frequency modes ( translations ,
rotations , and torsions ) .
we have previously
applied such a mixed quantum / classical approach successfully to linear
and 2d ir spectroscopy of liquid water and peptides in aqueous solution and at lipid membranes , as well as to sfg spectra of water at the
liquid / air interface . within this approach ,
the susceptibility for a multi - chromophore
system is given by10here , p and q index the amide i
chromophores , and apij(t ) and mqk(t ) are the time - dependent
10 transition polarizability and transition dipole elements
for chromophores p and q , respectively .
t1 is the amide i first excited - state lifetime
and is chosen to be 600 fs . importantly ,
the brackets now indicate an average over configurations from a classical
md simulation , making the calculation feasible .
f(t ) describes the time propagation of the hamiltonian:11where (t ) is the amide i hamiltonian
( divided by ):12here , p(t ) is the time - dependent local mode frequency for
chromophore p , and pq(t ) is the time - dependent coupling between
chromophores p and q. pq is the kronecker delta . to apply
these equations , it is necessary to calculate p(t ) , pq(t ) , mqk(t ) , and apij(t ) from an md simulation .
for proline frequencies and for modeling nearest - neighbor frequency
shifts and couplings including proline , however , methods by roy et
al . are used . to calculate apij(t ) , we follow others in employing the tensor determined by tsuboi et al .
for aspartame , a model amide .
in previous
works , this tensor has been presented in its diagonal form ; here ,
however , we present it in a molecular frame ( symbolized using x/y/z )
in which one of the axes is aligned with the transition dipole .
( we
do this so that the geometry of the chromophore in the simulation
frame can be easily described using euler angles of the transition
dipole in the next section . ) the transition dipole
which is
modeled as a point dipole has a location given by rc + ( 0.665 )nco + ( 0.258 )ncn , where rc is the position of the amide c , and nco and ncn are unit vectors pointing from the amide c to the amide
o and n , respectively .
the dipole lies in the con plane and forms
an angle of 10 with the oc vector . the molecular frame , then ,
is defined as follows : is aligned with the transition dipole ; x lies perpendicular to
and in the con plane such that the amide n has a positive x-coordinate ; and lies perpendicular to the con plane so as to form a right - handed
coordinate system . in this reference frame ,
depicted in figure 3 , the transition polarizability tensor is given
by13the equality yxz = xyz , stated in the previous
section , is a consequence of the symmetry of the transition polarizability
tensor , specifically the fact that ij = ji .
( note that any rotation
of this tensor , which can be represented as a unitary transformation ,
will preserve the symmetry of the tensor .
thus , the tensor is symmetric
not only in the molecule - fixed frame , but in the simulation frame
as well . )
the z-axis
forms an angle of 10 with the oc vector , and the x and z axes are defined to lie in
the con plane .
the y-axis ( not pictured )
lies orthogonal to the con plane so as to form a right - handed coordinate
system . for later analysis
, it is
useful to consider the sfg spectrum of
an isolated chromophore in the inhomogeneous limit ( i.e. , ignoring
both coupling and dynamical effects ) . the isolated - chromophore version
of eq 10 is14 in the
inhomogeneous limit , the time dependence
of the variables in the above expression is negligible , and we can
replace aij(t ) and ( ) with aij(0 ) and (0 ) , respectively .
if we ignore the lifetime
decay ( effectively letting t1
) , we can carry out the integration over t. we focus on the imaginary part of the result because we have chosen
to examine the heterodyne - detected signal .
this gives us the sfg spectral
density:15 for our model ,
the quantity aij(0)mk(0 ) has no explicit frequency
dependence ,
and it can be determined entirely from the geometry . following an
approach similar to that of laaser and zanni ( among others ) , we show in appendix 2 that the value of aij(0)mk(0 ) can be expressed
in terms of two euler angles : a tilt angle that describes
the angle between the transition dipole vector and the surface normal ,
and a twist angle that describes a subsequent rotation around
the transition dipole axis .
( that a third angle is not required is
a consequence of the azimuthal symmetry of the system . )
aijk(, )
is independent of the configurational average denoted by the angled
brackets and can therefore be moved outside the average .
we may also
take the integrals outside the average , with the result:17where p(,, )
= ( (0))(
(0)) is the joint
probability distribution describing the chromophore s frequency
and orientation .
note that in the bulk , the orientational part of p(,, ) is isotropic and independent
from the frequency distribution , such that p(,, )
= p()p(, ) = p( ) sin /4. equation 17 provides a means of determining
the effect of local - mode orientation on the sfg amplitude , independent
of dynamic or coupling effects . to do this ,
we determine p(,, ) from simulation and use the following theoretical
results ( derived in appendix 2 ) for a(,):181920note that the numerical prefactors in the
last two equations result from the substitution of values specific
to our models of the amide i local mode transition dipole and polarizability .
thus , these equations apply only to amide i local modes treated using
these models . in figure 4 , we depict
these results graphically .
note that whereas the ssp and ppp amplitudes depend mostly on the
tilt angle , the psp amplitude depends more strongly on the
twist angle . also , note that all three amplitudes are anti - symmetric
on reflection over the line = 90 , followed by a 180
shift in . this operation transforms x to x , to , and to ;
i.e. , it inverts the molecular orientation .
theoretical ssp ( top ) ,
psp ( middle ) , and ppp ( bottom ) amplitudes
as a function of transition dipole orientation for the amide i model
used in this study .
ssp contours run from a relative amplitude of 0.8 to 0.8 in
increments of 0.16 , psp contours run from 0.25 to 0.25 in
increments of 0.05 , and ppp contours run from 1.64 to 1.64
in increments of 0.328 .
a final important point concerns the fact that the ssp and
ppp
amplitudes are symmetric , and the psp amplitude anti - symmetric , on
reflection over the line = 90 or = 90.
the location of these lines of symmetry is a consequence of the fact
that we chose the y-axis to point out of
the amide plane , such that for our model , xy = yz = 0 , while xz is non - zero . because
of this , a 90 rotation in is required to align the amide
plane ( which for achiral molecules is the symmetry plane of the molecule )
with the surface normal for arbitrary . if we had instead chosen
the x-axis to point out of the amide plane ,
then yz would have been the non - zero element , and the lines of symmetry
would have been located at = 0 and = 180. in this
work , we calculate the raman spectrum of dilute nma in bulk water
in order to verify the transferability of the transition polarizability
tensor . within the mixed quantum / classical approximation , the polarization - dependent
raman line shape for a single chromophore
is given by21typically , raman spectra
are recorded either
in vv mode ( input and signal polarizations parallel ) or in vh mode
( input and signal polarizations perpendicular ) .
due to the isotropy
of the bulk system , the vv and vh spectra can be represented as spherical
averages over the constituent iijs:2223the depolarization
ratio is defined as24where2526 md simulations
are performed according to the following procedure .
first , the initial
( cubic ) box is constructed using the genbox utility
of gromacs4.5.3 .
the box contains a
single ( n - deuterated ) peptide of interest ( nma , vg ,
or gs10 ) , sufficient water ( d2o ) molecules to fill a box
with edge lengths of at least 4.05 nm ( 3004 for nma , 2217 for vg ,
and 2354 for gs10 ) , and , for gs10 , two cl counterions .
next ,
a continuation run is performed in the npt ensemble , and configurations
are output every 100 ps so as to create an ensemble of 20 initial
configurations for nma , and 50 initial configurations for vg and gs10 .
for each of these configurations ,
a pair of surfaces is created by
extending the z - dimension of the box to at least
three times its initial size .
equilibration runs are then performed
in the nvt ensemble for 5 ns for nma and vg , and for 15 ns for gs10 .
finally , nvt production runs are performed .
for nma , production runs
are 10 ns in length , for a total simulation time of 200 ns ; for vg ,
production runs are 25 ns , for a total of 1.25 s ; and for gs10 ,
production runs are 20 ns , for a total of 1 s . configurations
for production runs are output every 10 fs . for all runs ,
a
2 fs time step was employed , particle - mesh ewald was used for electrostatics ,
and a simple cutoff with a long - range dispersion correction was applied
for lennard - jones forces .
force - field parameters were taken from the
gromos96 53a6 parameter set , which employs
the spc water model .
for equilibration
runs , the berendsen thermostat and barostat were used ; for production runs , the nos hoover thermostat was used . although gs10 remains at a single interface in all
simulations ,
both nma and vg cross the box and find the other interface on occasion ;
to address this , we apply a switching function to the transition dipole
elements , as in previous work for water .
( the dipole elements are scaled based on the dipole position , as
parametrized by torii and tasumi . ) all reported spectra are averaged over the full length of the production
runs , with starting points for the calculation of tcfs taken every
50 fs .
all reported sfg spectra are normalized by the number of chromophores
and the number of starting points used , such that the amplitudes of
each are directly comparable .
to investigate
the transferability of the amide i transition polarizability tensor
obtained for aspartame , in figure 5 we show vv and vh raman spectra for bulk nma in
water , as well as experimental spectra from chen et al .
the theoretical and experimental spectra compare
favorably , and the theoretical depolarization ratio of 0.21 compares
reasonably to the experimental value of 0.16 ( see table 4 from the
experimental work ) , considering that
the theoretical tensor was taken from a different peptide .
experimental
( solid ) and theoretical ( dashed ) raman vv ( black )
and vh ( red ) line shapes for bulk , aqueous n - deuterated
nma .
experimental curves are taken from chen et al . and are scaled to match the reported depolarization ratio
of 0.16 .
experimental and theoretical curves are also normalized to
the maximum value for the vv line shape . in figure 6 , we show ssp and psp line
shapes
for nma ( from the surface simulations ) . as significant statistical
error is still present in our result even after 200 ns of md , we also
provide a 95% confidence interval for the results ( thin lines ) , calculated
by comparing the values for each frequency across the 20 independent
runs .
( this uncertainty , also present for vg dipeptide , seems to result
mainly from the fact that nma does not sit stably at one interface
but ventures into the bulk and even crosses the box to find the other
interface at several points in the simulations .
thus , portions of
the simulation are spent not describing the interfacial configurations ,
but rather averaging over bulk configurations . )
note that the psp
signal for this achiral molecule is 0 ( within the error ) , as expected .
nma s
frequency - dependent orientational distribution p(,, )
( determined from the md simulations ) is symmetrical on reflection
over the line = 90 at all frequencies , such that its
psp signal vanishes ( see figure 4 ) .
the distribution
is peaked near = 90 , but angles less than 90 are
weakly favored at low frequency , while angles greater than 90
are favored somewhat more strongly at high frequency .
thus , although
the peak in the frequency distribution p( )
is close to 1625 cm ( as in figure 5 ) , the sfg intensity is relatively weak at this frequency
because the average is very close to 90. instead , the
sfg intensity peaks at 1645 cm , where p( ) is smaller but a larger proportion of the transition
dipoles point in the same direction ( down ) .
theoretical ssp ( black )
and psp ( red ) sfg spectra ( imaginary part )
for nma .
thin lines mark the 95% confidence interval ( 2 standard
errors of the mean ) .
to understand the frequency - dependent sign of the ssp signal ,
recall
that in our model ( taken from torii and tasumi ) , the transition dipole points nearly opposite the co bond
vector . at low frequency , therefore , there is a slight tendency for
the co to point down .
this favors the formation of hydrogen bonds
between water and the carbonyl oxygen and lowers the amide i frequency .
at high frequency , meanwhile , the co tends to point up , decreasing
the number or strength of hydrogen bonds to water , and increasing
the frequency . in figure 7
, we show ssp and psp line shapes
for vg dipeptide ,
again with 95% confidence intervals .
vg differs in that it has a taller
positive peak and a shallower negative peak , which may indicate that
configurations with upward - pointing transition dipoles are overall
more favorable in this case . unlike nma ,
the chiral vg molecule generates
a significant psp signal , which is roughly as intense as the ssp signal
over the entire frequency range ( in the limit that the fresnel and
angular factors are ignored ) .
the presence and sign of the psp signal
can be understood from the fact that the transition dipole orientational
distribution p(, ) ( again calculated
from the simulation ) is peaked near = 95 , =
195 , in a region which figure 4 shows
to correspond to a positive psp signal .
visualization of this geometry
shows that it allows the carbonyl oxygen to point down , toward water ,
while allowing the valine side chain to point up , toward the vapor .
because of the chirality of the valine c , the adoption
of such a geometry requires specific tilt and twist angles .
the psp
signal can therefore be readily understood from the geometric constraints
imposed by the chirality of the molecule and the varying hydrophilicity
of its parts .
theoretical ssp ( black ) and psp ( red ) sfg spectra ( imaginary
part )
for vg dipeptide .
thin lines mark the 95% confidence interval ( 2
standard errors of the mean ) . before showing spectra
for gs10 , it is worth describing its important structural features ,
as well as its typical orientation at the interface .
gs10 , a cyclic
peptide with the sequence ( vklyp)2 , effectively possesses c2 symmetry because the two vklyp chains are
structurally equivalent .
( we say effectively because
any given simulation snapshot is unlikely to show c2 symmetry ; only the time - averaged structure does . )
gs10
has a primarily anti - parallel -sheet structure , with tight
turns at the pro side chains .
the eight amide groups whose n is not
donated by pro ( i.e. , all except the amides arising from the tyr
pro
peptide bond ) form four cross - strand hydrogen bonds . at the interface ,
gs10 orients such that the val and leu side chains point toward the
vapor , while the lys and tyr side chains point toward water .
this
interfacial configuration is quite stable , and the sfg spectra of
gs10 are well - converged after 1 s of simulation .
consequently ,
no confidence interval is shown for the sfg spectra of gs10 . in figure 8 ,
the ssp and psp spectra for gs10
are shown ( black ) , along with two limiting cases . in the first ( red )
,
the spectrum is calculated with all couplings artificially set to
zero . in the second ( blue )
, the spectral density is calculated using
eq 17 ( summing over the individual results for
each chromophore ) ; this result is insensitive to both coupling and
dynamical effects . comparing the spectra with and without coupling
( black and red )
, we see that coupling influences the spectra moderately :
the peak positions shift by 1015 cm , and
the peak shape changes somewhat for the psp spectrum . however , the
influence of the coupling is small enough that the line shapes can
be qualitatively explained without considering coupling effects .
meanwhile ,
comparing the line shape without coupling ( red ) to the spectral density
( blue ) reveals that dynamical effects such as motional narrowing have
very little effect on the spectrum .
thus , to a very reasonable approximation ,
we can understand the sfg spectra simply in terms of the orientations
of the individual amide i modes . for
a significant signal to be observed ,
the amides must adopt orientations that not only give rise to large
sfg amplitudes , but also do not significantly cancel each other . in
gs10 , for example , the ssp and psp signals are strongly negative for
only 2 of the 10 amides , causing the full - system sfg spectra to be
mostly positive .
the most important influence of secondary structure ,
then , appears not to be its influence on the couplings between amides ,
but rather its influence on the relative orientation of the amides .
theoretical
sfg spectra ( imaginary part , ssp and psp ) for gs10
peptide .
black line is the full calculation ; red line is the calculation
without coupling effects ; blue line is the spectral density calculated
using eq 17 .
it is notable that the sfg spectra of gs10 lack the characteristic
a and a peaks seen in the ir spectra
of many -peptides .
for instance , the ir spectrum of the 12-residue
-hairpin trpzip2 shows a low - frequency peak near 1636 cm and a high - frequency shoulder near 1673 cm in both experiment and theory .
first , gs10 is
even smaller than trpzip2 , and two of the four cross - strand hydogen
bonds ( the ones nearest the turns ) are somewhat unstable
. both of
these factors will cause gs10 s spectra to deviate from that
of an idealized -sheet .
second , the sfg spectrum includes contributions
from both the transition dipole and the transition polarizability .
density functional theory calculations on a large -sheet complex
by welch et al .
indicate that the a mode of -sheets
is significantly less active in raman spectroscopy than in ir spectroscopy , which suggests that the transition polarizability
associated with this mode is small . in the previous sections ,
we have presented sfg spectra for model peptides and explained how
their interfacial orientations determined using simulation give
rise to the observed spectral features .
it is also desirable to be
able to determine peptides orientations based simply on experimental
data . to this end , it is common to employ a simplified model for the
angular distribution , such that it is proportional to a two - dimensional
delta function , ()( ) .
in this way , the problem of determining the angular distribution is
reduced to the determination of two angles , and ,
a problem which can be solved using constraints from sfg or other
techniques , such as linear dichroism . in the context of our formalism , within this approximation
the triple joint distribution function is27if one is in the inhomogeneous limit , then
from eq 17,28the
important point is that if one forms the
ratio of i m for two different polarizations , then for
a given frequency , p cancels and one obtains
simply the ratio of the two a factors . for a given
ratio of signals from two different polarizations , one can therefore
use the expressions in eqs 1820 to determine , pairs
that are consistent with this ratio .
because the sfg amplitudes depend
on the frequency , data at different frequencies may produce different
curves .
furthermore , various choices of the two polarizations lead
to different functional forms for the ratio , generating different
sets of curves .
if the angular distribution is truly a delta function ,
then the curves for different polarization ratios will cross at certain
points , which represent values of and consistent with
the signal ratios . if the angular distribution is not a delta function
( it never truly is ) but is nevertheless sharply peaked , or if the
spectral density expression of eq 17 is not
an excellent approximation ( because of motional narrowing ) , then the
curves may not cross at the same point , but they should still approach
each other . in this section
, we explore this idea by using the
relative amplitudes
of ssp , psp , and ppp spectra of individual chromophore pairs in gs10
to determine constraints on those peptides interfacial orientations
consistent with a delta distribution .
we examine chromophore pairs
because the c2 symmetry of gs10 dictates
that each peptide in gs10 has a counterpart whose environment is identical ,
on average .
( e.g. , the two chromophores derived from val lys
peptide bonds are structurally equivalent . )
moreover , analysis of
the md simulations reveals that the orientational distributions p(, ) of each chromophore possess a single
dominant peak , indicating that for the most part , the two amides sample
similar orientations at any given time .
this allows us to interpret
the sfg spectra of each amide pair in terms of a single orientation . in figure 9 , we present ssp , psp , and ppp
spectra ( without coupling ) for the amide pairs arising from the val
we determine
the amplitude ratios for psp / ssp and ppp / ssp over a range of frequencies
including the peak : 16501680 cm for val lys
( ssp peak at 1667 cm ) , and 16201650 cm for tyr pro ( ssp peak at 1638 cm ) . for val
lys , the psp / ssp ratio varies from 0.787
to 0.597 , and the ppp / ssp ratio varies from 0.887 to 1.353 .
for tyr pro ,
the psp / ssp ratio varies from 0.196 to
0.059 , and the ppp / ssp ratio varies from 3.105 to 3.399 . as discussed
above ,
each amplitude ratio is consistent with a line of constraint
in and ; in figure 10 , these
lines of constraint are plotted .
solid lines show orientations consistent
with the psp / ssp ratios , while dashed lines show orientations consistent
with the ppp / ssp ratios .
theoretical ssp ( black ) , psp ( red ) , and
ppp ( blue ) spectra ( imaginary
part ) including only chromophores from val lys ( top ) or tyr
transition dipole orientational distribution p(, ) for val lys ( top ) and tyr
pro
( bottom )
amides of gs10 at an air / water interface , along with lines of constraint
derived by assuming a delta - function distribution consistent with
the psp / ssp ( solid ) and ppp / ssp ( dashed ) ratios at several frequencies .
for orientational distributions , blue indicates low probability , and
red indicates high probability .
lys lines of constraint
are determined at 1650 ( magenta ) , 1660 ( yellow ) , 1670 ( cyan ) , and
1680 ( gray ) ; tyr pro lines of constraint are determined at
1620 ( magenta ) , 1630 ( yellow ) , 1640 ( cyan ) , and 1650 ( gray ) . for val
lys ( top panel )
one sees that there two regions
in the plane where all the curves approach
each other .
however , we can rule out the solution
near = 65, = 125 because it corresponds
to incorrect signs for the different signals .
this leaves a single
possible solution near = 115 , = 235. we
can now compare this predicted orientation with the actual orientational
distribution , as determined by the simulation .
this distribution is
also shown in the top panel , and its dominant peak very nicely overlaps
the region of the solution determined using sfg .
again ,
although multiple solutions for the peptide orientation are possible ,
only one ( the correct one , near = 42 , = 98 )
is consistent with the correct sign for each signal and with the data
at each frequency .
therefore , we conclude that for a single
chromophore with a well - defined
orientation , this method could be used to determine one or a few possible
orientations from experimental heterodyne - detected signals using at
least three different polarizations ( giving at least two amplitude
ratios ) . in certain pathological situations ,
tyr chromophore has an average
tilt angle very close to 90 , such that its ssp and ppp
signals are small , and the ssp / ppp ratio does not offer a reliable
constraint . alternatively ,
if motional narrowing is important , or
if the chromophore is not isolated ( i.e. , it has significant couplings
to other chromophores ) , the results will worsen . finally , there is
the possibility that the angular distribution is not sharply peaked ,
in which case the method also will not work .
nonetheless , there would
seem to be many instances in which the method will work well , especially
if isotope - labeling is used to isolate single chromophores .
these results also emphasize the utility of examining the imaginary
part of ( using heterodyne detection ) rather than the magnitude
of ( using homodyne detection ) .
because homodyned results lack
sign information , they are not as useful for discriminating between
multiple solutions that are consistent with the psp / ssp and ppp / ssp
ratios , making it necessary to use physical intuition or theoretical
modeling to decide which result is correct .
although such results are useful in many instances ,
it is of course desirable to use the most detailed information possible
when interpreting experiments .
in this study , we have presented a theoretical protocol for the
calculation of peptide amide i sfg spectra ( including chiral
spectra ) from md simulations and applied it to study both achiral
and chiral single - chromophore molecules , as well as a 10-amino - acid
chiral peptide with an anti - parallel -sheet secondary structure .
we have shown that the ssp and psp spectra of these model systems
can be largely understood based simply on the orientational distributions
of the amide i modes involved , with only minimal effects due to coupling
or dynamics ; indeed , such an approach has already been applied to
static structures in previous studies ( e.g. , for the second harmonic
generation spectra of bacteriorhodopsin ) .
this implies that the main roles of secondary structure in determining
sfg amplitudes are simply to lock amide chromophores into tight orientational
distributions and to determine whether the spectral contributions
of different amide groups tend to reinforce or cancel each other . for gs10
, we made use of the psp / ssp and ppp / ssp amplitude ratios
to attempt to predict the amide orientations from the spectra by assuming
a delta - function distribution for the orientation of the amides at
the interface .
this method typically yields tilt and twist angles
consistent with the actual peak in the distribution .
this suggests
that as long as the orientational distribution is dominated by a single ,
relatively uniform peak , sfg amplitude ratios offer a reasonable means
of determining the interfacial orientations of amide chromophores .
despite some challenges , the development of experimental techniques
for heterodyne detection and isotope labeling holds great promise
for future studies in protein sfg spectroscopy . at present , most experiments
employ homodyne detection , which , while simple and reasonably robust ,
does not permit the reliable extraction of the imaginary part of the
resonant sfg signal ; these experiments are thus difficult to compare
to theoretical results , and do not provide a particularly strong basis
for judging the accuracy of md simulations .
heterodyne detection ,
meanwhile , directly probes the imaginary part of the signal and thus
allows for the extraction of detailed information related to the orientation
of interfacial chromophores .
meanwhile , isotope labeling provides a means of simplifying the
sfg spectra of complex systems by shifting the frequencies of labeled
chromophores away from the main amide i band.co labeling , for instance , typically lowers
the amide i frequency by 70 cm . by isolating
the spectral contributions of individual chromophores ,
their interfacial
orientations can be probed even more directly by sfg . through the combination of these techniques with computational
methods such as those outlined here , it becomes possible to obtain
a detailed understanding of protein interactions with interfaces and
to shed light on important processes such as the function of amps
and ion channels or the aggregation of hiapp . | vibrational sum frequency generation
( sfg ) has become a very promising
technique for the study of proteins at interfaces , and it has been
applied to important systems such as anti - microbial peptides , ion
channel proteins , and human islet amyloid polypeptide .
moreover , so - called
chiral sfg techniques , which rely on polarization
combinations that generate strong signals primarily for chiral molecules ,
have proven to be particularly discriminatory of protein secondary
structure . in this work ,
we present a theoretical strategy for calculating
protein amide i sfg spectra by combining line - shape theory with molecular
dynamics simulations .
we then apply this method to three model peptides ,
demonstrating the existence of a significant chiral sfg signal for
peptides with chiral centers , and providing a framework for interpreting
the results on the basis of the dependence of the sfg signal on the
peptide orientation .
we also examine the importance of dynamical and
coupling effects .
finally , we suggest a simple method for determining
a chromophore s orientation relative to the surface using ratios
of experimental heterodyne - detected signals with different polarizations ,
and test this method using theoretical spectra . | Introduction
Methods
Results
and Discussion
Conclusions | moreover , some peptides
are thought to malfunction via membrane interactions ; for example ,
the membrane interactions of human islet amyloid polypeptide ( hiapp )
may be important to its aggregation , a process speculated to damage
the membrane . to study them , therefore , it is desirable to employ
an experimental technique that is selectively sensitive to the structure
of interfacial proteins and that operates on the fast time scale of
protein conformational change . here
one powerful technique for determining the structures
of interfacial proteins in realistic environments is nuclear magnetic
resonance , which has been used to probe many systems , including amps
and proton channels in micelles and lipid membranes . electron paramagnetic resonance
with site - specific spin labeling , meanwhile , has allowed researchers
to examine particular elements of protein structure in detail and
to characterize their dynamics , and has provided detailed information
on protein conformational change in membranes . finally ,
infrared ( ir ) spectroscopic techniques provide a powerful means of
studying protein structure and dynamics due to the sensitivity of
vibrational line shapes to environmental influences and ( for time - domain
studies ) due to the sub - picosecond periods of ir pulses , which enable
these techniques to distinguish transient protein conformations . attenuated total reflection fourier
transform ir spectroscopy provides additional interface selectivity
due to the small penetration depth of the probe radiation , and it
has been used to determine the orientation of membrane proteins . vibrational sum frequency generation ( sfg )
spectroscopy offers
an attractive alternative to these techniques because of its intrinsic
surface selectivity ( described below ) , which obviates the need for
vesicles or desolvated systems , and its fast dynamical time scale . thus , it has become much - used for the study of interfacial proteins and has enabled researchers to study these systems in real time
and in situ . in particular ,
many recent experiments have focused on the amide
i ( primarily co - stretch ) mode , which has also been extensively used
for linear and 2d ir studies . in vibrational sfg spectroscopy ,
a resonant ir pulse and a non - resonant
visible pulse
are overlapped spatially and temporally on a sample
surface , and the signal is detected at the sum frequency of the incident
beams . typically ,
the pulses are controlled to propagate in a single plane ( perpendicular
to the sample surface ) , with polarizations either parallel ( p ) or
perpendicular ( s ) to that plane , giving eight possible polarization
combinations : ppp , pps , psp , spp , pss , sps , ssp , and sss ( letters
listed in decreasing frequency order sum frequency , then visible ,
then ir ) . in particular , yan
and co - workers have found that different secondary structures can
be distinguished on the basis of the existence of detectable psp signals
for the protein amide i and nh - stretch modes . for example , a parallel -sheet exhibited a distinct signal
only for the amide i mode , an -helix exhibited a signal only
for the nh - stretch mode , and an antiparallel -sheet exhibited
signals for both modes . commonly ,
vibrational sfg spectra are interpreted in terms of a
summation over independent contributions from amide vibrations in
fixed orientations relative to the surface . that is , the amide i vibrations
are described in terms of a limited number of local or normal modes ;
the local modes are assigned a fixed molecular - frame transition dipole
and transition polarizability ; and the protein is assumed to be fixed
in a single conformation relative to the surface ( i.e. in
order to determine the relative importance of each of these
factors ,
it is necessary to develop a theoretical framework within
which experimental results may be interpreted . several researchers
have made strides in this direction for peptides , applying theory
to static structures or to relatively short ( 2 ns
or less ) simulations of fairly large proteins , whose sfg spectra are
difficult to interpret in detail . in this work , we take a different approach , using long ( 200
ns1.25 s ) molecular dynamics ( md ) simulations to examine
in detail both chiral and non - chiral sfg spectra for relatively small
model systems . in previous works , we have developed a theoretical
strategy for
the calculation of peptide amide i spectra from md simulations and
have applied this strategy to study , e.g. here
, we extend this method to the calculation of sfg spectra , providing
a means to bridge sfg experiments and md simulation , and to interpret
experimental spectra in a detailed manner . we then apply this method
to study the ssp and psp spectra of three model systems , which are
displayed pictorially in figure 1 . next ,
we study a single - chromophore chiral molecule , vg dipeptide ( in its
zwitterionic form ) , and illustrate how the presence of a single chiral
center gives rise to a significant psp signal . we then calculate spectra
for gramicidin s ( gs10 ) , a cyclic decapeptide with the sequence ( vklyp)2 , which possesses overall c2 symmetry
and a highly stable anti - parallel -sheet structure . analysis of the results provides insight into the influence of secondary
structure , vibrational coupling , and dynamics on sfg spectra . we conclude
by suggesting a simple method for using ratios of experimental heterodyne - detected
signals for different polarizations to determine a chromophore s
orientation , and testing this method via our simulations . typically , the scattering plane is perpendicular to
the surface ; we therefore define the z - axis as the
surface normal , and the x z plane as the scattering
plane . the angles of the input and sum - frequency wavevectors
with respect to the surface normal are , vis , and s ; by convention , we always consider the
angle with respect to vectors pointing away from the surface , such
that is between 0 and 90 for all three beams . , , and k indicate
the polarization directions for the sum frequency , visible , and ir
beams , respectively ( here drawn for ppp polarization ) . we now focus our derivation on
the lab - frame ssp and psp signals ,
which consist of three types of factors : elements of the ( second - rank )
fresnel tensor l , which describe effects caused by the
different refractive indices of the media under study ; factors arising
from the angles of the input and sum - frequency beams ; and elements
of the complex third - rank tensor ( individual
elements are symbolized as ijk ) ,
which describe the intrinsic response of the sample . substituting these polarization vectors into eq 1
gives the results:23 in this work , we will assume = vis = 45 ;
because vis
and s sin s = vis sin vis + sin , we can also set s = 45.
within this limit , the angular terms merely contribute constant factors
to both sspeff and pspeff , and we will ignore these factors . accordingly , we identify the coordinate frame of the
simulation not with the lab frame ( x / y / z ) but with the surface - fixed frame ( x / y / z ) , for which we impose azimuthal
symmetry . in this work ,
we focus exclusively on the heterodyne - detected
signal , which is given by the imaginary part of ijkr( ) and is the most physically meaningful part of the response . ijkr( ) can be expressed as the following
quantum time - correlation function ( tcf):9here , the trace is over
all nuclear quantum
states , is the equilibrium density operator for the nuclear
hamiltonian , ij(t ) is an element of the ground electronic state 10 transition
polarizability tensor , and k(0 )
is an element of the 10 transition dipole . to simplify the analysis
, we divide
the system into a quantum subspace consisting of the amide i vibrations
and a classical bath consisting of low - frequency modes ( translations ,
rotations , and torsions ) . f(t ) describes the time propagation of the hamiltonian:11where (t ) is the amide i hamiltonian
( divided by ):12here , p(t ) is the time - dependent local mode frequency for
chromophore p , and pq(t ) is the time - dependent coupling between
chromophores p and q. pq is the kronecker delta . in previous
works , this tensor has been presented in its diagonal form ; here ,
however , we present it in a molecular frame ( symbolized using x/y/z )
in which one of the axes is aligned with the transition dipole . the transition dipole
which is
modeled as a point dipole has a location given by rc + ( 0.665 )nco + ( 0.258 )ncn , where rc is the position of the amide c , and nco and ncn are unit vectors pointing from the amide c to the amide
o and n , respectively . the isolated - chromophore version
of eq 10 is14 in the
inhomogeneous limit , the time dependence
of the variables in the above expression is negligible , and we can
replace aij(t ) and ( ) with aij(0 ) and (0 ) , respectively . if we ignore the lifetime
decay ( effectively letting t1
) , we can carry out the integration over t. we focus on the imaginary part of the result because we have chosen
to examine the heterodyne - detected signal . this gives us the sfg spectral
density:15 for our model ,
the quantity aij(0)mk(0 ) has no explicit frequency
dependence ,
and it can be determined entirely from the geometry . following an
approach similar to that of laaser and zanni ( among others ) , we show in appendix 2 that the value of aij(0)mk(0 ) can be expressed
in terms of two euler angles : a tilt angle that describes
the angle between the transition dipole vector and the surface normal ,
and a twist angle that describes a subsequent rotation around
the transition dipole axis . equation 17 provides a means of determining
the effect of local - mode orientation on the sfg amplitude , independent
of dynamic or coupling effects . to do this ,
we determine p(,, ) from simulation and use the following theoretical
results ( derived in appendix 2 ) for a(,):181920note that the numerical prefactors in the
last two equations result from the substitution of values specific
to our models of the amide i local mode transition dipole and polarizability . theoretical ssp ( top ) ,
psp ( middle ) , and ppp ( bottom ) amplitudes
as a function of transition dipole orientation for the amide i model
used in this study . a final important point concerns the fact that the ssp and
ppp
amplitudes are symmetric , and the psp amplitude anti - symmetric , on
reflection over the line = 90 or = 90.
the location of these lines of symmetry is a consequence of the fact
that we chose the y-axis to point out of
the amide plane , such that for our model , xy = yz = 0 , while xz is non - zero . if we had instead chosen
the x-axis to point out of the amide plane ,
then yz would have been the non - zero element , and the lines of symmetry
would have been located at = 0 and = 180. in this
work , we calculate the raman spectrum of dilute nma in bulk water
in order to verify the transferability of the transition polarizability
tensor . due to the isotropy
of the bulk system , the vv and vh spectra can be represented as spherical
averages over the constituent iijs:2223the depolarization
ratio is defined as24where2526 md simulations
are performed according to the following procedure . as significant statistical
error is still present in our result even after 200 ns of md , we also
provide a 95% confidence interval for the results ( thin lines ) , calculated
by comparing the values for each frequency across the 20 independent
runs . vg differs in that it has a taller
positive peak and a shallower negative peak , which may indicate that
configurations with upward - pointing transition dipoles are overall
more favorable in this case . this
interfacial configuration is quite stable , and the sfg spectra of
gs10 are well - converged after 1 s of simulation . consequently ,
no confidence interval is shown for the sfg spectra of gs10 . comparing the spectra with and without coupling
( black and red )
, we see that coupling influences the spectra moderately :
the peak positions shift by 1015 cm , and
the peak shape changes somewhat for the psp spectrum . meanwhile ,
comparing the line shape without coupling ( red ) to the spectral density
( blue ) reveals that dynamical effects such as motional narrowing have
very little effect on the spectrum . thus , to a very reasonable approximation ,
we can understand the sfg spectra simply in terms of the orientations
of the individual amide i modes . in
gs10 , for example , the ssp and psp signals are strongly negative for
only 2 of the 10 amides , causing the full - system sfg spectra to be
mostly positive . the most important influence of secondary structure ,
then , appears not to be its influence on the couplings between amides ,
but rather its influence on the relative orientation of the amides . it is notable that the sfg spectra of gs10 lack the characteristic
a and a peaks seen in the ir spectra
of many -peptides . in the previous sections ,
we have presented sfg spectra for model peptides and explained how
their interfacial orientations determined using simulation give
rise to the observed spectral features . in this way , the problem of determining the angular distribution is
reduced to the determination of two angles , and ,
a problem which can be solved using constraints from sfg or other
techniques , such as linear dichroism . in the context of our formalism , within this approximation
the triple joint distribution function is27if one is in the inhomogeneous limit , then
from eq 17,28the
important point is that if one forms the
ratio of i m for two different polarizations , then for
a given frequency , p cancels and one obtains
simply the ratio of the two a factors . in this section
, we explore this idea by using the
relative amplitudes
of ssp , psp , and ppp spectra of individual chromophore pairs in gs10
to determine constraints on those peptides interfacial orientations
consistent with a delta distribution . moreover , analysis of
the md simulations reveals that the orientational distributions p(, ) of each chromophore possess a single
dominant peak , indicating that for the most part , the two amides sample
similar orientations at any given time . this allows us to interpret
the sfg spectra of each amide pair in terms of a single orientation . in figure 9 , we present ssp , psp , and ppp
spectra ( without coupling ) for the amide pairs arising from the val
we determine
the amplitude ratios for psp / ssp and ppp / ssp over a range of frequencies
including the peak : 16501680 cm for val lys
( ssp peak at 1667 cm ) , and 16201650 cm for tyr pro ( ssp peak at 1638 cm ) . again ,
although multiple solutions for the peptide orientation are possible ,
only one ( the correct one , near = 42 , = 98 )
is consistent with the correct sign for each signal and with the data
at each frequency . therefore , we conclude that for a single
chromophore with a well - defined
orientation , this method could be used to determine one or a few possible
orientations from experimental heterodyne - detected signals using at
least three different polarizations ( giving at least two amplitude
ratios ) . in this study , we have presented a theoretical protocol for the
calculation of peptide amide i sfg spectra ( including chiral
spectra ) from md simulations and applied it to study both achiral
and chiral single - chromophore molecules , as well as a 10-amino - acid
chiral peptide with an anti - parallel -sheet secondary structure . we have shown that the ssp and psp spectra of these model systems
can be largely understood based simply on the orientational distributions
of the amide i modes involved , with only minimal effects due to coupling
or dynamics ; indeed , such an approach has already been applied to
static structures in previous studies ( e.g. for gs10
, we made use of the psp / ssp and ppp / ssp amplitude ratios
to attempt to predict the amide orientations from the spectra by assuming
a delta - function distribution for the orientation of the amides at
the interface . at present , most experiments
employ homodyne detection , which , while simple and reasonably robust ,
does not permit the reliable extraction of the imaginary part of the
resonant sfg signal ; these experiments are thus difficult to compare
to theoretical results , and do not provide a particularly strong basis
for judging the accuracy of md simulations . heterodyne detection ,
meanwhile , directly probes the imaginary part of the signal and thus
allows for the extraction of detailed information related to the orientation
of interfacial chromophores . meanwhile , isotope labeling provides a means of simplifying the
sfg spectra of complex systems by shifting the frequencies of labeled
chromophores away from the main amide i band.co labeling , for instance , typically lowers
the amide i frequency by 70 cm . | [
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a relation between hearing loss and diabetes was first reported by abel jordo in 1864.1 hearing loss in diabetic patients may be of vascular origin , due to atherosclerosis .
rust et al.,2 however , demonstrated a loss of external hair cells in diabetic rats , more intense in the ones that ingested rapidly absorbable sugars .
tintera and goldman4 ( 1956 ) , goldman5 ( 1962 ) , and powers6 ( 1972 ) analyzed cases of menire 's disease associated to hypoglycemia .
updegraff,7 in 1977 , proposed routine insulin titration for examining patients with auditory and/or vestibular disorders that presented a clinical possibility of altered carbohydrate metabolism .
updegraff 's proposal was based on the observations of joseph r. kraft,8 who , in 1975 , published his database with 3,000 glucose tolerance tests , including simultaneous insulin titration .
he introduced the concept of diabetes in situ , or occult diabetes , to describe types of changes that would evolve gradually to clinical diabetes . in his book ,
published in 2008,9 he concluded that , based on more than 14,000 tests , his original observations were correct .
fukuda,10 in 1982 , and mangabeira - albernaz and fukuda,11 in 1984 , using kraft 's method , analyzed a group of patients with metabolic problems and inner ear disorders , and concluded that hyperinsulinemia was the most common finding .
they confirmed that the insulin levels were more sensitive indicators of carbohydrate metabolism changes than glucose levels . in 1985 ,
12 described for the first time vestibular disorders caused by hypoglycemia resulting from defective enzyme mechanisms of the small intestine ( brush border membrane disease ) .
d'avila and lavinsky , in 2005 , studied the glucose and insulin profiles in patients with menire 's disease14 and zuma , maia and lavinsky , in 2006 , studied otoacoustic emissions in an animal model of induced hyperinsulinemia.15 ramos , ramos et al .
revised the topic of metabolic vertigo in 2014.16
the stria vascularis is the essential metabolic center of the inner ear .
it is responsible for the endocochlear potential and the maintenance of the chemical structure of endolymph , removing its sodium and controlling its high concentration of potassium .
this is accomplished by { na - k } atpase , commonly called sodium - potassium pump , an enzyme that is well - distributed in the organism and present in the stria in high concentrations.17
koide et al.,18 in 1960 , studied the action of insulin in the inner ear of guinea pigs .
they noticed that a reduction of cochlear microphonics occurred three hours after the administration of 80 ui of insulin per kilogram of body weight .
the administration of glucose or other krebs ' cycle substrates resulted in a partial recovery .
mendelsohn and roderique,19 in 1972 , administered insulin to guinea pigs and reported changes in the chemical composition of endolymph : a progressive reduction of the potassium concentration and progressive increase in sodium concentration .
it is well known that glucose and oxygen are essential nutrients to the sodium - potassium pump ; however , glucose transport mechanisms in the cochlea are not so well known .
there are studies showing that glucose transport is accomplished through communication junctions ( nexus ) with the help of a transporter ( glut1 ) , the presence of which has been demonstrated in the basal cells of the stria vascularis in mice .
immunochemical tests have shown that glut1 , ocludine ( a protein that closes the nexus ) and the proteins conexin 26 and conexin 30 have also been demonstrated in the basal cells of the stria , also in mice.20
the + 80 mv endocochlear potential is essential for normal hearing and is intimately related to the mechanisms for potassium transportation.21
22 sodium is also metabolized by the stria vascularis by means of selective channels.23
in patients with brush border membrane disease , the high concentrations of lactose and saccharose in the small intestine create an osmotic unbalance that results in transposition of blood glucose to the intestine , producing hypoglycemia .
the unabsorbed sugars proceed to the colon and are digested by intestinal bacteria , resulting in abdominal distension and , often , gas and diarrhea.13
changes in glucose and insulin blood concentration affect cochlear homeostasis by changing the chemical structure of endolymph .
the increase in sodium concentration and reduction of potassium increase osmotic pressure , requiring more water in the endolymphatic space .
it is known that endolymphatic hydrops is the substrate of menire 's disease , but we also know that hydrops occurs in other inner ear disorders . on the other hand , hydrops may derive from excessive production or lack of absorption of the endolymph and these mechanisms are unrelated with metabolic disorders .
this research was approved by the ethics committee of the hospital albert einstein ( caae : 36936314.8.0000.0071 ) .
this is a retrospective study , including 376 patients ( 155 males and 221 females ) sequentially seen in a clinic , from january 2004 to june 2014 , under suspicion of metabolic inner ear problems .
their ages varied from 14 to 81 years , with a mean of 46.90 years .
all of the patients underwent a clinical examination , a neurotological evaluation and laboratory procedures .
all of the patients underwent a general physical examination and ear , nose , and throat evaluation .
next , patients underwent audiological tests , including pure tone audiometry , speech discrimination tests and immitance tests .
were performed , as well as tests for spontaneous and gaze nystagmus , eyetracking , optokinetic nystagmus , torsion swing tests , and air caloric tests .
the glucose tolerance test with simultaneous insulin titration is the most important test for this group of patients . in the last years
, three - hour curves are obtained , collecting blood at 0 ( fast ) , 30 , 60 , 90 , 120 , and 180 minutes . immediately after the fast sample ,
authors considered a value equal to or smaller than 55 mg / dl of glucose at any point as hypoglycemia .
values of more than 200 mg / dl at any point were considered indicative of diabetes mellitus type ii .
resistance to insulin was measured by taking into account the fast values of glucose and insulin , using a calculator for homa ir ( homeostasis model assessment insulin resistance ) .
the interpretation of the insulin curves was conducted according to the principles established by kraft .
the normal fast levels are 5 to 30 ui / ml and the peak values occur either at 30 or 60 minutes .
the maximum value for the sample obtained at 120 minutes is 50 ui / ml and the sum of the value for 120 and 180 minutes shall not exceed 60 ui / ml . in kraft 's type ii curve , peak levels also occur at 30 or 60 minutes ; however , the values at 120 and 180 minutes are higher , characterizing a normal peak and slow recovery .
in kraft 's type iii curve
the peak may occur at 120 minutes subtype iiia or at 180 minutes subtype iiib .
kraft 's type iv curve is characterized by high fast values of insulin ( 50 ui / ml or more ) and are seen in diabetic patients .
the patients with fast values of 30 ui or more with one or more glucose values above 200 mg / dl were included in this category . kraft 's type v curve shows low values of insulin at all times and is suggestive of intestinal sugar malabsorption .
fifty - one of the patients with a type v curve were submitted to an oral lactose tolerance test , with samples collected at fast , then 30 , 60 , and 90 minutes after the ingestion of 50 g of lactose .
the increase in the glucose blood levels is normally higher than 20 mg / dl ; lesser levels suggest brush border membrane disease , a small intestine disorder affecting the production of disaccharidases , particularly lactase.12
13 the test is also considered positive when the patient has diarrhea after the lactose intake .
the test was not ordered when the patient had a clinical history of frequent diarrhea and gas episodes . for four patients ,
only the oral lactose tolerance test was ordered , in view of their gastrointestinal symptoms .
more recently , a genetic test for lactose intolerance that does not require a lactose intake has been also employed.24
25
for some patients other laboratory tests related to their clinical history , such as t4 and tsh , cholesterol and its fractions , triglycerides , liver function tests , cortisol , and dehydroepiandrosterone .
imaging .
computerized tomography scans are important in the differential diagnosis of structural cochlear defects such as mondini 's dysplasia , dilated vestibular aqueducts , superior semicircular canal dehiscence and malformations of the internal acoustic meatus .
magnetic resonance imaging with paramagnetic contrast was required in patients with unilateral deafness to discard vestibular schwannomas and other tumors .
the patients in this study presented cochlear symptoms , vestibular symptoms or a combination of both types .
cochlear symptoms alone were observed in 77 patients ( 20.42% ) ; vestibular symptoms in 155 ( 41.11% ) .
typical meniere 's syndrome was seen in 29 patients ( 7.69% ) ; 28 patients ( 7.42% ) had migraine ; intraaural pressure was present in 16 patients ( 4.24% ) .
a history of drop attacks was referred by 9 patients ( 2.38% ) and 12 patients ( 3.18% ) had panic syndrome .
some patients complained of intermittent blocking of the ears , similar to that observed during an airplane 's descent .
the most common vestibular symptom was the sensation of floating , like walking on a soft rubber foam floor .
many patients described a sudden need to ingest foods rich in sugar ; this usually coincided with moments of hypoglycemia . some of them had parents or relatives with type ii diabetes .
some patients complained of digestive problems such as constipation , diarrhea , and abdominal distention .
it is worth noting that many patients presented more than one of the symptoms listed .
the patients were divided into groups i , ii , iiia , iiib , iv , and v , according to the type of their insulin curves , using kraft 's classification .
the fast levels for glucose ranged from 67 to 146 mg / dl , with a mean of 85.93 and a standard deviation of 16.99 .
thirty - nine patients presented glucose levels of 200 mg / dl or more during the three - hour glucose tolerance tests .
table 3 shows a statistical evaluation of the significance of the differences in glucose blood levels found for groups ii , iiia , iiib , iv , and v in relation to group i ( normal ) .
student t tests were employed to establish the significance of the results ; the values of the expected t correspond to a level of confidence of 0.05 ( 5.00% ) .
the values for the insulin curves , including the means and the standard deviations , are shown in table 4 .
table 5 shows a statistical evaluation of the significance of the differences in insulin blood levels found for groups ii , iiia , iiib , iv , and v in relation to group i ( normal ) . since all of the patients with insulin curve types ii , iiia , iiib , and iv present hyperinsulinemia
, they were compared together with the patients with type v curves , who present intestinal malabsorption of carbohydrates ( brush border membrane disease ) .
table 6 shows mean glucose blood levels for the patients with hyperglycemia ( groups ii , iiia , iiib , and iv together ) and the corresponding standard deviations .
table 7 shows mean glucose blood levels for the patients with brush border membrane disease ( group v ) and the corresponding standard deviations .
table 8 shows mean insulin blood levels for the patients with hyperinsulinemia ( groups ii , iiia , iiib , and iv ) and the corresponding standard deviations .
table 9 shows mean insulin blood levels for the patients with brush border membrane disease ( group v ) and the corresponding standard deviations .
3 shows a comparison of mean glucose levels in patients with hyperinsulinemia and brush border membrane disease .
mean glucose levels in patients with hyperinsulinemia ( hi ) and brush border membrane disease ( bbmd ) .
4 shows a comparison of mean insulin levels in patients with hyperinsulinemia and brush border membrane .
mean insulin levels in patients with hyperinsulinemia ( hi ) and brush border membrane disease ( bbmd ) .
table 10 shows a statistical evaluation of the significance of the differences in insulin blood levels found for the patents with hyperinsulinemia ( groups ii , iiia , iiib , iv , together ) and brush border membrane disease ( group v ) .
table 11 shows the means and standard deviations of the glucose levels of the 55 patients submitted to an oral lactose tolerance test .
table 12 shows the homa ir values for the patients with hyperinsulinemia , showing the percentage of cases in each type of kraft curve that showed a value above the established limit of 2.7 .
the author found that one patient had a 1.5 mm intracanalicular vestibular schwannoma in the left ear .
immitance tests were usually normal . in some cases , electrocochleography showed changes in the relation between summation and action potentials .
the patients were divided into groups i , ii , iiia , iiib , iv , and v , according to the type of their insulin curves , using kraft 's classification .
abbreviations : n , number of individuals ; std , standard deviation . the fast levels for glucose ranged from 67 to 146 mg / dl , with a mean of 85.93 and a standard deviation of 16.99 .
thirty - nine patients presented glucose levels of 200 mg / dl or more during the three - hour glucose tolerance tests .
table 3 shows a statistical evaluation of the significance of the differences in glucose blood levels found for groups ii , iiia , iiib , iv , and v in relation to group i ( normal ) .
student t tests were employed to establish the significance of the results ; the values of the expected t correspond to a level of confidence of 0.05 ( 5.00% ) .
the values for the insulin curves , including the means and the standard deviations , are shown in table 4 .
table 5 shows a statistical evaluation of the significance of the differences in insulin blood levels found for groups ii , iiia , iiib , iv , and v in relation to group i ( normal ) . since all of the patients with insulin curve types ii , iiia , iiib , and iv present hyperinsulinemia
, they were compared together with the patients with type v curves , who present intestinal malabsorption of carbohydrates ( brush border membrane disease ) .
table 6 shows mean glucose blood levels for the patients with hyperglycemia ( groups ii , iiia , iiib , and iv together ) and the corresponding standard deviations .
table 7 shows mean glucose blood levels for the patients with brush border membrane disease ( group v ) and the corresponding standard deviations .
table 8 shows mean insulin blood levels for the patients with hyperinsulinemia ( groups ii , iiia , iiib , and iv ) and the corresponding standard deviations .
table 9 shows mean insulin blood levels for the patients with brush border membrane disease ( group v ) and the corresponding standard deviations .
3 shows a comparison of mean glucose levels in patients with hyperinsulinemia and brush border membrane disease .
mean glucose levels in patients with hyperinsulinemia ( hi ) and brush border membrane disease ( bbmd ) .
4 shows a comparison of mean insulin levels in patients with hyperinsulinemia and brush border membrane .
mean insulin levels in patients with hyperinsulinemia ( hi ) and brush border membrane disease ( bbmd ) .
table 10 shows a statistical evaluation of the significance of the differences in insulin blood levels found for the patents with hyperinsulinemia ( groups ii , iiia , iiib , iv , together ) and brush border membrane disease ( group v ) .
table 11 shows the means and standard deviations of the glucose levels of the 55 patients submitted to an oral lactose tolerance test .
table 12 shows the homa ir values for the patients with hyperinsulinemia , showing the percentage of cases in each type of kraft curve that showed a value above the established limit of 2.7 .
the author found that one patient had a 1.5 mm intracanalicular vestibular schwannoma in the left ear . all other ear and/or
taking into consideration the series of studies previously performed by the present author and his associates,10
11
26 a total of 1377 patients have been investigated over a period of 32 years , providing a significant amount of clinical experience .
there are no clear explanations for the fact that some patients have only cochlear symptoms , some have only vestibular symptoms whereas others have both cochlear and vestibular symptoms . since the endolymph in the vestibular portion of the system does not have the 80 mv electric charge , it would be reasonable to infer that the cochlea should be more sensitive to changes in fluids ' chemical composition , but this is not the case .
mention must be made of symptoms not related to the inner ear , such as metabolic migraine , caused by excessive accumulation of sodium in the brain cells and blurred vision , caused by loss of glycogen in the lens in moments of intense hypoglycemia , recognized as a cause of cataracts in young persons .
patients with hyperinsulinemia ( kraft curves type ii , iiia , iiib , and iv ) and patients with brush border membrane disease ( kraft curve type v ) exhibit some clinical differences . patients with excessive liberation of insulin have a tendency to present low glucose blood levels during certain hours of the day .
the foods that cause excessive insulin liberation in sensitive people are the rapid absorption carbohydrates , such as purified sucrose and fructose .
overweight patients have high levels of blood insulin caused by the increased peripheral insulin resistance due to blocked receptors .
5 shows the marked differences in glucose and insulin blood levels in a 41-year - old female patient that underwent bariatric surgery .
the glucose pre - operative curve shows a high fast blood level that rises to a delayed peak at 120 minutes .
glucose ( left ) and insulin ( right ) blood levels in a patient before and after bariatric surgery .
the post - operative glucose curve shows a normal fast level , a normal peak at 30 minutes and reactive hypoglycemia at 120 minutes .
the pre - operative insulin curve shows a delayed peak at 180 minutes , showing that the pancreas is unable to react to the sudden increase in glucose blood level .
brush border membrane disease , on the other hand , causes a fluctuation of the glucose level due to the osmotic unbalance caused by unabsorbed sugars ( usually lactose ) in the small intestine .
the limit established by kraft is 30 ui / ml , but the author observed that some patients with confirmed disaccharidases had values up to 50 ui / ml .
a double - peaked insulin curve , parallel to the glucose curve , was also found to be common in patients with intestinal carbohydrate malabsorption .
for these patients , the hypoglycemic peaks usually occur one to two hours after the glucose ingestion ; whereas , in patients with hyperinsulinemia , it usually occurs three hours or more after the ingestion .
the fact that patients with brush border membrane disease have symptoms similar to those with hyperinsulinemia supports the concept that the inner ear symptoms are actually related to hypoglycemia .
the involvement of the transport proteins that carry the monosaccharides into the blood stream after the enzymatic break - up of the disaccharides is usually affected by the disorder ; therefore , it is also important to restrict sucrose to a certain extent .
maltase production is very rarely affected ; it was only seen in rare cases of marathon runners , who would only eat carbohydrates for long periods of time .
contrary to patients with hyperinsulinemia , patients with brush border membrane disease very seldom become diabetic . in our original study12 biopsies of the jejunal mucosa of the patients with suspected brush border membrane disease were obtained and we confirmed reduction or absence of lactase by microchemical assay .
this was performed to exclude other types of intestinal disorders that could eventually produce similar symptoms .
most of the time , the hearing thresholds for low and high tones show some fluctuation . as a rule , there is no complaint of hearing loss , since the thresholds for the speech frequencies are almost always within the normal social range ; however , they often report the sensation of intermittent blocking , which they compare to what one experiences in a descending airplane . furthermore
the fluctuant hearing loss and the sensitivity to noise are usually reversible with treatment . in some untreated cases ,
the episodic sensation of floating , or walking over foam , is the most frequent vestibular symptom , although many patients refer to episodes of vertigo .
vestibular hyperrelexia is the most common finding in vestibular examination , and is probably related to the changes in the density of endolymph , caused by the increased sodium concentration and water accumulation to correct osmotic changes .
tension and stress often aggravate the symptoms by increasing the consumption of glucose by central nervous system cells .
the diagnosis of altered carbohydrate metabolism is confirmed by the oral glucose tolerance test with simultaneous insulin titration .
most clinicians rely only on the glucose fast levels for this diagnosis , but this is a test of low sensitivity , even if it is interpreted together with the glycated hemoglobin test . in this series of 376 patients ,
only 56 ( 14.89% ) had glucose fast levels of 100 mg / dl or more .
homa ir was abnormal ( 2.7 or greater ) only in 25 patients ( 6.72% ) , suggesting that most of the patients in this study have intolerance to rapidly absorbable carbohydrates and do not show resistance to insulin .
the insulin curves were found to be twice as sensitive as the glucose curves.11 it is interesting to note that there is a reluctance on the part of many clinicians and endocrinologists to accept the predictions based on kraft 's classification , in spite of the clinical and experimental data available.9
11
18
19
26 our findings confirm kraft 's observations that insulin curves do , in fact , allow for the prediction of future diabetes.8
9
on the other hand , only 31 patients ( 8.22% ) in this study had a normal ( kraft type i ) insulin curve , indicating the value of the clinical history and neurological evaluation as a basis for the diagnosis of impaired carbohydrate metabolism .
the insulin curves were used to divide the patients into six groups , according to kraft 's classification .
taking in consideration that the patients were selected on the basis of their clinical symptoms and results of neurotological tests , an actual control group is unavailable .
for this reason the statistical tests were performed by comparing the patients with kraft 's curve types ii , iiia , iiib , iv , and v with the patients with type i ( normal ) curves . in spite of the fact that the groups followed the classification of their insulin curves ,
it will be noted ( tables 2 and 3 ) that the glucose curves of the patients in groups ii , iiia , iiib , iv , and v showed statistically significant differences in relation to those of group i. this indicates that the glucose blood levels at different times are essentially dependent on the amount of liberated insulin .
a comparison between the glucose and insulin levels of patients with hyperinsulinemia ( groups ii , iia iiib , and iv , together , compared with group v ) also showed statistically significant differences .
the hearing losses that result from impaired carbohydrate metabolism are among the few that can be adequately treated and may return to normal .
the same can be said of the vestibular symptoms , that disappear in most patients .
in fact , controlling glucose blood levels and the amount of insulin liberated by the pancreas are effective not only to control inner ear symptoms , but also to identify patients that are prone to become diabetic and , possibly , delaying the onset of the disease .
these patients must reduce the intake of quickly absorbable sugars and reduce intervals between their meals , eating every three hours of the day .
the ideal food for the intermediate meals are fruits ; the fructose in fruit is not rapidly absorbed , being attached to the pulp of the fruit . as an alternative , unsweetened cereal bars or crackers can be consumed , or a light yogurt .
large molecule carbohydrates ( i.e. , bread , pasta , rice , beans ) may be consumed , up to 100 g per day .
obese patients are strongly advised to lose weight to reduce the effect of insulin resistance due to the blocking of insulin receptors .
the alternative is bariatric surgery , when indicated . in patients with brush border membrane disease ,
commercially available lactase may also be used to help digest milk and derivatives . in view of the frequent involvement of the transport proteins that carry monosaccharides into the blood stream , sucrose
the amount of sugar intake must be determined by each patient using a trial - and - error approach .
such diets usually take 45 to 90 days to regulate the patient 's glucose levels .
those that suffer from severe vestibular disorders are given medications for a short period of time , until audiological tests show that hearing has been stabilized .
many vasodilator drugs increase the glucose consumption by the central nervous system cells ; these medications must be avoided .
the follow - up of patients with carbohydrate metabolism disorders of the inner ear is quite complex .
occasionally , patients ' friends , or patients referred by them , relay that they are well .
some patients that abandoned their diets occasionally return when their symptoms get worse ; most of them have been found to be diabetic.26
the identification of these problems , however , is very important for the patients .
it provides them with a better quality of life , correcting their inner ear symptoms and preventing , or at least postponing for many years , the occurrence of clinical diabetes .
the inner ears are very sensitive organs and , in many patients , are the first to show signs of carbohydrate metabolism disorders .
the cochlear and vestibular symptoms resulting from these disorders are very common , can be diagnosed and treated , and are often reversible . | introduction
metabolic activity of the inner ear is very intense , and makes it sensitive to changes in the body homeostasis .
this study involves a group of patients with inner ear disorders related to carbohydrate metabolism disturbances , including hearing loss , tinnitus , dizziness , and episodes of vertigo.objectives
to describe the symptoms of metabolic inner ear disorders and the examinations required to establish diagnoses .
these symptoms are often the first to allow for an early diagnosis of metabolic disorders and diabetes.methods
retrospective study of 376 patients with inner ear symptoms suggestive of disturbances of carbohydrate metabolism .
the authors present patient clinical symptoms and clinical evaluations , with emphasis on the glucose and insulin essays.results
authors based their conclusions on otolaryngological findings , diagnostic procedures and treatment principles .
they found that auditory and vestibular symptoms usually occur prior to other manifestations of metabolic changes , leading to an early diagnosis of hyperinsulinemia , intestinal sugar malabsorption or diabetes .
previously undiagnosed diabetes mellitus type ii was found in 39 patients.conclusions
the identification of carbohydrate metabolism disturbances is important not only to minimize the patients ' clinical symptoms , but also to help maintain their general health . | Introduction
Methods
Results
Audiological Findings
Vestibular Examination
Glucose and Insulin Assays
Imaging
Discussion
Conclusions | a relation between hearing loss and diabetes was first reported by abel jordo in 1864.1 hearing loss in diabetic patients may be of vascular origin , due to atherosclerosis . tintera and goldman4 ( 1956 ) , goldman5 ( 1962 ) , and powers6 ( 1972 ) analyzed cases of menire 's disease associated to hypoglycemia . updegraff,7 in 1977 , proposed routine insulin titration for examining patients with auditory and/or vestibular disorders that presented a clinical possibility of altered carbohydrate metabolism . updegraff 's proposal was based on the observations of joseph r. kraft,8 who , in 1975 , published his database with 3,000 glucose tolerance tests , including simultaneous insulin titration . he introduced the concept of diabetes in situ , or occult diabetes , to describe types of changes that would evolve gradually to clinical diabetes . fukuda,10 in 1982 , and mangabeira - albernaz and fukuda,11 in 1984 , using kraft 's method , analyzed a group of patients with metabolic problems and inner ear disorders , and concluded that hyperinsulinemia was the most common finding . they confirmed that the insulin levels were more sensitive indicators of carbohydrate metabolism changes than glucose levels . in 1985 ,
12 described for the first time vestibular disorders caused by hypoglycemia resulting from defective enzyme mechanisms of the small intestine ( brush border membrane disease ) . d'avila and lavinsky , in 2005 , studied the glucose and insulin profiles in patients with menire 's disease14 and zuma , maia and lavinsky , in 2006 , studied otoacoustic emissions in an animal model of induced hyperinsulinemia.15 ramos , ramos et al . revised the topic of metabolic vertigo in 2014.16
the stria vascularis is the essential metabolic center of the inner ear . it is responsible for the endocochlear potential and the maintenance of the chemical structure of endolymph , removing its sodium and controlling its high concentration of potassium . this is accomplished by { na - k } atpase , commonly called sodium - potassium pump , an enzyme that is well - distributed in the organism and present in the stria in high concentrations.17
koide et al.,18 in 1960 , studied the action of insulin in the inner ear of guinea pigs . mendelsohn and roderique,19 in 1972 , administered insulin to guinea pigs and reported changes in the chemical composition of endolymph : a progressive reduction of the potassium concentration and progressive increase in sodium concentration . it is well known that glucose and oxygen are essential nutrients to the sodium - potassium pump ; however , glucose transport mechanisms in the cochlea are not so well known . there are studies showing that glucose transport is accomplished through communication junctions ( nexus ) with the help of a transporter ( glut1 ) , the presence of which has been demonstrated in the basal cells of the stria vascularis in mice . immunochemical tests have shown that glut1 , ocludine ( a protein that closes the nexus ) and the proteins conexin 26 and conexin 30 have also been demonstrated in the basal cells of the stria , also in mice.20
the + 80 mv endocochlear potential is essential for normal hearing and is intimately related to the mechanisms for potassium transportation.21
22 sodium is also metabolized by the stria vascularis by means of selective channels.23
in patients with brush border membrane disease , the high concentrations of lactose and saccharose in the small intestine create an osmotic unbalance that results in transposition of blood glucose to the intestine , producing hypoglycemia . the unabsorbed sugars proceed to the colon and are digested by intestinal bacteria , resulting in abdominal distension and , often , gas and diarrhea.13
changes in glucose and insulin blood concentration affect cochlear homeostasis by changing the chemical structure of endolymph . it is known that endolymphatic hydrops is the substrate of menire 's disease , but we also know that hydrops occurs in other inner ear disorders . on the other hand , hydrops may derive from excessive production or lack of absorption of the endolymph and these mechanisms are unrelated with metabolic disorders . this is a retrospective study , including 376 patients ( 155 males and 221 females ) sequentially seen in a clinic , from january 2004 to june 2014 , under suspicion of metabolic inner ear problems . all of the patients underwent a clinical examination , a neurotological evaluation and laboratory procedures . all of the patients underwent a general physical examination and ear , nose , and throat evaluation . were performed , as well as tests for spontaneous and gaze nystagmus , eyetracking , optokinetic nystagmus , torsion swing tests , and air caloric tests . the glucose tolerance test with simultaneous insulin titration is the most important test for this group of patients . in the last years
, three - hour curves are obtained , collecting blood at 0 ( fast ) , 30 , 60 , 90 , 120 , and 180 minutes . values of more than 200 mg / dl at any point were considered indicative of diabetes mellitus type ii . resistance to insulin was measured by taking into account the fast values of glucose and insulin , using a calculator for homa ir ( homeostasis model assessment insulin resistance ) . the interpretation of the insulin curves was conducted according to the principles established by kraft . the maximum value for the sample obtained at 120 minutes is 50 ui / ml and the sum of the value for 120 and 180 minutes shall not exceed 60 ui / ml . in kraft 's type ii curve , peak levels also occur at 30 or 60 minutes ; however , the values at 120 and 180 minutes are higher , characterizing a normal peak and slow recovery . the patients with fast values of 30 ui or more with one or more glucose values above 200 mg / dl were included in this category . kraft 's type v curve shows low values of insulin at all times and is suggestive of intestinal sugar malabsorption . fifty - one of the patients with a type v curve were submitted to an oral lactose tolerance test , with samples collected at fast , then 30 , 60 , and 90 minutes after the ingestion of 50 g of lactose . the increase in the glucose blood levels is normally higher than 20 mg / dl ; lesser levels suggest brush border membrane disease , a small intestine disorder affecting the production of disaccharidases , particularly lactase.12
13 the test is also considered positive when the patient has diarrhea after the lactose intake . more recently , a genetic test for lactose intolerance that does not require a lactose intake has been also employed.24
25
for some patients other laboratory tests related to their clinical history , such as t4 and tsh , cholesterol and its fractions , triglycerides , liver function tests , cortisol , and dehydroepiandrosterone . computerized tomography scans are important in the differential diagnosis of structural cochlear defects such as mondini 's dysplasia , dilated vestibular aqueducts , superior semicircular canal dehiscence and malformations of the internal acoustic meatus . the patients in this study presented cochlear symptoms , vestibular symptoms or a combination of both types . some patients complained of intermittent blocking of the ears , similar to that observed during an airplane 's descent . some patients complained of digestive problems such as constipation , diarrhea , and abdominal distention . it is worth noting that many patients presented more than one of the symptoms listed . the patients were divided into groups i , ii , iiia , iiib , iv , and v , according to the type of their insulin curves , using kraft 's classification . table 3 shows a statistical evaluation of the significance of the differences in glucose blood levels found for groups ii , iiia , iiib , iv , and v in relation to group i ( normal ) . student t tests were employed to establish the significance of the results ; the values of the expected t correspond to a level of confidence of 0.05 ( 5.00% ) . the values for the insulin curves , including the means and the standard deviations , are shown in table 4 . table 5 shows a statistical evaluation of the significance of the differences in insulin blood levels found for groups ii , iiia , iiib , iv , and v in relation to group i ( normal ) . since all of the patients with insulin curve types ii , iiia , iiib , and iv present hyperinsulinemia
, they were compared together with the patients with type v curves , who present intestinal malabsorption of carbohydrates ( brush border membrane disease ) . table 6 shows mean glucose blood levels for the patients with hyperglycemia ( groups ii , iiia , iiib , and iv together ) and the corresponding standard deviations . table 7 shows mean glucose blood levels for the patients with brush border membrane disease ( group v ) and the corresponding standard deviations . table 8 shows mean insulin blood levels for the patients with hyperinsulinemia ( groups ii , iiia , iiib , and iv ) and the corresponding standard deviations . table 9 shows mean insulin blood levels for the patients with brush border membrane disease ( group v ) and the corresponding standard deviations . mean glucose levels in patients with hyperinsulinemia ( hi ) and brush border membrane disease ( bbmd ) . table 11 shows the means and standard deviations of the glucose levels of the 55 patients submitted to an oral lactose tolerance test . table 12 shows the homa ir values for the patients with hyperinsulinemia , showing the percentage of cases in each type of kraft curve that showed a value above the established limit of 2.7 . the author found that one patient had a 1.5 mm intracanalicular vestibular schwannoma in the left ear . in some cases , electrocochleography showed changes in the relation between summation and action potentials . the patients were divided into groups i , ii , iiia , iiib , iv , and v , according to the type of their insulin curves , using kraft 's classification . table 3 shows a statistical evaluation of the significance of the differences in glucose blood levels found for groups ii , iiia , iiib , iv , and v in relation to group i ( normal ) . student t tests were employed to establish the significance of the results ; the values of the expected t correspond to a level of confidence of 0.05 ( 5.00% ) . the values for the insulin curves , including the means and the standard deviations , are shown in table 4 . table 5 shows a statistical evaluation of the significance of the differences in insulin blood levels found for groups ii , iiia , iiib , iv , and v in relation to group i ( normal ) . since all of the patients with insulin curve types ii , iiia , iiib , and iv present hyperinsulinemia
, they were compared together with the patients with type v curves , who present intestinal malabsorption of carbohydrates ( brush border membrane disease ) . table 6 shows mean glucose blood levels for the patients with hyperglycemia ( groups ii , iiia , iiib , and iv together ) and the corresponding standard deviations . table 7 shows mean glucose blood levels for the patients with brush border membrane disease ( group v ) and the corresponding standard deviations . table 8 shows mean insulin blood levels for the patients with hyperinsulinemia ( groups ii , iiia , iiib , and iv ) and the corresponding standard deviations . table 9 shows mean insulin blood levels for the patients with brush border membrane disease ( group v ) and the corresponding standard deviations . mean glucose levels in patients with hyperinsulinemia ( hi ) and brush border membrane disease ( bbmd ) . mean insulin levels in patients with hyperinsulinemia ( hi ) and brush border membrane disease ( bbmd ) . table 10 shows a statistical evaluation of the significance of the differences in insulin blood levels found for the patents with hyperinsulinemia ( groups ii , iiia , iiib , iv , together ) and brush border membrane disease ( group v ) . table 11 shows the means and standard deviations of the glucose levels of the 55 patients submitted to an oral lactose tolerance test . table 12 shows the homa ir values for the patients with hyperinsulinemia , showing the percentage of cases in each type of kraft curve that showed a value above the established limit of 2.7 . the author found that one patient had a 1.5 mm intracanalicular vestibular schwannoma in the left ear . there are no clear explanations for the fact that some patients have only cochlear symptoms , some have only vestibular symptoms whereas others have both cochlear and vestibular symptoms . since the endolymph in the vestibular portion of the system does not have the 80 mv electric charge , it would be reasonable to infer that the cochlea should be more sensitive to changes in fluids ' chemical composition , but this is not the case . mention must be made of symptoms not related to the inner ear , such as metabolic migraine , caused by excessive accumulation of sodium in the brain cells and blurred vision , caused by loss of glycogen in the lens in moments of intense hypoglycemia , recognized as a cause of cataracts in young persons . patients with hyperinsulinemia ( kraft curves type ii , iiia , iiib , and iv ) and patients with brush border membrane disease ( kraft curve type v ) exhibit some clinical differences . patients with excessive liberation of insulin have a tendency to present low glucose blood levels during certain hours of the day . 5 shows the marked differences in glucose and insulin blood levels in a 41-year - old female patient that underwent bariatric surgery . brush border membrane disease , on the other hand , causes a fluctuation of the glucose level due to the osmotic unbalance caused by unabsorbed sugars ( usually lactose ) in the small intestine . the limit established by kraft is 30 ui / ml , but the author observed that some patients with confirmed disaccharidases had values up to 50 ui / ml . a double - peaked insulin curve , parallel to the glucose curve , was also found to be common in patients with intestinal carbohydrate malabsorption . for these patients , the hypoglycemic peaks usually occur one to two hours after the glucose ingestion ; whereas , in patients with hyperinsulinemia , it usually occurs three hours or more after the ingestion . the fact that patients with brush border membrane disease have symptoms similar to those with hyperinsulinemia supports the concept that the inner ear symptoms are actually related to hypoglycemia . contrary to patients with hyperinsulinemia , patients with brush border membrane disease very seldom become diabetic . in our original study12 biopsies of the jejunal mucosa of the patients with suspected brush border membrane disease were obtained and we confirmed reduction or absence of lactase by microchemical assay . as a rule , there is no complaint of hearing loss , since the thresholds for the speech frequencies are almost always within the normal social range ; however , they often report the sensation of intermittent blocking , which they compare to what one experiences in a descending airplane . furthermore
the fluctuant hearing loss and the sensitivity to noise are usually reversible with treatment . in some untreated cases ,
the episodic sensation of floating , or walking over foam , is the most frequent vestibular symptom , although many patients refer to episodes of vertigo . vestibular hyperrelexia is the most common finding in vestibular examination , and is probably related to the changes in the density of endolymph , caused by the increased sodium concentration and water accumulation to correct osmotic changes . the diagnosis of altered carbohydrate metabolism is confirmed by the oral glucose tolerance test with simultaneous insulin titration . most clinicians rely only on the glucose fast levels for this diagnosis , but this is a test of low sensitivity , even if it is interpreted together with the glycated hemoglobin test . in this series of 376 patients ,
only 56 ( 14.89% ) had glucose fast levels of 100 mg / dl or more . homa ir was abnormal ( 2.7 or greater ) only in 25 patients ( 6.72% ) , suggesting that most of the patients in this study have intolerance to rapidly absorbable carbohydrates and do not show resistance to insulin . the insulin curves were found to be twice as sensitive as the glucose curves.11 it is interesting to note that there is a reluctance on the part of many clinicians and endocrinologists to accept the predictions based on kraft 's classification , in spite of the clinical and experimental data available.9
11
18
19
26 our findings confirm kraft 's observations that insulin curves do , in fact , allow for the prediction of future diabetes.8
9
on the other hand , only 31 patients ( 8.22% ) in this study had a normal ( kraft type i ) insulin curve , indicating the value of the clinical history and neurological evaluation as a basis for the diagnosis of impaired carbohydrate metabolism . the insulin curves were used to divide the patients into six groups , according to kraft 's classification . taking in consideration that the patients were selected on the basis of their clinical symptoms and results of neurotological tests , an actual control group is unavailable . for this reason the statistical tests were performed by comparing the patients with kraft 's curve types ii , iiia , iiib , iv , and v with the patients with type i ( normal ) curves . in spite of the fact that the groups followed the classification of their insulin curves ,
it will be noted ( tables 2 and 3 ) that the glucose curves of the patients in groups ii , iiia , iiib , iv , and v showed statistically significant differences in relation to those of group i. this indicates that the glucose blood levels at different times are essentially dependent on the amount of liberated insulin . a comparison between the glucose and insulin levels of patients with hyperinsulinemia ( groups ii , iia iiib , and iv , together , compared with group v ) also showed statistically significant differences . the hearing losses that result from impaired carbohydrate metabolism are among the few that can be adequately treated and may return to normal . the same can be said of the vestibular symptoms , that disappear in most patients . in fact , controlling glucose blood levels and the amount of insulin liberated by the pancreas are effective not only to control inner ear symptoms , but also to identify patients that are prone to become diabetic and , possibly , delaying the onset of the disease . in patients with brush border membrane disease ,
commercially available lactase may also be used to help digest milk and derivatives . in view of the frequent involvement of the transport proteins that carry monosaccharides into the blood stream , sucrose
the amount of sugar intake must be determined by each patient using a trial - and - error approach . many vasodilator drugs increase the glucose consumption by the central nervous system cells ; these medications must be avoided . the follow - up of patients with carbohydrate metabolism disorders of the inner ear is quite complex . some patients that abandoned their diets occasionally return when their symptoms get worse ; most of them have been found to be diabetic.26
the identification of these problems , however , is very important for the patients . it provides them with a better quality of life , correcting their inner ear symptoms and preventing , or at least postponing for many years , the occurrence of clinical diabetes . the inner ears are very sensitive organs and , in many patients , are the first to show signs of carbohydrate metabolism disorders . the cochlear and vestibular symptoms resulting from these disorders are very common , can be diagnosed and treated , and are often reversible . | [
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voltage - dependent ion channels underlie the electrical signaling that allows for neurotransmission within neurons , and allow for various modes of ca influx critical to coupling this electrical activity to diverse physiological events ( hille , 2001 ) . as such , voltage - gated ion channel activity
neuronal ion channels exist as supramolecular protein complexes composed of pore - forming transmembrane principal or subunits ( yu et al . , 2005 ) , auxiliary or regulatory subunits ( hanlon and wallace , 2002 ) , and a diverse array of interacting proteins ( dai et al . , 2009 ) .
diverse posttranslational events acting on each of these components dynamically regulate the expression , localization , and function of neuronal ion channels ( levitan , 2006 ) .
while numerous noncovalent mechanisms such as ligand binding , sensing of transmembrane voltage , and interaction with other proteins are known to play prominent roles in regulating neuronal ion channels , direct covalent modification of the component subunits of these multiprotein ion channel complexes by phosphorylation has long been recognized as a widely used and potent mechanism for neurons to achieve dynamic and reversible changes in ion channel function , and to impact their contribution to neuronal signaling ( levitan , 1985 ) .
phosphorylation constitutes a common covalent post - translational modification in eukaryotes ( cohen , 2001 ) , with ( as of early 2009 ) up to 25,000 described phosphorylation sites ( or phosphosites ) on 7,000 human proteins , out of an estimated 500,000 potential phosphosites that exist in a cellular proteome ( lemeer and heck , 2009 ) . in neurons
, reversible activity - dependent phosphorylation represents a major mechanism of dynamic regulation of synaptic development ( saneyoshi et al . , 2010 ) , as well as synaptic potentiation , depression , and homeostatic plasticity ( turrigiano , 2008 ) , through phosphorylation of a large number of synaptic proteins including ligand - gated ion channels ( collins and grant , 2007 ) .
neurons also exhibit cellular plasticity at the level of intrinsic excitability , achieved through phosphorylation of components of ion channel subunits , for example of voltage - gated sodium or nav ( cantrell and catterall , 2001 ) and potassium or kv ( schulz et al . , 2008 ) channels , which localize in distinct neuronal compartments ( vacher et al . , 2008
as opposed to the classical approaches of in vivo or in vitro radiolabeling with p , peptide mapping and/or sequencing , and site - directed mutagenesis ( e.g. , costa et al . , 1982 ; costa and catterall , 1984 ) , mass spectrometry ( ms)based phosphoproteomic techniques have recently emerged as the primary tool for the identification of phosphorylation on ion channel subunits ( cerda and trimmer , 2010 ) . while many of these studies continue to rely on effective purification of the target ion channel before analysis ,
a set of recent studies from the proteomics field , aimed at defining the global phosphoproteome of mouse brain samples with high complexity , and without a focus on ion channels per se , have yielded a dataset that is extremely valuable to the ion channel community . here
we provide an overview of these studies , as well as the subset of these databases that pertain to voltage - gated ion channel subunits .
these studies provide important insights to the ion channel community on the extent and nature of phosphorylation of mammalian brain ion channels , and a wealth of phosphosites that can be tested for their specific role in regulating these ion channels through dynamic and reversible phosphorylation of their principal pore - forming and voltage - sensing subunits .
recent innovations in proteomics and bioinformatics have expanded our knowledge to include nearly 10,000 mammalian brain proteins ( wang et al . , 2006 ) .
data from such high - throughput proteomic approaches represents information on ion channel expression patterns that could be of great use to the ion channel community , but may not be as readily accessible to the average channelologist .
however , the number of publications describing such global analyses is large and ever increasing , and sifting through huge databases to gain information on the spatial and temporal expression patterns of your favorite channel can be tedious and time consuming , although the resultant information can reveal important insights .
more recently , analogous high - throughput studies have provided an even rarer gem , the determination of brain peptides chemically modified with phosphate , and the site of phosphorylation within these peptides ( lemeer and heck , 2009 ) .
such in vivo studies have yielded an enormous dataset of phosphosites , including those on mammalian brain ion channels .
) what in the past would take a tremendous amount of effort in purifying the ion channel proteins from brain preparations , and then identifying the phosphosites ( using techniques that often used multiple millicuries of p ) is now available to the average channelologist at the click of the mouse .
that said , one first needs to be aware of the existence of these studies , then search through the accompanying datasets for the presence of your favorite channel , and then perform analyses to determine the site of phosphorylation within specific channel isoforms .
this can present a challenge for those more accustomed to a patch clamp rig than a large and complex proteomics dataset . in this brief review
, we attempt to first bring these recent studies to the attention of members of the ion channel community , to highlight the wealth of the data being generated by these approaches , and second to detail the subset of the data related to voltage - gated ion channels .
the goal is to ensure that the existing data , and others that will emerge from future studies , can be effectively used to gain mechanistic insights into how neuronal ion channels are regulated by reversible phosphorylation at these in vivo phosphosites .
note that this synopsis is assuredly not comprehensive , as there are likely other high - throughput studies that have yielded data leading to identification of ion channel phosphosites that we have not included in our analyses , which focuses only on data from four recent global analyses from mouse brain ( munton et al .
, 2007 ; trinidad et al . , 2008 ; tweedie - cullen et al . , 2009 ; wisniewski et al . , 2010 ) .
moreover , we should mention that the recent establishment of open source curated databases of posttranslational modifications , such as phosphositeplus ( http://www.phosphosite.org ) , phosida ( http://141.61.102.18/phosida/index.aspx , which currently contains 23,387 proteins and 69,790 identified phosphorylation sites across eukaryotes ; gnad et al . , 2007 ) , phospho.elm ( http://phospho.elm.eu.org ; current version 9.0 contains 8,718 substrate proteins from different species covering 42,573 phosphorylation sites ; diella et al . , 2004 ; diella et al . ,
2008 ) , and human protein reference database ( http://www.hprd.org , containing 30,047 proteins and 93,710 posttranslational modifications ; amanchy et al . , 2007 ; prasad et al . ,
2009 ) , provides investigators additional opportunities for mining large phospho - sites databases for ion channel phosphorylation sites .
( 2010 ) analyzed phosphosites on whole brain tissue with parallel whole protein and peptide level fractionation methods .
a crude brain homogenate solubilized in detergent buffer was digested using a recently developed filter - assisted sample preparation method , rather than conventional in - solution or in - gel digestion methods ( wisniewski et al . , 2009 ) .
the resultant peptides were directly subjected to protein size exclusion chromatography , or applied to anti - phosphotyrosine antibody beads .
phosphopeptides were then enriched by binding to tio2 beads in the presence of dihydroxybenzoic acid to avoid nonspecific binding , and/or further fractionated by strong anion exchange ( sax ) chromatography . using an ltq - orbitrap ms instrument ,
the 10 most intense precursors at a resolution of 60,000 were selected , and the precursors were fragmented through collision - induced dissociation ( cid ) , one of the most widely used peptide fragmentation methods .
peptide identification is accomplished by matching the acquired cid mass information with a segment of the sequence from a protein database .
for all of the cid tandem mass spectra , the mascot search engine was used to search a mouse protein sequence database ( i.e. , mouse ipi database , version 3.46 ) . note that such large - scale peptide and protein identification approaches often contain nonspecific matching with a false segment of protein sequence , resulting in incorrect peptide identification . to assess the identification in large - scale data , a modified protein sequence database was applied , which contains forward ( real ) and reverse ( virtual ) protein sequences ( total 110,572 entries ) .
a false - positive rate was calculated , based on the proportion of incorrect identifications assigned to reverse sequences relative to the total identifications ( forward and reverse databases ) . in this study , false - positive rates for both peptides and proteins were 1% .
the search yielded 12,035 unique phosphosites on 4,579 proteins , including 231 different phosphosites on 42 different voltage - gated ion channel subunits , as detailed in table i. phosphorylation sites identified on voltage - gated ion channel subunits note additional sites identified on slo1/kcnma1 q08460 - 2 : t670 ( w ) and s672 ( w , t ) , corresponding to t709 and s711 in q08460 - 1 .
, trinidad et al . , 2008 ; m , munton et al . , 2007 ; t , tweedie - cullen et al . , 2009 .
trinidad et al . ( 2008 ) analyzed phosphosites on postsynaptic density fractions generated from four different mouse brain regions by differential centrifugation and sucrose gradient density centrifugation .
after in - solution digestions , the digested peptide samples from cortex , midbrain , cerebellum , and hippocampus were individually labeled with four different itraq reagents ( 114 , 115 , 116 , and 117 d , respectively ) , to allow for the subsequent identification of peptides originating from the different brain regions . a mixture with equal amounts of each sample
was subjected to strong cation exchange ( scx ) chromatography , which yielded over 90 fractions .
eluted phosphopeptides were analyzed on a hybrid quadrupole time - of - flight ms ( qstar pulsar ) instrument .
all cid tandem mass spectra were searched by analyst qs software against a uniprot mus musculus database containing original and randomized protein sequences ( 64,717 entries ) .
phosphopeptide identification and site assignment was manually performed with diagnostic peaks ( e.g. , high abundant neutral loss peak ) when appropriate , and quantification of itraq - labeled phosphopeptides was performed using proteinprospector software .
this study yielded 1,564 unique phosphosites on 831 proteins , including 52 different phosphosites on 19 different voltage - gated ion channel subunits ( table i ) .
tryptic peptides generated by in - solution digestion were subjected to scx chromatography , and five of the scx fractions were further enriched by immobilized metal ion chromatography ( imac ) .
eluted material was analyzed on maldi tof - tof ms , ion trap lcq , and ltq - ft ms instruments with a reject mass list for common air contaminants .
all cid tandem mass spectra from these synaptosomal phosphoproteomes were searched using mascot against an ebi mouse protein database ( 32,849 entries ) .
this study yielded 974 unique phosphosites on 499 proteins , including 32 different phosphosites on 16 different voltage - gated ion channel subunits ( table i ) .
tweedie - cullen et al . , ( 2009 ) analyzed phosphosites on a synaptic fraction prepared by differential centrifugation and sucrose gradient density centrifugation .
isolated nuclear , synaptic , and histone proteins were digested in solution , and the resultant tryptic peptides were subjected to scx chromatography , and the early eluting phosphopeptide - rich fractions applied to tio2 or imac beads .
eluted phosphopeptide samples were analyzed by three different types of ms instruments : maldi tof / tof , ltq - ft , or ltq - orbitrap . in the case of the mass analysis with the ltq - orbitrap instrument ,
database analysis of the total cid tandem mass spectra was performed against an ebi mouse protein database ( 42,656 entries ) .
all assigned tandem spectra with phosphorylation modifications were manually inspected and filtered out with strict criteria ( mascot expected value below 0.05 ) .
normalized delta ion scores were calculated with scores between two top ranking peptides and used for precise site assignment ( < 0.4 ) .
this study yielded 1,341 unique phosphosites on 638 proteins , including 32 different phosphosites on 14 different voltage - gated ion channel subunits ( table i ) . while the phosphosites identified in the above studies
were confirmed using the most strict proteomic criteria and manual inspections , the bulk of the phosphosites obtained from these high - throughput approaches were not experimentally validated by other means .
recent proteomic technological innovations using ms have lead to a wealth of newly identified phosphosites on cellular proteins but , to date , there exist few alternative approaches to effectively cross - validate phosphosite identification .
therefore , the current method of determining the false - positive rate of these high - throughput data are based entirely on the dataset from that particular mass spectrometric analysis , and not any independent evaluation .
for example , a recent study of the yeast phosphoproteome showed that applying a single false discovery rate for all peptide identifications significantly overestimates the occurrence of rare modifications such as tyrosine phosphorylation ( there are no dedicated tyrosine kinases in yeast ) ( gnad et al . , 2009 ) .
an appropriate false discovery rate threshold confined to phosphopeptides , among all peptides identified , is adjustable for more sensitive or more reliable data analysis .
whether the identified phosphosites are located in structurally accessible protein regions may be a further consideration when determining false positives ; this is rarely an option for ion channel subunits , for which few structures are available , and those that are ( e.g. , kv1.2 ; long et al . , 2005 )
effective validation of the biological role of any identified phosphosites still involves using biochemical , cell biological , and electrophysiological analyses of individual proteins expressing site - specific mutations in the identified phosphosites .
an overview of the dataset ( table i ) focusing on the location of the identified phosphosites on the 24 transmembrane segment ion channel subunits reveals that for cav channel subunits , phosphorylation is primarily split between the i d ii - iii linker and the c terminus ( fig .
1 ) , whereas for nav channel subunits , phosphorylation is primarily ( 90% ) on the i d i - ii linker ( fig .
1 ) , as suggested by previous studies ( cantrell and catterall , 2001 ) .
2 ) have the majority of their phosphosites on the c terminus , although this varies from 90% for kv7 and slo1-bk channel family members , to 65% for kv10 and hcn family members .
schematic of phosphosites identified in high - throughput studies on 24 transmembrane segment ion channel subunits .
areas of the green circles represent the percentage of phosphosites found within a specific cytoplasmic domain ( n - terminal , i d i - ii , id - ii - iii , or c - terminal ) .
numbers adjacent to circles represent the total number of unique phosphosites identified within that domain for all members of that family ( cav : six members ; nav : four members ) .
schematic of phosphosites identified in high - throughput studies on six transmembrane segment ion channel subunits
. areas of the green circles represent the percentage of phosphosites found within a specific cytoplasmic domain ( n or c terminus ) .
numbers adjacent to circles represent the total number of unique phosphosites identified within that domain for all members of that family ( kv1 - 4 : 14 members ; kv7 : 3 members ; kv10 : 4 members ; slo1-bk : 1 member ; hcn : 4 members ; trp : 5 members ) .
an overall number of unique phosphosites identified shows that the largest ion channel subunits , namely those within the 24 transmembrane segment family of cav and nav channel subunits , have the most extensive phosphorylation , with cav2.1 ( 24 phosphosites ) , nav1.2 ( 23 phosphosites ) , and cav2.3 ( 22 phosphosites ) topping the list ( table i ) .
this may be expected given the large size of these polypeptides ( 2,0002,400 amino acids ) and that only one subunit is present per channel complex . among the six transmembrane segment family members , which are smaller polypeptides and assemble as tetramers in the final channel complex , kv7.2 ( 15 phosphosites ) , hcn2 ( 14 phosphosites ) ,
kv2.1 ( 13 phosphosites ) , and slo1-bk ( 10 phosphosites ) are notable in their extensive representation , suggesting a high degree of phosphorylation - dependent modulation of these channels .
note that while these data suggest extensive multisite phosphorylation exists on individual molecules of ion channel subunits , these studies have not provided conclusive data as these phosphosites come from relatively small tryptic fragments of the larger polypeptides .
as such , except for the small subset of neighboring phosphosites found within the same multiply phosphorylated tryptic peptide , it is formally possible that the multiple phospho - sites identified in these ion channel subunits are stochastically distributed in different molecules . note that representation on the list of most phosphorylated ion channel subunits is likely a combination of not only the actual extent of phosphorylation , but also nature of the phosphorylation , as the techniques employed ( ms - based analyses of tryptic peptides ) bias the analysis toward phosphosites on tryptic peptides with a chemical nature ( e.g. , size , charge , hydropathy , etc . )
of course , the overall abundance of a specific ion channel subunit in a given sample , whether it is whole brain or a subcellular fraction thereof , will also affect its representation in samples with high proteomic and phosphoproteomic complexity , such as those used for global analyses .
as such , enriching for ion channel subunits is an attractive approach to increase coverage of their phosphoproteome .
this raises questions as to the implications for the rat and human brain orthologues of these mouse brain ion channel subunits .
if conservation of the phosphoacceptor site itself is any indication , then these mouse brain data are likely to have strong implications for studies in other mammals .
focusing on the ion channel subunits within a subfamily that exhibit the highest number of identified phosphosites ( cav2.1 , nav1.2 , kv2.1 , kv7.2 , and trpm3 ) , we found that , with the exception of mouse and human cav2.1 ( 87% overall identity ) , overall sequence identity between mouse , rat , and human ion channel subunit orthologues was 93% or higher .
as to the phosphosites themselves ( 85 in total ) , there was 100% conservation of every site in mouse , rat , and human nav1.2 ( 23 phosphosites ) , kv2.1 ( 13 phosphosites ) , kv7.2 ( 15 phosphosites ) , and trpm3 ( 8 phosphosites ) , and for human cav2.1 , 92% conservation ( 22/24 ) .
it should be noted that conservation of the phospho - acceptor site itself may not in itself suggest that the site is phosphorylated , as changes in neighboring amino acids in the primary , secondary , or tertiary structure may alter protein kinase consensus motifs ( ubersax and ferrell , 2007 ) .
also note that with the exception of the study by munton et al . , these studies focused on samples prepared from animals under basal conditions , and may not contain examples of sites whose phosphorylation requires specific stimuli .
there are numerous examples of phosphorylation at defined sites on specific ion channels dynamically regulating how these ion channels impact cell function .
one way that phosphorylation can act is through altering the abundance of specific ion channels in specific membrane domains .
this can occur via changes in the proportion of ion channels in intracellular membranes versus the plasma membrane , by impacting the rates of channel exocytosis and endocytosis , or by affecting the density of channels in a specific plasma membrane subdomain via effects on channel clustering .
phosphorylation can also affect the gating of ion channels , altering the probability that an ion channel will be open under a given set of conditions . a large literature exists providing a large number of examples of stimuli impacting ion channel abundance , distribution , and gating .
as many of these stimuli are known to act through signaling pathways leading to activation of protein kinases or protein phosphatases , the assumption has been that the effects are meditated through changes in the phosphorylation state of the ion channel subunits themselves . in some cases
, regulation can be obtained for recombinant ion channels expressed in heterologous cells , in which case candidate phosphosites can be identified using phosphosite prediction software ( e.g. , http://www.cbs.dtu.dk/services/netphosk/ ; http://scansite.mit.edu/motifscan_seq.phtml ; http://www.hprd.org/phosphomotif_finder ) , and mutations generated that lock specific candidate phosphosites in either a dephospho- or phospho - mimetic state ( e. g. , ser to ala or asp mutations , respectively ) .
data obtained from these approaches have allowed for the identification of specific sites critical to phosphorylation - dependent modulation of channel abundance , distribution , or function . however , rarely have these studies directly shown that the impact of the mutations is via altering phosphorylation state , and that the mutation only altered phosphorylation at the mutated site .
moreover , there are few cases where the candidate phosphosite being studied is known to be modified by phosphorylation in native cells . as such ,
we are left with a large number of candidate phosphosites whose mutation impacts the channel , but whose physiological relevance is unknown .
the recent high - throughput approaches described here have yielded , for the most part , a dataset completely complementary to these types of studies , in providing a large amount of information as to which sites are found phosphorylated in native cells , but without any functional insights .
in a few cases , which we detail below , phosphosites identified in these recent phosphoproteomics studies have provided in vivo relevance to studies already performed in heterologous cells .
future studies aimed at determining the functional impact of the phosphosites we highlight here will serve to increase the number of examples of phosphosites that appear in vivo , and functional relevance is known .
phosphorylation of cav2.2 1 subunits , which regulate neurotransmitter release from nerve terminals , in their di - dii linker region has been proposed to play a crucial role in diverse aspects of cav channel function .
for example , mutation of rat cav2.2 at the site identified in mouse brain ( using accession no .
o55017 ) as s424 mediates pkc - dependent cav2.2 inhibition ( fang et al . , 2006 ; rajagopal et al . ,
erk - dependent up - regulation of cav2.2 channels is mediated through phosphorylation of the site identified in mouse brain as s446 ( martin et al . , 2006 ) .
in the dii - diii linker , pkc phosphorylates the sites identified in mouse brain as s773 and s892 , and camkii at s783 , and impacts syntaxin1a cav2.2 interaction ( yokoyama et al . , 2005 ) .
pkc - dependent phosphorylation on s892 was also found to disrupt interaction with syntaxin 1a and abolish the hyperpolarizing shift in the steady - state inactivation caused by this protein
protein interaction ( jarvis and zamponi , 2001 ) . the predominant sodium channel in brain ,
nav1.2 , is a substrate for pka phosphorylation in its di - dii linker domain ( cantrell et al . , 1997 ;
nav1.2 at the in vivo mouse brain sites s573 , s610 , and s623 , calcineurin dephosphorylates these three phosphosites , and protein phosphatase 2a ( pp2a ) dephosphorylates s610 ( smith and goldin , 1997 ) .
pka causes a decrease of the nav peak current triggered by dopaminergic stimulation of rat hippocampal neurons , but does not alter the voltage dependence or kinetic parameters .
the s573a mutation abolishes the dopamine - induced pka effects ( cantrell et al . , 1997 ) .
kv1.2 channel subunits are expressed in axons and nerve terminals , and modulate action potential propagation and nerve terminal excitation .
site - directed mutagenesis studies showed that the s440a and s441a mutations diminish kv1.2 surface expression and whole cell current ( yang et al . , 2007 ) .
the s449a substitution causes a decrease in phosphorylation at s440 and s441 , again leading to diminished surface expression ( yang et al . , 2007 ) .
kv2.1 is expressed in large clusters in the somatodendritic membrane of most neurons ( trimmer , 1991 ) .
kv2.1 is extensively phosphorylated in vivo , and increased neuronal excitability leads to calcineurin - mediated kv2.1 dephosphorylation , a loss of clustering , and hyperpolarizing shifts in voltage - dependent gating ( misonou et al . , 2004 , 2005 , 2006 ) .
a mass spectrometry based analysis of kv2.1 purified from rat brain identified 16 phosphosites ( park et al .
, 2006 ; mohapatra et al . , 2007 ) , of which five ( s15 , s484 , s520 , s567 , s655 , and s804 )
were also identified in high - throughput approaches detailed here ( table i ) . of the phosphosites identified in the high throughput studies , mutations at s15 , s567 , and s655 partially mimic and occlude the effects of dephosphorylation in causing hyperpolarizing shifts in the voltage - dependent gating .
in addition , phosphorylation on s804 via p38mapk increases the surface expression of kv2.1 , leading to neuronal apoptosis ( redman et al . , 2007 ) .
kv channels containing kv4.2 subunits mediate transient a - type k currents in neurons , which are critically involved in the regulation of dendritic excitability and plasticity ( shah et al . , 2010 ) . in hippocampal slices , pka activation by forskolin and other pharmacological agents results in kv4.2 phosphorylation at s552 ( anderson et al . , 2000 ) .
pka - dependent phosphorylation of this residue results in a depolarizing shift of kv4.2 activation , but only in presence of the auxiliary subunit kchip3 ( schrader et al . , 2002 ) .
pka phosphorylation at s552 also mediates ampa - stimulated kv4.2 internalization from dendritic spines ( hammond et al . , 2008 ) .
note that a previous mass spectrometry based analysis of kv4.2 purified from rat brain ( seikel and trimmer , 2009 ) identified four phosphosites ( s548 , s552 , s572 , and s575 ) of which two ( s548 and s552 ) were found in the high - throughput analyses of mouse brain ( table i ) .
kv7 ( kcnq ) subunits form neuronal kv channels that underlie the m - current ( so - called due to its modulation by muscarinic signaling ) .
these channels localize in axon initial segments , nodes of ranvier , and somata throughout the nervous system . of the residues found phosphorylated in mouse brain ( table i ) ,
pka phosphorylation of kcnq2 at s52 has been shown to enhance kcnq2 currents in heterologous cells ( schroeder et al . , 1998 ) . a previous mass spectrometric based analysis identified phosphorylation at either s578 or t579 on recombinant human kcnq3 expressed in heterologous cells ( surti et al . , 2005 ) .
mutations at these positions in kcnq3 eliminate functional expression of kcnq2/3 channels ( surti et al . , 2005 ) .
these sites are analogous to in vivo phosphosites identified in mouse brain proteomic studies on kv7.3 at s579 and t580 .
large conductance ca - activated k ( slo1-bk ) channels are involved in neuronal repolarization and rapid afterhyperpolarization .
pkc - dependent phosphorylation on s695 of the bovine slo1-bk subunit ( corresponding to s711 of the mouse isoform shown in table i ) decreases the open probability of the channel , an effect countered by protein phosphatase 1 ( zhou et al . , 2010 ) .
pkg - dependent phosphorylation on s855 together with s869 ( s869 corresponding to s924 in the mouse isoform used here ) enhances the current amplitude of the human slo1-bk channel in xenopus oocytes ( nara et al . , 2000 ) . to gain insights into whether these data provide insights into the predominance of ion channel phosphorylation by a particular protein kinase , we have analyzed the phosphosites on the ion channel subunits with the most extensive phosphorylation ( cav2.1 , nav1.2 , kv2.1 , kv7.2 , and trpm3 ) for consensus motifs using netphosk ( http://www.cbs.dtu.dk/services/netphosk/ ) . of the 83 phosphosites analyzed ,
the most predominant motifs recognized were for the proline - directed protein kinases p38map kinase , gsk3 and cdk5 , with 30 phosphosites being scored for phosphorylation by one or more of these protein kinases .
there were 13 phosphosites that scored as consensus motifs for pka , and 10 for pkc .
there were 19 phosphosites that did not score as sites for any consensus protein kinase motif .
these results are somewhat consistent with our previous analyses of phosphosites found on nav1.2 ( berendt et al . , 2010 ) and kv2.1 ( park et al . , 2006 )
purified from rat brain , which revealed a predominance of phosphosites for proline - directed protein kinases , as well as several sites not recognized by any prediction program . in this regard
it is interesting to compare the data obtained from these high throughput global analyses versus those from studies on purified ion channel subunits ( table ii ) .
recent studies from our laboratory have used this approach for nav1.2 ( berendt et al . , 2010 ) , kv2.1 ( park et al . , 2006 ) , and slo1-bk ( in collaboration with fakler et al . )
note that in each case , the analyses of purified ion channel subunits were from rat brain , while the global analyses were performed on mouse brain . for nav1.2 , of the 23 phosphosites identified in these global analyses ,
10 were found in the single - target analysis of purified nav1.2 ( berendt et al . , 2010 ) .
however , the ms - based analyses of purified nav1.2 identified six phosphosites not seen in the global analyses . in the case of kv2.1 , of the 13 phosphosites identified in the global analyses of mouse brain , six
were observed in the analysis of kv2.1 purified from rat brain , which also revealed the presence of 10 additional phosphosites not seen in the global analyses ( park et al . , 2006 ) . for slo1-bk , of the 10 unique phosphosites identified in the global analyses ,
all but two were also observed in the analyses of the purified slo1-bk , which also provided an additional 20 phosphosites ( yan et al . , 2008 ) .
results from high versus low - throughput analyses these comparisons suggest that while the dataset from these global analyses represents a wealth of information for ion channel researchers , parallel approaches using enrichment of the target ion channel subunits provide datasets that are oftentimes more extensive , revealing additional phosphosites that may play important roles in the modulation of these channels .
what these datasets do suggest is that neither approach used in isolation has yet to provide a comprehensive view of phosphosites on any given ion channel subunit .
of course , phosphorylation is by nature dynamic , such that variations in the exact state of the animal subject , details of sample preparation , and other factors that are difficult to precisely control can influence the dataset obtained .
moreover , these analyses were primarily performed on samples derived from whole adult rat or mouse brains , such that phosphosites highly represented in other temporal and spatial contexts may be underrepresented .
it is also important to stress that any site identified in phosphoproteomic screens needs to be directly confirmed by at least one independent method , for example multiple reaction monitoring ( which can also provide quantitative information ; rinehart et al . , 2009 ) , immunoblotting with phosphospecific antibodies ( park et al . , 2006 ) , or other approaches .
the wealth of data obtained from these studies raises the question as to whether we in the ion channel field should simply sit back and wait as these types of global analyses , being performed by our colleagues in the proteomics field , provide us with intriguing and extensive sets of in vivo phosphosites .
we would argue that we should accept such bountiful gifts with gratitude and appreciation , but that we in the ion channel field need to continue our complementary efforts to identify in vivo phosphosites through focused analyses of individual ion channel subunits purified from brain and other tissues .
together , these approaches are likely to yield a comprehensive dataset that will allow for a more complete picture of the nature and extent of in vivo phosphosites crucial to dynamic regulation of native ion channel expression , localization , and function .
wisniewski et al . ( 2010 ) analyzed phosphosites on whole brain tissue with parallel whole protein and peptide level fractionation methods .
a crude brain homogenate solubilized in detergent buffer was digested using a recently developed filter - assisted sample preparation method , rather than conventional in - solution or in - gel digestion methods ( wisniewski et al . , 2009 ) .
the resultant peptides were directly subjected to protein size exclusion chromatography , or applied to anti - phosphotyrosine antibody beads .
phosphopeptides were then enriched by binding to tio2 beads in the presence of dihydroxybenzoic acid to avoid nonspecific binding , and/or further fractionated by strong anion exchange ( sax ) chromatography . using an ltq - orbitrap ms instrument ,
the 10 most intense precursors at a resolution of 60,000 were selected , and the precursors were fragmented through collision - induced dissociation ( cid ) , one of the most widely used peptide fragmentation methods .
peptide identification is accomplished by matching the acquired cid mass information with a segment of the sequence from a protein database .
for all of the cid tandem mass spectra , the mascot search engine was used to search a mouse protein sequence database ( i.e. , mouse ipi database , version 3.46 ) . note that such large - scale peptide and protein identification approaches often contain nonspecific matching with a false segment of protein sequence , resulting in incorrect peptide identification . to assess the identification in large - scale data , a modified protein sequence database was applied , which contains forward ( real ) and reverse ( virtual ) protein sequences ( total 110,572 entries ) .
a false - positive rate was calculated , based on the proportion of incorrect identifications assigned to reverse sequences relative to the total identifications ( forward and reverse databases ) . in this study ,
the search yielded 12,035 unique phosphosites on 4,579 proteins , including 231 different phosphosites on 42 different voltage - gated ion channel subunits , as detailed in table i. phosphorylation sites identified on voltage - gated ion channel subunits note additional sites identified on slo1/kcnma1 q08460 - 2 : t670 ( w ) and s672 ( w , t ) , corresponding to t709 and s711 in q08460 - 1 .
, 2008 ; m , munton et al . , 2007 ; t , tweedie - cullen et al . , 2009 .
trinidad et al . ( 2008 ) analyzed phosphosites on postsynaptic density fractions generated from four different mouse brain regions by differential centrifugation and sucrose gradient density centrifugation .
after in - solution digestions , the digested peptide samples from cortex , midbrain , cerebellum , and hippocampus were individually labeled with four different itraq reagents ( 114 , 115 , 116 , and 117 d , respectively ) , to allow for the subsequent identification of peptides originating from the different brain regions . a mixture with equal amounts of each sample
was subjected to strong cation exchange ( scx ) chromatography , which yielded over 90 fractions .
eluted phosphopeptides were analyzed on a hybrid quadrupole time - of - flight ms ( qstar pulsar ) instrument .
all cid tandem mass spectra were searched by analyst qs software against a uniprot mus musculus database containing original and randomized protein sequences ( 64,717 entries ) .
phosphopeptide identification and site assignment was manually performed with diagnostic peaks ( e.g. , high abundant neutral loss peak ) when appropriate , and quantification of itraq - labeled phosphopeptides was performed using proteinprospector software .
this study yielded 1,564 unique phosphosites on 831 proteins , including 52 different phosphosites on 19 different voltage - gated ion channel subunits ( table i ) .
tryptic peptides generated by in - solution digestion were subjected to scx chromatography , and five of the scx fractions were further enriched by immobilized metal ion chromatography ( imac ) .
eluted material was analyzed on maldi tof - tof ms , ion trap lcq , and ltq - ft ms instruments with a reject mass list for common air contaminants .
all cid tandem mass spectra from these synaptosomal phosphoproteomes were searched using mascot against an ebi mouse protein database ( 32,849 entries ) .
this study yielded 974 unique phosphosites on 499 proteins , including 32 different phosphosites on 16 different voltage - gated ion channel subunits ( table i ) .
tweedie - cullen et al . , ( 2009 ) analyzed phosphosites on a synaptic fraction prepared by differential centrifugation and sucrose gradient density centrifugation .
isolated nuclear , synaptic , and histone proteins were digested in solution , and the resultant tryptic peptides were subjected to scx chromatography , and the early eluting phosphopeptide - rich fractions applied to tio2 or imac beads . eluted phosphopeptide samples were analyzed by three different types of ms instruments : maldi tof / tof , ltq - ft , or ltq - orbitrap . in the case of the mass analysis with the ltq - orbitrap instrument ,
database analysis of the total cid tandem mass spectra was performed against an ebi mouse protein database ( 42,656 entries ) .
all assigned tandem spectra with phosphorylation modifications were manually inspected and filtered out with strict criteria ( mascot expected value below 0.05 ) .
normalized delta ion scores were calculated with scores between two top ranking peptides and used for precise site assignment ( < 0.4 ) .
this study yielded 1,341 unique phosphosites on 638 proteins , including 32 different phosphosites on 14 different voltage - gated ion channel subunits ( table i ) . while the phosphosites identified in the above studies
were confirmed using the most strict proteomic criteria and manual inspections , the bulk of the phosphosites obtained from these high - throughput approaches were not experimentally validated by other means .
recent proteomic technological innovations using ms have lead to a wealth of newly identified phosphosites on cellular proteins but , to date , there exist few alternative approaches to effectively cross - validate phosphosite identification .
therefore , the current method of determining the false - positive rate of these high - throughput data are based entirely on the dataset from that particular mass spectrometric analysis , and not any independent evaluation .
for example , a recent study of the yeast phosphoproteome showed that applying a single false discovery rate for all peptide identifications significantly overestimates the occurrence of rare modifications such as tyrosine phosphorylation ( there are no dedicated tyrosine kinases in yeast ) ( gnad et al . , 2009 ) . an appropriate false discovery rate threshold confined to phosphopeptides , among all
whether the identified phosphosites are located in structurally accessible protein regions may be a further consideration when determining false positives ; this is rarely an option for ion channel subunits , for which few structures are available , and those that are ( e.g. , kv1.2 ; long et al . , 2005 )
effective validation of the biological role of any identified phosphosites still involves using biochemical , cell biological , and electrophysiological analyses of individual proteins expressing site - specific mutations in the identified phosphosites .
an overview of the dataset ( table i ) focusing on the location of the identified phosphosites on the 24 transmembrane segment ion channel subunits reveals that for cav channel subunits , phosphorylation is primarily split between the i d ii - iii linker and the c terminus ( fig .
1 ) , whereas for nav channel subunits , phosphorylation is primarily ( 90% ) on the i d i - ii linker ( fig .
1 ) , as suggested by previous studies ( cantrell and catterall , 2001 ) .
2 ) have the majority of their phosphosites on the c terminus , although this varies from 90% for kv7 and slo1-bk channel family members , to 65% for kv10 and hcn family members .
schematic of phosphosites identified in high - throughput studies on 24 transmembrane segment ion channel subunits .
areas of the green circles represent the percentage of phosphosites found within a specific cytoplasmic domain ( n - terminal , i d i - ii , id - ii - iii , or c - terminal ) .
numbers adjacent to circles represent the total number of unique phosphosites identified within that domain for all members of that family ( cav : six members ; nav : four members ) .
schematic of phosphosites identified in high - throughput studies on six transmembrane segment ion channel subunits .
areas of the green circles represent the percentage of phosphosites found within a specific cytoplasmic domain ( n or c terminus ) .
numbers adjacent to circles represent the total number of unique phosphosites identified within that domain for all members of that family ( kv1 - 4 : 14 members ; kv7 : 3 members ; kv10 : 4 members ; slo1-bk : 1 member ; hcn : 4 members ; trp : 5 members ) . an overall number of unique phosphosites identified shows that the largest ion channel subunits , namely those within the 24 transmembrane segment family of cav and nav channel subunits , have the most extensive phosphorylation , with cav2.1 ( 24 phosphosites ) , nav1.2 ( 23 phosphosites ) , and cav2.3 ( 22 phosphosites ) topping the list ( table i ) .
this may be expected given the large size of these polypeptides ( 2,0002,400 amino acids ) and that only one subunit is present per channel complex . among the six transmembrane segment family members , which are smaller polypeptides and assemble as tetramers in the final channel complex , kv7.2 ( 15 phosphosites ) , hcn2 ( 14 phosphosites ) , kv2.1 ( 13 phosphosites ) , and slo1-bk ( 10 phosphosites ) are notable in their extensive representation , suggesting a high degree of phosphorylation - dependent modulation of these channels .
note that while these data suggest extensive multisite phosphorylation exists on individual molecules of ion channel subunits , these studies have not provided conclusive data as these phosphosites come from relatively small tryptic fragments of the larger polypeptides .
as such , except for the small subset of neighboring phosphosites found within the same multiply phosphorylated tryptic peptide , it is formally possible that the multiple phospho - sites identified in these ion channel subunits are stochastically distributed in different molecules .
note that representation on the list of most phosphorylated ion channel subunits is likely a combination of not only the actual extent of phosphorylation , but also nature of the phosphorylation , as the techniques employed ( ms - based analyses of tryptic peptides ) bias the analysis toward phosphosites on tryptic peptides with a chemical nature ( e.g. , size , charge , hydropathy , etc . ) that makes them amenable to detection by mass spectrometers .
of course , the overall abundance of a specific ion channel subunit in a given sample , whether it is whole brain or a subcellular fraction thereof , will also affect its representation in samples with high proteomic and phosphoproteomic complexity , such as those used for global analyses .
as such , enriching for ion channel subunits is an attractive approach to increase coverage of their phosphoproteome .
this raises questions as to the implications for the rat and human brain orthologues of these mouse brain ion channel subunits . if conservation of the phosphoacceptor site itself is any indication , then these mouse brain data are likely to have strong implications for studies in other mammals . focusing on the ion channel subunits within a subfamily that exhibit the highest number of identified phosphosites ( cav2.1 , nav1.2 , kv2.1 , kv7.2 , and trpm3 ) , we found that , with the exception of mouse and human cav2.1 ( 87% overall identity ) , overall sequence identity between mouse , rat , and human ion channel subunit orthologues was 93% or higher .
as to the phosphosites themselves ( 85 in total ) , there was 100% conservation of every site in mouse , rat , and human nav1.2 ( 23 phosphosites ) , kv2.1 ( 13 phosphosites ) , kv7.2 ( 15 phosphosites ) , and trpm3 ( 8 phosphosites ) , and for human cav2.1 , 92% conservation ( 22/24 ) .
it should be noted that conservation of the phospho - acceptor site itself may not in itself suggest that the site is phosphorylated , as changes in neighboring amino acids in the primary , secondary , or tertiary structure may alter protein kinase consensus motifs ( ubersax and ferrell , 2007 ) .
also note that with the exception of the study by munton et al . , these studies focused on samples prepared from animals under basal conditions , and may not contain examples of sites whose phosphorylation requires specific stimuli .
there are numerous examples of phosphorylation at defined sites on specific ion channels dynamically regulating how these ion channels impact cell function .
one way that phosphorylation can act is through altering the abundance of specific ion channels in specific membrane domains .
this can occur via changes in the proportion of ion channels in intracellular membranes versus the plasma membrane , by impacting the rates of channel exocytosis and endocytosis , or by affecting the density of channels in a specific plasma membrane subdomain via effects on channel clustering .
phosphorylation can also affect the gating of ion channels , altering the probability that an ion channel will be open under a given set of conditions .
a large literature exists providing a large number of examples of stimuli impacting ion channel abundance , distribution , and gating .
as many of these stimuli are known to act through signaling pathways leading to activation of protein kinases or protein phosphatases , the assumption has been that the effects are meditated through changes in the phosphorylation state of the ion channel subunits themselves . in some cases
, regulation can be obtained for recombinant ion channels expressed in heterologous cells , in which case candidate phosphosites can be identified using phosphosite prediction software ( e.g. , http://www.cbs.dtu.dk/services/netphosk/ ; http://scansite.mit.edu/motifscan_seq.phtml ; http://www.hprd.org/phosphomotif_finder ) , and mutations generated that lock specific candidate phosphosites in either a dephospho- or phospho - mimetic state ( e. g. , ser to ala or asp mutations , respectively ) .
data obtained from these approaches have allowed for the identification of specific sites critical to phosphorylation - dependent modulation of channel abundance , distribution , or function . however , rarely have these studies directly shown that the impact of the mutations is via altering phosphorylation state , and that the mutation only altered phosphorylation at the mutated site . moreover , there are few cases where the candidate phosphosite being studied is known to be modified by phosphorylation in native cells . as such ,
we are left with a large number of candidate phosphosites whose mutation impacts the channel , but whose physiological relevance is unknown .
the recent high - throughput approaches described here have yielded , for the most part , a dataset completely complementary to these types of studies , in providing a large amount of information as to which sites are found phosphorylated in native cells , but without any functional insights .
in a few cases , which we detail below , phosphosites identified in these recent phosphoproteomics studies have provided in vivo relevance to studies already performed in heterologous cells .
future studies aimed at determining the functional impact of the phosphosites we highlight here will serve to increase the number of examples of phosphosites that appear in vivo , and functional relevance is known .
phosphorylation of cav2.2 1 subunits , which regulate neurotransmitter release from nerve terminals , in their di - dii linker region has been proposed to play a crucial role in diverse aspects of cav channel function .
for example , mutation of rat cav2.2 at the site identified in mouse brain ( using accession no .
o55017 ) as s424 mediates pkc - dependent cav2.2 inhibition ( fang et al . , 2006 ; rajagopal et al . ,
erk - dependent up - regulation of cav2.2 channels is mediated through phosphorylation of the site identified in mouse brain as s446 ( martin et al . , 2006 ) .
in the dii - diii linker , pkc phosphorylates the sites identified in mouse brain as s773 and s892 , and camkii at s783 , and impacts syntaxin1a cav2.2 interaction ( yokoyama et al . , 2005 ) .
pkc - dependent phosphorylation on s892 was also found to disrupt interaction with syntaxin 1a and abolish the hyperpolarizing shift in the steady - state inactivation caused by this protein
protein interaction ( jarvis and zamponi , 2001 ) . the predominant sodium channel in brain
, nav1.2 , is a substrate for pka phosphorylation in its di - dii linker domain ( cantrell et al . , 1997 ;
nav1.2 at the in vivo mouse brain sites s573 , s610 , and s623 , calcineurin dephosphorylates these three phosphosites , and protein phosphatase 2a ( pp2a ) dephosphorylates s610 ( smith and goldin , 1997 ) .
pka causes a decrease of the nav peak current triggered by dopaminergic stimulation of rat hippocampal neurons , but does not alter the voltage dependence or kinetic parameters .
the s573a mutation abolishes the dopamine - induced pka effects ( cantrell et al . , 1997 ) .
kv1.2 channel subunits are expressed in axons and nerve terminals , and modulate action potential propagation and nerve terminal excitation .
site - directed mutagenesis studies showed that the s440a and s441a mutations diminish kv1.2 surface expression and whole cell current ( yang et al . , 2007 ) .
the s449a substitution causes a decrease in phosphorylation at s440 and s441 , again leading to diminished surface expression ( yang et al . , 2007 ) .
kv2.1 is expressed in large clusters in the somatodendritic membrane of most neurons ( trimmer , 1991 ) .
kv2.1 is extensively phosphorylated in vivo , and increased neuronal excitability leads to calcineurin - mediated kv2.1 dephosphorylation , a loss of clustering , and hyperpolarizing shifts in voltage - dependent gating ( misonou et al . , 2004 , 2005 , 2006 ) .
a mass spectrometry based analysis of kv2.1 purified from rat brain identified 16 phosphosites ( park et al .
, 2006 ; mohapatra et al . , 2007 ) , of which five ( s15 , s484 , s520 , s567 , s655 , and s804 ) were also identified in high - throughput approaches detailed here ( table i ) . of the phosphosites identified in the high throughput studies , mutations at s15 , s567 , and s655 partially mimic and occlude the effects of dephosphorylation in causing hyperpolarizing shifts in the voltage - dependent gating .
in addition , phosphorylation on s804 via p38mapk increases the surface expression of kv2.1 , leading to neuronal apoptosis ( redman et al . , 2007 ) .
kv channels containing kv4.2 subunits mediate transient a - type k currents in neurons , which are critically involved in the regulation of dendritic excitability and plasticity ( shah et al . , 2010 ) . in hippocampal slices , pka activation by forskolin and other pharmacological agents results in kv4.2 phosphorylation at s552 ( anderson et al . , 2000 ) .
pka - dependent phosphorylation of this residue results in a depolarizing shift of kv4.2 activation , but only in presence of the auxiliary subunit kchip3 ( schrader et al . , 2002 ) .
pka phosphorylation at s552 also mediates ampa - stimulated kv4.2 internalization from dendritic spines ( hammond et al . , 2008 ) .
note that a previous mass spectrometry based analysis of kv4.2 purified from rat brain ( seikel and trimmer , 2009 ) identified four phosphosites ( s548 , s552 , s572 , and s575 ) of which two ( s548 and s552 ) were found in the high - throughput analyses of mouse brain ( table i ) .
kv7 ( kcnq ) subunits form neuronal kv channels that underlie the m - current ( so - called due to its modulation by muscarinic signaling ) .
these channels localize in axon initial segments , nodes of ranvier , and somata throughout the nervous system . of the residues found phosphorylated in mouse brain
( table i ) , pka phosphorylation of kcnq2 at s52 has been shown to enhance kcnq2 currents in heterologous cells ( schroeder et al . , 1998 ) .
a previous mass spectrometric based analysis identified phosphorylation at either s578 or t579 on recombinant human kcnq3 expressed in heterologous cells ( surti et al . , 2005 ) .
mutations at these positions in kcnq3 eliminate functional expression of kcnq2/3 channels ( surti et al . , 2005 ) .
these sites are analogous to in vivo phosphosites identified in mouse brain proteomic studies on kv7.3 at s579 and t580 .
large conductance ca - activated k ( slo1-bk ) channels are involved in neuronal repolarization and rapid afterhyperpolarization .
pkc - dependent phosphorylation on s695 of the bovine slo1-bk subunit ( corresponding to s711 of the mouse isoform shown in table i ) decreases the open probability of the channel , an effect countered by protein phosphatase 1 ( zhou et al . , 2010 ) .
pkg - dependent phosphorylation on s855 together with s869 ( s869 corresponding to s924 in the mouse isoform used here ) enhances the current amplitude of the human slo1-bk channel in xenopus oocytes ( nara et al .
to gain insights into whether these data provide insights into the predominance of ion channel phosphorylation by a particular protein kinase , we have analyzed the phosphosites on the ion channel subunits with the most extensive phosphorylation ( cav2.1 , nav1.2 , kv2.1 , kv7.2 , and trpm3 ) for consensus motifs using netphosk ( http://www.cbs.dtu.dk/services/netphosk/ ) . of the 83 phosphosites analyzed , the most predominant motifs recognized were for the proline - directed protein kinases p38map kinase , gsk3 and cdk5 , with 30 phosphosites being scored for phosphorylation by one or more of these protein kinases .
there were 13 phosphosites that scored as consensus motifs for pka , and 10 for pkc .
there were 19 phosphosites that did not score as sites for any consensus protein kinase motif .
these results are somewhat consistent with our previous analyses of phosphosites found on nav1.2 ( berendt et al . , 2010 ) and kv2.1 ( park et al . , 2006 )
purified from rat brain , which revealed a predominance of phosphosites for proline - directed protein kinases , as well as several sites not recognized by any prediction program . in this regard
it is interesting to compare the data obtained from these high throughput global analyses versus those from studies on purified ion channel subunits ( table ii ) .
recent studies from our laboratory have used this approach for nav1.2 ( berendt et al . , 2010 ) , kv2.1 ( park et al . , 2006 ) , and slo1-bk ( in collaboration with fakler et al . )
note that in each case , the analyses of purified ion channel subunits were from rat brain , while the global analyses were performed on mouse brain .
for nav1.2 , of the 23 phosphosites identified in these global analyses , 10 were found in the single - target analysis of purified nav1.2 ( berendt et al . , 2010 ) .
however , the ms - based analyses of purified nav1.2 identified six phosphosites not seen in the global analyses . in the case of kv2.1 , of the 13 phosphosites identified in the global analyses of mouse brain , six
were observed in the analysis of kv2.1 purified from rat brain , which also revealed the presence of 10 additional phosphosites not seen in the global analyses ( park et al . , 2006 ) .
for slo1-bk , of the 10 unique phosphosites identified in the global analyses , all but two were also observed in the analyses of the purified slo1-bk , which also provided an additional 20 phosphosites ( yan et al . , 2008 ) .
results from high versus low - throughput analyses these comparisons suggest that while the dataset from these global analyses represents a wealth of information for ion channel researchers , parallel approaches using enrichment of the target ion channel subunits provide datasets that are oftentimes more extensive , revealing additional phosphosites that may play important roles in the modulation of these channels .
what these datasets do suggest is that neither approach used in isolation has yet to provide a comprehensive view of phosphosites on any given ion channel subunit .
of course , phosphorylation is by nature dynamic , such that variations in the exact state of the animal subject , details of sample preparation , and other factors that are difficult to precisely control can influence the dataset obtained .
moreover , these analyses were primarily performed on samples derived from whole adult rat or mouse brains , such that phosphosites highly represented in other temporal and spatial contexts may be underrepresented .
it is also important to stress that any site identified in phosphoproteomic screens needs to be directly confirmed by at least one independent method , for example multiple reaction monitoring ( which can also provide quantitative information ; rinehart et al . , 2009 ) , immunoblotting with phosphospecific antibodies ( park et al . , 2006 ) , or other approaches .
the wealth of data obtained from these studies raises the question as to whether we in the ion channel field should simply sit back and wait as these types of global analyses , being performed by our colleagues in the proteomics field , provide us with intriguing and extensive sets of in vivo phosphosites .
we would argue that we should accept such bountiful gifts with gratitude and appreciation , but that we in the ion channel field need to continue our complementary efforts to identify in vivo phosphosites through focused analyses of individual ion channel subunits purified from brain and other tissues .
together , these approaches are likely to yield a comprehensive dataset that will allow for a more complete picture of the nature and extent of in vivo phosphosites crucial to dynamic regulation of native ion channel expression , localization , and function . | voltage - gated ion channels underlie electrical activity of neurons and are dynamically regulated by diverse cell signaling pathways that alter their phosphorylation state .
recent global mass spectrometric based analyses of the mouse brain phosphoproteome have yielded a treasure trove of new data as to the extent and nature of phosphorylation of numerous ion channel principal or subunits in mammalian brain . here
we compile and review data on 347 phosphorylation sites ( 261 unique ) on 42 different voltage - gated ion channel subunits that were identified in these recent studies .
researchers in the ion channel field can now begin to explore the role of these novel in vivo phosphorylation sites in the dynamic regulation of the localization , activity , and expression of brain ion channels through multisite phosphorylation of their principal subunits . | INTRODUCTION
Recent high-throughput analyses of the mouse brain phosphoproteome
Analysis of the obtained datasets for ion channel phosphosites
Functional implication of phosphosites identified in high-throughput approaches
The overall nature of ion channel phosphorylation revealed by high-throughput analyses | voltage - dependent ion channels underlie the electrical signaling that allows for neurotransmission within neurons , and allow for various modes of ca influx critical to coupling this electrical activity to diverse physiological events ( hille , 2001 ) . as such , voltage - gated ion channel activity
neuronal ion channels exist as supramolecular protein complexes composed of pore - forming transmembrane principal or subunits ( yu et al . diverse posttranslational events acting on each of these components dynamically regulate the expression , localization , and function of neuronal ion channels ( levitan , 2006 ) . while numerous noncovalent mechanisms such as ligand binding , sensing of transmembrane voltage , and interaction with other proteins are known to play prominent roles in regulating neuronal ion channels , direct covalent modification of the component subunits of these multiprotein ion channel complexes by phosphorylation has long been recognized as a widely used and potent mechanism for neurons to achieve dynamic and reversible changes in ion channel function , and to impact their contribution to neuronal signaling ( levitan , 1985 ) . , 2010 ) , as well as synaptic potentiation , depression , and homeostatic plasticity ( turrigiano , 2008 ) , through phosphorylation of a large number of synaptic proteins including ligand - gated ion channels ( collins and grant , 2007 ) . neurons also exhibit cellular plasticity at the level of intrinsic excitability , achieved through phosphorylation of components of ion channel subunits , for example of voltage - gated sodium or nav ( cantrell and catterall , 2001 ) and potassium or kv ( schulz et al . while many of these studies continue to rely on effective purification of the target ion channel before analysis ,
a set of recent studies from the proteomics field , aimed at defining the global phosphoproteome of mouse brain samples with high complexity , and without a focus on ion channels per se , have yielded a dataset that is extremely valuable to the ion channel community . here
we provide an overview of these studies , as well as the subset of these databases that pertain to voltage - gated ion channel subunits . these studies provide important insights to the ion channel community on the extent and nature of phosphorylation of mammalian brain ion channels , and a wealth of phosphosites that can be tested for their specific role in regulating these ion channels through dynamic and reversible phosphorylation of their principal pore - forming and voltage - sensing subunits . more recently , analogous high - throughput studies have provided an even rarer gem , the determination of brain peptides chemically modified with phosphate , and the site of phosphorylation within these peptides ( lemeer and heck , 2009 ) . such in vivo studies have yielded an enormous dataset of phosphosites , including those on mammalian brain ion channels .
) what in the past would take a tremendous amount of effort in purifying the ion channel proteins from brain preparations , and then identifying the phosphosites ( using techniques that often used multiple millicuries of p ) is now available to the average channelologist at the click of the mouse . that said , one first needs to be aware of the existence of these studies , then search through the accompanying datasets for the presence of your favorite channel , and then perform analyses to determine the site of phosphorylation within specific channel isoforms . in this brief review
, we attempt to first bring these recent studies to the attention of members of the ion channel community , to highlight the wealth of the data being generated by these approaches , and second to detail the subset of the data related to voltage - gated ion channels . the goal is to ensure that the existing data , and others that will emerge from future studies , can be effectively used to gain mechanistic insights into how neuronal ion channels are regulated by reversible phosphorylation at these in vivo phosphosites . note that this synopsis is assuredly not comprehensive , as there are likely other high - throughput studies that have yielded data leading to identification of ion channel phosphosites that we have not included in our analyses , which focuses only on data from four recent global analyses from mouse brain ( munton et al . the search yielded 12,035 unique phosphosites on 4,579 proteins , including 231 different phosphosites on 42 different voltage - gated ion channel subunits , as detailed in table i. phosphorylation sites identified on voltage - gated ion channel subunits note additional sites identified on slo1/kcnma1 q08460 - 2 : t670 ( w ) and s672 ( w , t ) , corresponding to t709 and s711 in q08460 - 1 . this study yielded 1,564 unique phosphosites on 831 proteins , including 52 different phosphosites on 19 different voltage - gated ion channel subunits ( table i ) . this study yielded 974 unique phosphosites on 499 proteins , including 32 different phosphosites on 16 different voltage - gated ion channel subunits ( table i ) . this study yielded 1,341 unique phosphosites on 638 proteins , including 32 different phosphosites on 14 different voltage - gated ion channel subunits ( table i ) . , 2005 )
effective validation of the biological role of any identified phosphosites still involves using biochemical , cell biological , and electrophysiological analyses of individual proteins expressing site - specific mutations in the identified phosphosites . schematic of phosphosites identified in high - throughput studies on six transmembrane segment ion channel subunits
. an overall number of unique phosphosites identified shows that the largest ion channel subunits , namely those within the 24 transmembrane segment family of cav and nav channel subunits , have the most extensive phosphorylation , with cav2.1 ( 24 phosphosites ) , nav1.2 ( 23 phosphosites ) , and cav2.3 ( 22 phosphosites ) topping the list ( table i ) . among the six transmembrane segment family members , which are smaller polypeptides and assemble as tetramers in the final channel complex , kv7.2 ( 15 phosphosites ) , hcn2 ( 14 phosphosites ) ,
kv2.1 ( 13 phosphosites ) , and slo1-bk ( 10 phosphosites ) are notable in their extensive representation , suggesting a high degree of phosphorylation - dependent modulation of these channels . note that while these data suggest extensive multisite phosphorylation exists on individual molecules of ion channel subunits , these studies have not provided conclusive data as these phosphosites come from relatively small tryptic fragments of the larger polypeptides . as such , except for the small subset of neighboring phosphosites found within the same multiply phosphorylated tryptic peptide , it is formally possible that the multiple phospho - sites identified in these ion channel subunits are stochastically distributed in different molecules . note that representation on the list of most phosphorylated ion channel subunits is likely a combination of not only the actual extent of phosphorylation , but also nature of the phosphorylation , as the techniques employed ( ms - based analyses of tryptic peptides ) bias the analysis toward phosphosites on tryptic peptides with a chemical nature ( e.g. this raises questions as to the implications for the rat and human brain orthologues of these mouse brain ion channel subunits . focusing on the ion channel subunits within a subfamily that exhibit the highest number of identified phosphosites ( cav2.1 , nav1.2 , kv2.1 , kv7.2 , and trpm3 ) , we found that , with the exception of mouse and human cav2.1 ( 87% overall identity ) , overall sequence identity between mouse , rat , and human ion channel subunit orthologues was 93% or higher . as many of these stimuli are known to act through signaling pathways leading to activation of protein kinases or protein phosphatases , the assumption has been that the effects are meditated through changes in the phosphorylation state of the ion channel subunits themselves . in a few cases , which we detail below , phosphosites identified in these recent phosphoproteomics studies have provided in vivo relevance to studies already performed in heterologous cells . future studies aimed at determining the functional impact of the phosphosites we highlight here will serve to increase the number of examples of phosphosites that appear in vivo , and functional relevance is known . ,
erk - dependent up - regulation of cav2.2 channels is mediated through phosphorylation of the site identified in mouse brain as s446 ( martin et al . in the dii - diii linker , pkc phosphorylates the sites identified in mouse brain as s773 and s892 , and camkii at s783 , and impacts syntaxin1a cav2.2 interaction ( yokoyama et al . of the phosphosites identified in the high throughput studies , mutations at s15 , s567 , and s655 partially mimic and occlude the effects of dephosphorylation in causing hyperpolarizing shifts in the voltage - dependent gating . note that a previous mass spectrometry based analysis of kv4.2 purified from rat brain ( seikel and trimmer , 2009 ) identified four phosphosites ( s548 , s552 , s572 , and s575 ) of which two ( s548 and s552 ) were found in the high - throughput analyses of mouse brain ( table i ) . to gain insights into whether these data provide insights into the predominance of ion channel phosphorylation by a particular protein kinase , we have analyzed the phosphosites on the ion channel subunits with the most extensive phosphorylation ( cav2.1 , nav1.2 , kv2.1 , kv7.2 , and trpm3 ) for consensus motifs using netphosk ( http://www.cbs.dtu.dk/services/netphosk/ ) . note that in each case , the analyses of purified ion channel subunits were from rat brain , while the global analyses were performed on mouse brain . for nav1.2 , of the 23 phosphosites identified in these global analyses ,
10 were found in the single - target analysis of purified nav1.2 ( berendt et al . however , the ms - based analyses of purified nav1.2 identified six phosphosites not seen in the global analyses . in the case of kv2.1 , of the 13 phosphosites identified in the global analyses of mouse brain , six
were observed in the analysis of kv2.1 purified from rat brain , which also revealed the presence of 10 additional phosphosites not seen in the global analyses ( park et al . for slo1-bk , of the 10 unique phosphosites identified in the global analyses ,
all but two were also observed in the analyses of the purified slo1-bk , which also provided an additional 20 phosphosites ( yan et al . results from high versus low - throughput analyses these comparisons suggest that while the dataset from these global analyses represents a wealth of information for ion channel researchers , parallel approaches using enrichment of the target ion channel subunits provide datasets that are oftentimes more extensive , revealing additional phosphosites that may play important roles in the modulation of these channels . the wealth of data obtained from these studies raises the question as to whether we in the ion channel field should simply sit back and wait as these types of global analyses , being performed by our colleagues in the proteomics field , provide us with intriguing and extensive sets of in vivo phosphosites . we would argue that we should accept such bountiful gifts with gratitude and appreciation , but that we in the ion channel field need to continue our complementary efforts to identify in vivo phosphosites through focused analyses of individual ion channel subunits purified from brain and other tissues . together , these approaches are likely to yield a comprehensive dataset that will allow for a more complete picture of the nature and extent of in vivo phosphosites crucial to dynamic regulation of native ion channel expression , localization , and function . in this study ,
the search yielded 12,035 unique phosphosites on 4,579 proteins , including 231 different phosphosites on 42 different voltage - gated ion channel subunits , as detailed in table i. phosphorylation sites identified on voltage - gated ion channel subunits note additional sites identified on slo1/kcnma1 q08460 - 2 : t670 ( w ) and s672 ( w , t ) , corresponding to t709 and s711 in q08460 - 1 . this study yielded 1,564 unique phosphosites on 831 proteins , including 52 different phosphosites on 19 different voltage - gated ion channel subunits ( table i ) . this study yielded 974 unique phosphosites on 499 proteins , including 32 different phosphosites on 16 different voltage - gated ion channel subunits ( table i ) . this study yielded 1,341 unique phosphosites on 638 proteins , including 32 different phosphosites on 14 different voltage - gated ion channel subunits ( table i ) . while the phosphosites identified in the above studies
were confirmed using the most strict proteomic criteria and manual inspections , the bulk of the phosphosites obtained from these high - throughput approaches were not experimentally validated by other means . , 2005 )
effective validation of the biological role of any identified phosphosites still involves using biochemical , cell biological , and electrophysiological analyses of individual proteins expressing site - specific mutations in the identified phosphosites . schematic of phosphosites identified in high - throughput studies on 24 transmembrane segment ion channel subunits . among the six transmembrane segment family members , which are smaller polypeptides and assemble as tetramers in the final channel complex , kv7.2 ( 15 phosphosites ) , hcn2 ( 14 phosphosites ) , kv2.1 ( 13 phosphosites ) , and slo1-bk ( 10 phosphosites ) are notable in their extensive representation , suggesting a high degree of phosphorylation - dependent modulation of these channels . note that while these data suggest extensive multisite phosphorylation exists on individual molecules of ion channel subunits , these studies have not provided conclusive data as these phosphosites come from relatively small tryptic fragments of the larger polypeptides . as such , except for the small subset of neighboring phosphosites found within the same multiply phosphorylated tryptic peptide , it is formally possible that the multiple phospho - sites identified in these ion channel subunits are stochastically distributed in different molecules . note that representation on the list of most phosphorylated ion channel subunits is likely a combination of not only the actual extent of phosphorylation , but also nature of the phosphorylation , as the techniques employed ( ms - based analyses of tryptic peptides ) bias the analysis toward phosphosites on tryptic peptides with a chemical nature ( e.g. this raises questions as to the implications for the rat and human brain orthologues of these mouse brain ion channel subunits . focusing on the ion channel subunits within a subfamily that exhibit the highest number of identified phosphosites ( cav2.1 , nav1.2 , kv2.1 , kv7.2 , and trpm3 ) , we found that , with the exception of mouse and human cav2.1 ( 87% overall identity ) , overall sequence identity between mouse , rat , and human ion channel subunit orthologues was 93% or higher . as many of these stimuli are known to act through signaling pathways leading to activation of protein kinases or protein phosphatases , the assumption has been that the effects are meditated through changes in the phosphorylation state of the ion channel subunits themselves . in a few cases , which we detail below , phosphosites identified in these recent phosphoproteomics studies have provided in vivo relevance to studies already performed in heterologous cells . ,
erk - dependent up - regulation of cav2.2 channels is mediated through phosphorylation of the site identified in mouse brain as s446 ( martin et al . in the dii - diii linker , pkc phosphorylates the sites identified in mouse brain as s773 and s892 , and camkii at s783 , and impacts syntaxin1a cav2.2 interaction ( yokoyama et al . of the phosphosites identified in the high throughput studies , mutations at s15 , s567 , and s655 partially mimic and occlude the effects of dephosphorylation in causing hyperpolarizing shifts in the voltage - dependent gating . note that a previous mass spectrometry based analysis of kv4.2 purified from rat brain ( seikel and trimmer , 2009 ) identified four phosphosites ( s548 , s552 , s572 , and s575 ) of which two ( s548 and s552 ) were found in the high - throughput analyses of mouse brain ( table i ) . to gain insights into whether these data provide insights into the predominance of ion channel phosphorylation by a particular protein kinase , we have analyzed the phosphosites on the ion channel subunits with the most extensive phosphorylation ( cav2.1 , nav1.2 , kv2.1 , kv7.2 , and trpm3 ) for consensus motifs using netphosk ( http://www.cbs.dtu.dk/services/netphosk/ ) . note that in each case , the analyses of purified ion channel subunits were from rat brain , while the global analyses were performed on mouse brain . for nav1.2 , of the 23 phosphosites identified in these global analyses , 10 were found in the single - target analysis of purified nav1.2 ( berendt et al . in the case of kv2.1 , of the 13 phosphosites identified in the global analyses of mouse brain , six
were observed in the analysis of kv2.1 purified from rat brain , which also revealed the presence of 10 additional phosphosites not seen in the global analyses ( park et al . for slo1-bk , of the 10 unique phosphosites identified in the global analyses , all but two were also observed in the analyses of the purified slo1-bk , which also provided an additional 20 phosphosites ( yan et al . results from high versus low - throughput analyses these comparisons suggest that while the dataset from these global analyses represents a wealth of information for ion channel researchers , parallel approaches using enrichment of the target ion channel subunits provide datasets that are oftentimes more extensive , revealing additional phosphosites that may play important roles in the modulation of these channels . the wealth of data obtained from these studies raises the question as to whether we in the ion channel field should simply sit back and wait as these types of global analyses , being performed by our colleagues in the proteomics field , provide us with intriguing and extensive sets of in vivo phosphosites . we would argue that we should accept such bountiful gifts with gratitude and appreciation , but that we in the ion channel field need to continue our complementary efforts to identify in vivo phosphosites through focused analyses of individual ion channel subunits purified from brain and other tissues . together , these approaches are likely to yield a comprehensive dataset that will allow for a more complete picture of the nature and extent of in vivo phosphosites crucial to dynamic regulation of native ion channel expression , localization , and function . | [
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] |
they are strikingly abundant in neurons and in particular at chemical synapses ( palay , 1956 ) where neuronal communication takes place .
it has long been recognized that mitochondria are important for the maintenance of neuronal integrity and function , as diseases caused by a mitochondrial failure often display a neurodegenerative component affecting the central or peripheral nervous system ( dimauro and schon , 2008 ) .
conversely , mitochondrial defects are frequently observed in tissue samples collected from patients with a neurodegenerative disorder ( chen and chan , 2009 ) and the involvement of mitochondrial dysfunction in a large number of diseases likely remains gravely underestimated ( table 1 ) .
these observations suggest an intimate involvement of mitochondria in maintaining neuronal integrity , possibly in part by controlling neurotransmission , yet the exact function of these organelles in synaptic activity has long remained elusive .
disorders are listed in this table along with their most characteristic phenotype , when known , their causative gene(s ) and the mitochondrial features reported to be defective .
the mitochondrial defect might be the direct cause of the disease or the result of an upstream dysfunction in the pathology .
mitochondria are dynamic organelles that divide , fuse and are redistributed within the cell in response to various physiological cues . besides their predominant role in energy metabolism ( figure 1 ) , they are involved in many other cellular processes that are also relevant for neuronal function , including the regulation of calcium homeostasis and a form of programmed cell death ( or apoptosis ) .
defects in all these functions as well as in mitochondrial fusion or fission are predicted to affect various aspects of neuronal physiology ( dimauro and schon , 2008 ; chen and chan , 2009 ) .
however , unraveling the link between a mitochondrial defect and its consequences on neuronal function or survival has proven a complex task , mostly because different mitochondrial functions are interdependent ( see for example niemann et al . , 2005 ; graier et al . , 2007
experimental results obtained using drugs or mutations that interfere with one aspect of mitochondrial physiology thus need to be interpreted with care , as they may have numerous side effects .
( 1 ) mitochondria are dynamic organelles , able to move bidirectionally along microtubules tracts with the help of motor proteins ( kinesin for anterograde and dynein for retrograde axonal transport , respectively ) and adaptor proteins ( e.g. , milton and the associated rho - gtpase miro or syntabulin ) .
short - distance transport along actin filaments is mediated by myosin motors ( not shown ) .
( 2 ) the krebs cycle takes place in the mitochondrial matrix , generating -ketoglutarate and electron donors ( nadh and succinate ) .
( 3 ) transamination of -ketoglutarate yields the neurotransmitter glutamate , which can also be formed by glutamine hydrolysis .
( 4 ) the electron transport chain ( etc ) , consisting of 4 multiprotein complexes , mediates electron transfer ( blue arrow ) from nadh and succinate to the electron acceptor o2 .
numerous drugs target components of the etc , for example mptp ( 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ) and rotenone affect complex i while antimycin a1 inhibits complex iii .
electron transfer is coupled with proton extrusion out of the matrix toward the intermembrane space , producing an electrochemical gradient across the inner membrane ( about 200 mv ) .
( 5 ) protons ultimately return to the matrix through the atp synthase , using the free energy produced by the etc to drive atp synthesis .
( 6 ) mitochondria are also involved in calcium homeostasis , and ca ions can be sequestered in the matrix under the form of a reversible phosphate complex .
while the outer mitochondrial membrane is rather permeable to calcium , ca entry across the inner mitochondrial membrane is mediated by a uniporter , and its extrusion largely relies on a sodium / calcium exchanger .
this review focuses on the role of presynaptic mitochondria in the control of synaptic vesicle ( sv ) function . in general ,
svs can be attributed to three functionally different pools , termed the readily releasable pool , the recycling pool and the reserve pool ( rp ) ( rizzoli and betz , 2005 ) .
together , the readily releasable pool and the recycling pool , sometimes referred to as the exo - endo cycling pool ( ecp ) , typically represent about 1020% of the svs and are released under moderate or intense neuronal activity , whereas vesicles from the rp are recruited only upon high frequency stimulation ( figure 2 ) ( kuromi and kidokoro , 2005 ; rizzoli and betz , 2005 ) .
based on these characteristics , different stimulation protocols have been developed that allow the specific mobilization of svs from different pools as well as their labeling with fluorescent dyes ( kuromi and kidokoro , 2002 ; verstreken et al . , 2008 ) . because the synaptic vesicle cycle involves numerous atp - consuming steps and is tightly controlled by the cytosolic calcium concentration ( [ ca]cyto ) , vesicle cycling appears to be highly dependent on mitochondrial function . in the following sections we will review recent reports indicating how mitochondria can affect the distribution in pools and release properties of svs .
in particular , we reexamine experimental evidence which together indicate that these organelles are essential for synaptic vesicle release under conditions of intense neuronal activity through vesicle mobilization from the rp .
current studies dedicated to this link between mitochondria and the sv cycle will hopefully lead to new therapeutic strategies for the handling of neurological disorders characterized by a mitochondrial defect and which have a tremendous social impact , like alzheimer 's , huntington 's and parkinson 's diseases .
( 1 ) synaptic vesicles ( svs ) from the exo - endo cycling pool ( ecp ) participate in neurotransmitter release at active zones ( in red ) . upon arrival of an action potential at the nerve terminal ,
( 2 ) in contrast , svs from the reserve pool ( rp ) , which contains the majority of vesicles in most synapses , are tethered to cytoskeletal elements and are only recruited to the active zone during intense stimulation .
( 3 ) the atp produced by the mitochondria also plays an important role for preynaptic ca buffering in the endoplasmic reticulum ( er ) .
high ca , induced by exo - endocytosis , activate calcineurin that in turn activates and dephosphorylates drp1 , resulting in mitochondrial fission and transport .
in excitable cells , depolarization of the plasma membrane triggers the opening of voltage - gated calcium channels , leading to ca entry and a subsequent rise of the [ ca]cyto that initiates various downstream cellular responses . in neurons ,
calcium signaling is involved in neurotransmitter release from the presynaptic terminals , in vesicle recycling via endocytosis and in long - term neuronal responses like neurite growth and degeneration , or modulation of synaptic strength ( ghosh and greenberg , 1995 ; zucker , 1999 ; jonas , 2006 ; yao et al . ,
similarly to the endoplasmic reticulum ( er ) , mitochondria are able to sequester and release ca ions and thereby modulate calcium - dependent signaling events .
finally , mitochondria may also contribute to calcium homeostasis by providing atp to fuel the plasma membrane ca - atpase ( zenisek and matthews , 2000 ) .
considerable effort has thus been invested in studying how presynaptic mitochondria control the time - course and the amplitude of calcium signaling .
mitochondrial calcium uptake is driven by the negative potential established across the inner mitochondrial membrane ( figure 1 ) and can be inhibited by drugs that dissipate the proton gradient responsible for this potential ( gunter et al .
the outer mitochondrial membrane is relatively permeable to calcium ions , so that ca accumulation in the mitochondrial matrix mainly depends on a mitochondrial calcium uniporter ( mcu ) that resides in the inner membrane ( figure 1 ) .
the mcu shows a very low affinity for calcium ions ( km estimated between 1 and 189 m in different mitochondrial preparations ) ( gunter and sheu , 2009 ) , that can be overcome by the proximity between mitochondria and the ca source , i.e. , the cytosolic orifice of calcium channels at the mitochondria - er ( see below ) or mitochondria - plasma membrane contact sites , where local subdomains of high ca concentration can be generated ( rizzuto et al . , 1999 ; montero et al . , 2000 ) . in heart and liver cells ,
an additional calcium uptake mechanism that does not rely on the mcu , termed rapid uptake mode , has also been described ( buntinas et al . , 2001 ) .
once accumulated in the matrix , calcium ions can be buffered by forming reversible ca - phosphate complexes , or be exported back to the cytosol by a na / ca exchanger ( figure 1 ) ( gunter and sheu , 2009 ) .
interestingly , mitochondrial transport along microtubule tracks is negatively regulated by calcium ( yi et al . , 2004 )
( see also below ) , and in drosophila ca ions were found to disrupt the interaction between the motor protein kinesin and the miro gtpase , which is required for the transport of these organelles along microtubules ( figure 1 ) ( macaskill et al . , 2009 ) .
local subcellular regions of elevated [ ca]cyto building up during the opening of voltage - gated plasma membrane ca channel as observed upon arrival of an action potential at the nerve terminal might thus cause mitochondria to pause and transiently accumulate close to active zones where svs will fuse . in neurons , [ ca]cyto peaks stimulate sv exocytosis and hence neurotransmitter release .
this effect is mediated by synaptotagmin , a ca sensor that resides in the vesicle membrane and promotes snare - mediated fusion with the presynaptic membrane ( tucker et al .
by sequestering ca , presynaptic mitochondria can rapidly buffer the [ ca]cyto and thus diminish the sv release probability , thereby accelerating recovery of neurotransmission after nerve stimulation .
this role of mitochondria in ca buffering is prominent in chromaffin cells ( montero et al . , 2000 ) and in certain types of synapses , for example at the rat calyx of held ( billups and forsythe , 2002 ) and at the mouse neuromuscular junction ( nmj ) ( david and barrett , 2003 ) . in these synapses ,
pharmacological inhibition of mitochondrial ca uptake slows down the decay of the [ ca]cyto peak observed after nerve stimulation .
this in turn affects neurotransmission , as synaptic depression is more rapid ( david and barrett , 2003 ) and under these conditions recovery from synaptic depression after a stimulus train is slowed down ( billups and forsythe , 2002 ) , suggesting that the prolonged presence of free calcium ions in the cytosol stimulates the depletion of the pool of available svs .
a first effect of mitochondrial calcium uptake is thus to restrict the period of neurotransmitter release after the arrival of an action potential at the nerve terminal .
stimulation of sv exocytosis by cytosolic calcium ions is also at the heart of the so - called residual calcium hypothesis , according to which residual calcium ions present in the cytoplasm during the decay of the [ ca]cyto peak after each action potential increase the probability of neurotransmitter release ( katz and miledi , 1968 ) .
i.e. , the er and/or mitochondria after the stimulus end causes a persistent elevation of [ ca]cyto that in turn stimulates sv exocytosis ( tang and zucker , 1997 ; yang et al .
this has been well described at the crayfish nmj , where inhibition of mitochondrial ca uptake leads to a higher [ ca]cyto during tetanic stimulation , as in the work from the groups of forsythe and barrett described above , and increases short - term augmentation ( tang and zucker , 1997 ) .
in contrast , decay of the [ ca]cyto peak after tetanus is faster than in untreated neurons , thereby reducing long - term potentiation ( tang and zucker , 1997 ) .
recently , the importance of mitochondria in controlling presynaptic [ ca]cyto and hence in synaptic plasticity has been illustrated in mammals as well .
defective calcium buffering at mouse hippocampal presynaptic terminals , resulting from a lack of the microtubule - binding protein syntaphilin required for axonal docking of mitochondria ( figure 1 ) , was shown to cause an elevation of [ ca]cyto during intense stimulation , prolonging the facilitation phenomenon observed in control neurons ( kang et al . , 2008 ) .
in short , a second consequence of calcium buffering by mitochondria is that neurotransmitter release can be prolonged after the stimulus end as ca ions are progressively unloaded from the mitochondrial matrix into the cytosol .
there are thus both a negative and a positive effect of mitochondrial calcium sequestration on synaptic activity .
according to the residual calcium hypothesis , a defect in calcium buffering is expected to favor recovery from depression , as indeed observed by several groups ( dittman and regehr , 1998 ; wang and kaczmarek , 1998 ) .
this however contrasts with the results from the groups of barrett and forsythe discussed above , according to which mitochondrial calcium buffering shortens synaptic depression ( billups and forsythe , 2002 ; david and barrett , 2003 ) .
this apparent discrepancy is most likely due to the use of different stimulation protocols in various model systems , and to the different approaches used to observe calcium buffering , either via mobile calcium buffers ( dittman and regehr , 1998 ; wang and kaczmarek , 1998 ) or specifically by the mitochondria ( billups and forsythe , 2002 ; david and barrett , 2003 ) .
more importantly , this controversy also stresses the versatile role of calcium in the sv cycle .
indeed , ca ions not only influence exocytosis of svs but also their recycling via endocytosis ( yao et al . ,
the [ ca]cyto required for proper endocytosis at synapses is much lower than the concentration needed to cause sv exocytosis and is actually close to the intra - axonal [ ca]cyto ( shupliakov , 2009 ) , but there is a positive correlation between the intracellular calcium concentration and the endocytic rate ( wu et al . , 2009 ) .
experimental data collected e.g. , from the frog nmj ( ceccarelli and hurlbut , 1980 ) , giant lamprey synapse ( gad et al . , 1998 ) and rat hippocampal neurons ( balaji et al
. , 2008 ) suggest that extracellular ca might actually be required only to increase the endocytic capacity of the presynaptic membrane under conditions of intense stimulation , in order to compensate the large influx of exocytic material ( dittman and ryan , 2009 ) .
the molecular mechanism of this regulation is only starting to be elucidated , but it appears that the calcium - binding protein calmodulin initiates endocytosis at the synapse and facilitates both plasma membrane invagination and fission ( wu et al . , 2009 ) .
moreover , other ca - binding endocytic proteins including synaptotagmin and endophilin have been identified ( brose et al . , 1992 ; chen et al .
the overall effect of ca ions on neurotransmitter release should thus be regarded as a balance between the stimulation of sv exocytosis , which can promote either recovery from depression or sv depletion depending on the experimental conditions , and a positive effect on sv recycling .
when mitochondria are overloaded with ca , they undergo a dramatic event termed mitochondrial permeability transition ( mpt ) , i.e. , the formation of non - selective pores in the inner mitochondrial membrane that allows the entry of any molecule smaller than 1,500 dalton into the mitochondrial matrix , causing an increase in osmotic pressure leading to mitochondrial swelling and rupture of the outer mitochondrial membrane ( norenberg and rao , 2007 ) .
strikingly , synaptic mitochondria are able to buffer less ca ions and are more prone to display mpt than non - synaptic mitochondria isolated from the same sample of rat cortical brain tissue ( brown et al . , 2006 ) or mouse sympathetic neurons ( nez et al . , 2007 )
the reason for this difference in ca - handling capacity remains unclear , but it might favor ca - dependent exocytosis at the presynaptic sites by maintaining a higher [ ca]cyto . yet a reduced ca - buffering capacity of synaptic mitochondria , caused under pathogenic conditions or by aging , may presumably increase their susceptibility to mpt and subsequent release of cell - death molecules ( see below ) , leading to synapse degeneration as observed e.g. , in alzheimer 's disease ( selkoe , 2002 ) , schizophrenia ( glantz et al . , 2006 ) or huntington 's disease ( damiano et al . , 2010
, the drosophila nmj mitochondrial contribution to ca buffering appears to be only minor and/or easily compensated by alternative mechanisms , at least during mild stimulation ( guo et al .
2006 ) . inhibiting mitochondrial ca uptake in fly motor nerve terminals by adding tpp ( tetraphenylphosphonium ) ( verstreken et al . , 2005 ) or antimycin a1 ( lnenicka et al . , 2006 ) ( figure 1 ) does indeed not significantly influence calcium clearance or sv mobilization and recycling after a 10 hz train stimulus .
in contrast , decay of the [ ca]cyto peak is significantly slowed down when the ca atpase mediating calcium uptake into the er is pharmacologically blocked or , to an even greater extent , when the plasma membrane ca atpase ( pmca ) is inhibited ( lnenicka et al . , 2006 ) . although ca clearance is thus mainly ensured by the pmca ( lnenicka et al . , 2006 ) ,
calcium storage in the er lumen contributes to evoked neurotransmitter release at the drosophila nmj , supposedly by allowing the [ ca]cyto to remain elevated after the stimulus end ( sanyal et al . , 2005 ) .
similarly , in frog motor nerve terminals intracellular ca release from the er after cessation of stimulation leads to post - tetanic potentiation ( ptp ) ( narita et al . , 2000 ) . in this context , it is worth noting that a very close physical connection between the er and mitochondria can be observed in many cell types , including neurons ( mironov and symonchuk , 2006 ) . such contacts , termed mitochondria - associated er membrane ( mam ) , allow the bidirectional exchange of phospholipids and ca ions between these organelles ( hayashi et al .
2009 ) . in mouse respiratory neurons , where both the er and mitochondria contribute to calcium - dependent modulation of synaptic activity , some of the ca ions transiently accumulated within er vesicles in response to a stimulus are subsequently transferred to the mitochondrial matrix ( mironov and symonchuk , 2006 ) .
if this transfer is prevented , the peak of [ ca]mito observed after stimulation is smaller compared to controls and neurotransmitter release is reduced ( mironov and symonchuk , 2006 ) .
given the low affinity of the mitochondrial calcium uptake system , mams may thus play a critical role in the capacity of mitochondria to promptly respond to a change in [ ca]cyto and thereby affect many aspects of mitochondrial biology ( hayashi et al . , 2009b ) , including the control of sv exocytosis and/or endocytosis . finally ,
in addition to their direct effects on exocytosis and endocytosis , ca ions can also regulate the sv cycle indirectly by modulating mitochondrial bioenergetics .
three enzymes of the krebs cycle residing in the mitochondrial matrix are indeed activated in a calcium - dependent manner ( duchen , 1992 ) , so that mitochondrial calcium uptake increases the flux of electrons through the electron transport chain ( etc ) and hence atp production ( figure 1 ) ( graier et al . , 2007 ; gunter and sheu , 2009 ) . as a consequence
, drugs preventing mitochondrial calcium uptake also negatively affect atp production , thereby complicating the interpretation of experimental data .
thus it is advisable to look at the effects of these drugs under conditions where the cytosolic atp level is artificially preserved . in conclusion
, ca ions play a versatile role in several steps of the sv cycle through different ca sensors .
mitochondria thus affect exocytosis and endocytosis of svs through their ability to regulate ca homeostasis .
however , the relative contribution of the er and mitochondria to synaptic ca - buffering appears to vary between different neuronal systems and might be influenced by the integration of different signaling pathways ( rizzuto , 2003 ) .
the most well - known function of mitochondria is to produce energy , and in neurons they generate the majority of atp ( about 90 % ) via aerobic metabolism ( figure 1 ) , the rest being provided by glycolysis in the cytosol ( mathews and van holde , 1995 ) .
this energy supply is critical since proper neuronal function requires atp in order to fuel ion pumps , to organize cytoskeletal components , to support numerous phosphorylation reactions ( attwell and laughlin , 2001 ) and for many steps in the sv cycle including myosin - dependent axonal transport of svs , their snare - mediated fusion with the plasma membrane , membrane scission , vesicle uncoating and refilling of vesicles with neurotransmitter ( murthy and camilli , 2003 ) . because of the highly specialized architecture of neurons , with their long processes contacting cells located far away from the cell body , atp diffusion from a central location to the distal synapses is thought to be too slow to meet these energy requirements ( kuiper et al . , 2008 ) .
accordingly , in neurons mitochondrial biogenesis occurs mostly in the soma ( davis and clayton , 1996 ) but these organelles can subsequently be transported along cytoskeleton tracks in the neuronal processes ( figure 1 ) ( hollenbeck and saxton , 2005 ) , where they show saltatory bidirectional movement , with frequent pauses and changes of direction ( cai and sheng , 2009 ) .
mitochondria are then specifically retained in regions of the axon where the energy demand is high ( hollenbeck , 2005 ) , including synapses ( palay , 1956 ; nguyen et al . , 1997 ) and in particular near the active zones where svs are released ( rowland et al .
moreover , there is a positive correlation between anterograde axonal transport of mitochondria and the electrical potential across their inner membrane ( miller and sheetz , 2004 ) which drives atp production ( alfonzo and racker , 1979 ) ( figure 1 ) , and mitochondria with a high potential tend to accumulate at synapses ( lee and peng , 2006 ) .
finally , there appears to be a direct relationship between the frequency and/or the intensity of activity in a given synapse and the number and metabolic activity of presynaptic mitochondria , both under physiological and pathological conditions ( atwood et al .
, 1986 ; nguyen et al . , 1997 ; brodin et al . , 1999 ; dubinsky , 2009 ) .
all these observations point to a central role of mitochondrial atp production in neuronal transmission , but the exact molecular mechanisms involved in this regulation are only beginning to be deciphered .
the number of presynaptic mitochondria is partially regulated by drp1 ( dynamin related protein ) ( verstreken et al . , 2005 ) .
this protein mediates mitochondrial fission ( praefcke and mcmahon , 2004 ; rube and van der bliek , 2004 ) and its activity is controlled by calcineurin , a ca responsive phosphatase ( cereghetti et al . , 2008 ) .
high ca leads to calcineurin activation ( guerini , 1997 ) and in turn to dephosphorylation and activation of drp1 and thus mitochondrial fission , which has been associated with the transport of mitochondria into the synapse ( verstreken et al . , 2005 ; cereghetti et al . ,
, these data support a model where dynamic mitochondria distribution in neurons is adjusted to meet the energy requirement for proper neuronal communication .
several studies now start to shed light on the exact nature of the presynaptic events that most critically depend on mitochondrial atp production . at drosophila
nmj synapses , svs can be assigned to different , but spatially mixed functional vesicle pools , the ecp and rp ( kuromi and kidokoro , 2005 ) .
several mutants have been used to examine how these two vesicles pools are affected by mitochondrial dysfunction .
the first observation that was made is that synaptic communication is rapidly inhibited when the activity of the mitochondrial atp translocase is switched off by means of the thermosensitive mutation sesb ( stress sensitive b ) , suggesting that the cytosolic pool of atp at synaptic terminals is turned over rapidly under basal conditions ( rikhy et al . , 2003 ; trotta et al . , 2004 ) ,
further studies showed that these mutants have problems maintaining sv cycling during periods of high demand .
vesicle trafficking defects are also observed when mitochondrial morphology is altered due to impaired fission in thermosensitive drp1 mutant flies ( rikhy et al .
, these observations suggest that mitochondria extensively control neuronal function , maybe by influencing sv pools . because both the sesb and drp1 mutations enhance the phenotype of the paralytic mutant shibire , where endocytosis is known to be blocked at the restrictive temperature ( hinshaw and schmid , 1995 ) , one possibility proposed by the authors is that reduced atp levels could affect endocytosis , thereby preventing efficient sv recycling ( rikhy et al . , 2003 , 2007 ) .
remarkably , in the absence of presynaptic mitochondria in drp1 or miro ( mitochondrial rho - gtpase ) loss - of - function mutant flies basal synaptic activity at the drosophila neuromuscular junction ( nmj ) appears not to be significantly altered . in these flies
mitochondrial fission or axonal mitochondrial transport are inhibited , respectively , resulting in synapses largely devoid of mitochondria ( guo et al . , 2005 ; verstreken et al .
in contrast , these mutants show impaired neurotransmission during tetanic stimulation ( guo et al . , 2005 ;
verstreken et al . , 2005 ) and this phenotype can be largely restored by supplementing synapses with exogenous atp ( verstreken et al . ,
2005 ) , suggesting that energy production by presynaptic mitochondria is essential only during intense neuronal activity .
failure of the drp1 mutant to sustain neuronal activity during intense stimulation is due to a defect in rp mobilization , while basal exocytosis and endocytosis of ecp vesicles is unaffected ( verstreken et al .
similar effects on rp vesicle mobilization are observed at wild - type nmjs treated with oligomycin , an inhibitor of the mitochondrial atp synthase ( figure 1 ) , and forward - filling drp1 motor neurons with atp restores the vesicle trafficking defects in the rp ( verstreken et al . , 2005 ) .
thus , mitochondrial atp production is specifically required for mobilization of the rp , a process believed to underlie ptp ( kuromi and kidokoro , 2005 ) .
in particular , atp appears to be needed for rp vesicle propulsion along actin filaments mediated by the myosin atpase complex ( verstreken et al . ,
an additional role of atp in rp mobilization is exerted after its conversion into camp by phosphodiesterase and adenylate cyclase .
camp indeed activates the pka kinase , which mediates unthetering of rp vesicles and allows their subsequent recruitment at the active zone ( kuromi and kidokoro , 2005 ) .
a very similar dependence of neurotransmission on mitochondrial atp production has recently been observed in mammalian neurons , where anterograde transport of mitochondria along the axon relies on the adaptor protein syntabulin , linking these organelles to the kinesin heavy chain ( figure 1 ) ( cai et al . ,
knock - down of syntabulin or disruption of its interaction with kinesin impair neurotransmitter release under intense stimulation conditions and this defect is partially rescued by exogenous atp addition ( ma et al . , 2009 ) , paralleling the effects of the fly drp1 mutation ( verstreken et al . , 2005 ) .
interestingly , in rat hippocampal neurons the gtpase activity of drp1 appears to be positively regulated by the antiapoptotic protein bcl - xl ( li et al . , 2008 ) .
by activating drp1 , bcl - xl promotes mitochondrial fission and recruitment at developing synaptic sites , and is believed to thereby increase atp availability to fuel synaptogenesis and further synaptic activity ( jonas et al . , 2003 ; li et al . , 2008 ) .
overexpression of either bcl - xl or drp1 in rat hippocampal neurons leads to the formation of larger sv clusters compared to controls .
conceivably , the presence of an increased number of mitochondria at the synapses may stimulate the formation of a larger rp and/or favor sv recycling ( li et al . , 2008 ) .
this is likely to be attributed to increased atp release from mitochondria since , at least at the squid giant synapse , the positive effects of bcl - xl overexpression on neurotransmission can be mimicked by atp injection ( jonas et al . , 2003 ) . that mitochondrial energy production is required for the mobilization of svs is also supported by numerous studies investigating the spatial organization of presynaptic mitochondria and svs . in the cat calyx of held presynaptic mitochondria
are positioned about 200 nm away from the plasma membrane , next to the active zones , within a characteristic structure named mitochondrion - associated adherens complex ( mac ) ( rowland et al . , 2000 ) .
compared to other mitochondria in the same neuron , mac mitochondria display a higher density of crista - membranes , suggesting increased metabolic activity ( perkins et al . , 2010 ) .
strikingly , dense populations of svs are connected to cytoskeletal elements emanating from macs , which appear to form a barrier between vesicle pools associated with neighboring active zones ( perkins et al . , 2010 ) .
as suggested by the authors , this physical separation might contribute to the different vesicle release probability observed between different active zones . at the calyx of held from rat ,
presynaptic mitochondria are located within so - called swellings , where they are surrounded by active zones and a large population of svs thought to represent the rp ( wimmer et al . , 2006 ) .
in both types of calyces , this particular distribution of mitochondria relative to synaptic vesicles might contribute to trap atp and glutamate which is generated in mitochondria from precursors emanating from the krebs cycle or from glutamine produced by glial cells ( figure 1 ) in restricted areas , thereby facilitating vesicle refilling and mobilization , ensuring sustained synaptic transmission during intense neuronal activity .
in conclusion , although most processes involved in the sv cycle are known to be atp - dependent , experimental evidence point to a critical role of mitochondrial atp production mostly in the establishment of large sv pools and in sustained neurotransmitter release during intense neuronal activity , i.e. , for mobilization of rp vesicles and for vesicle recycling via endocytosis .
in contrast , low atp levels produced by glycolysis in the cytosol appear to be sufficient to fuel other steps of the sv cycle ( calupca et al .
, 2001 ) , and this is likely accomplished by optimal positioning of glycolytic enzymes close to the atp consumption sites ( ishida et al . , 2009 ) .
as described above , energy generation by mitochondria is of major importance for the sv cycle in neurons , and one can expect that disturbed atp production may have deleterious consequences for neurons in health and disease .
defects in most of the complexes of the etc ( figure 1 ) are indeed associated with different neurodegenerative diseases and numerous mutations in mitochondrial - associated proteins have been linked to a variety of neurological diseases ( ly and verstreken , 2006 ) .
the first evidence that mitochondria are involved in parkinson 's disease ( pd ) came from the observation that mitochondrial drugs like mptp and rotenone that specifically affect complex i of the etc ( figure 1 ) cause pd - like symptoms , including degeneration of dopaminergic neurons ( german et al .
it was later shown that mutations in the parkinson related gene pink1 similarly lead to a reduced complex i activity ( gautier et al . , 2008 ; morais et al . , 2009 ) .
the product of two other parkinson related genes , parkin and dj-1 , also appear to be involved in the regulation of mitochondrial functions ( bonifati et al . , 2003 ;
furthermore , amyotrophic lateral sclerosis ( als ) , leigh 's syndrome and multiple sclerosis can also be caused by defects in several complexes of the etc ( kalman et al .
, 2007 ; finsterer , 2008 ) . aside from direct etc dysfunction that leads to reduced atp production , disturbed mitochondrial transport to sites of high energy demand , including synapses , may also lead to local atp depletion and thus synaptic transmission deficits . as discussed before , drp1 , a regulator of mitochondrial fission appears to be a key regulatory switch controlling mitochondrial transport and thus synaptic atp production , possibly in response to calcium influx and neuronal activity ( lee et al . , 2004 ; verstreken et al .
while to date no inherited disease resulting directly from mutations in genes regulating fission are known ; increased levels of drp1 , and thus presumably also increased mitochondrial fission and synaptic transport has been implicated in alleviating parkinson disease - like phenotypes in flies , yet it is still unclear whether these effects are direct or not ( morais et al . , 2009 ; poole et al . ,
indeed , increased drp1 activity is predicted to augment synaptic atp production by concentrating mitochondria at active synapses , in turn facilitating sv mobilization . the converse process , mitochondrial fusion , results in lower mitochondrial membrane potential and thus reduced atp production ( chen and chan , 2005 ) and is associated with neurodegeneration .
three proteins are required for mitochondrial fusion : opa1 , mitofusin 1 ( mfn1 ) and mitofusin 2 ( mfn2 ) ( chen and chan , 2005 ) .
the latter two proteins are also required for axonal transport of mitochondria ( misko et al . , 2010 ) .
mfn2 has been identified as one of the genes causing charcot - marie - tooth type 2a ( cmt2a ) syndrome .
cmt2a is a neuropathy that affects sensory and motor neurons of the distal extremities and is characterized by progressive weakness of the muscles followed by muscular atrophy ( zuchner et al . , 2004 ) .
mutations in opa1 cause autosomal dominant optic atrophy ( doa ) , characterized by a degeneration of retinal ganglia cells leading to optic nerve atrophy ( alexander et al . , 2000 ;
it might seem that mutations in two genes encoding proteins involved in mitochondrial fusion cause two diseases with differential phenotypes . yet cmt2a patients have been described to suffer from optic atrophy ( banchs et al . , 2008 ) .
likewise , some doa patients express additional symptoms including peripheral motor - sensory neuropathy ( amati - bonneau et al . , 2008 ) .
linking these mitochondrial fusion related diseases to defects in neuronal function , the reduced atp levels as a consequence of less mitochondrial fusion may be responsible for defects in sv release and/or recycling under intense stimulation . taken together , these data support the hypothesis that atp production may play a critical role in neuronal communication during intense stimulation and that mitochondria play a specific role in regulating synaptic strength , suggesting that neurological disorders with defects in presynaptic mitochondrial function or mitochondrial transport also display abnormalities in the sv cycle .
one puzzling feature common to all the diseases discussed here is the selective tissue vulnerability associated with mitochondrial defects .
first of all , the particular mode of mitochondrial inheritance might contribute to the appearance of clinical manifestations only in a specific cell population , since stochastic distribution of mitochondria between daughter cells can lead to very different ratio of mutant to wild - type organelles after cytokinesis ( chan , 2006 ) .
such a genetic drift has indeed been observed for some mitochondrial encephalomyopathies where mitochondrial dna carrying the causative mutation accumulates in a specific region of the brain ( kurenai et al . , 2000 ;
this is most likely related to their very high energy demands and their specific architecture , as mitochondria must travel long distances from the cell body to reach regions of intense atp consumption like the presynaptic sites .
defective mitochondrial fusion and/or fission and hence altered mitochondria recruitment at the nerve terminals is thus particularly detrimental for these cells ( guo et al .
this is expected to be even more critical for neurons possessing a very long axon , like those innervating distal muscles that are affected in patients with cmt disease ( misko et al . , 2010 ) .
finally , in addition to this cellular context the tissue context might also influence the sensitivity of some neurons to mitochondrial dysfunction .
for example , dopaminergic neurons which are progressively lost in pd are innervated by glutamatergic neurons , likely making them more susceptible to glutamate excitotoxicity and hence to calcium overload ( caudle and zhang , 2009 ) . a defect in mitochondrial calcium handling capacity and/or bioenergetics would in this model predispose dopaminergic neurons to excitotoxic damage and be particularly detrimental for them compared to other cell types ( greene and greenamyre , 1996 ; nicholls et al . , 2007 ) .
we thus surmise that dysfunction or degeneration of specific neuronal populations in specific neurological diseases may be a consequence of both cell autonomous effects that inherently regulate mitochondrial function and cell non - autonomous effects , where the particular cellular context a neuron finds itself in may ( over- ) stress mitochondrial function . in conclusion
, there is now accumulating evidence that the capacity of presynaptic mitochondria to produce atp and to buffer ca is of major importance to maintain neuronal communication , at least in part by controlling svs mobilization and recycling .
mitochondrial dysfunctions linked to neurodegenerative diseases are thus likely to exert their effect at least in part through an alteration of the sv cycle . in the light of the literature
detailed above , it seems reasonable to speculate that disease - related mutations that eventually cause a reduction of mitochondrial atp production and calcium buffering capacity in the nerve terminals would negatively affect neurotransmission by preventing replenishment of the ecp and vesicle mobilization from the rp , as indeed observed in pink1 mutant drosophila where expression of a clinical mutant fails to rescue rp mobilization ( morais et al . , 2009 ) .
this model does not exclude that other mechanisms such as reactive oxygen species production or enhanced neuronal death are involved .
these aspects lie outside the scope of this review and have been excellently reviewed by others ( see for example yuan and yankner , 2000 ; benn and woolf , 2004 ; naoi et al . , 2009 ) .
further work is now needed to shed light on the exact implications of altered mitochondrial metabolism and dynamics in the pathogenesis of neuronal disorders , and hopefully to generate more efficient treatments for these diseases .
the authors declare the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | cell types rich in mitochondria , including neurons , display a high energy demand and a need for calcium buffering .
the importance of mitochondria for proper neuronal function is stressed by the occurrence of neurological defects in patients suffering from a great variety of diseases caused by mutations in mitochondrial genes . genetic and pharmacological evidence
also reveal a role of these organelles in various aspects of neuronal physiology and in the pathogenesis of neurodegenerative disorders .
yet the mechanisms by which mitochondria can affect neurotransmission largely remain to be elucidated . in this review
we focus on experimental data that suggest a critical function of synaptic mitochondria in the function and organization of synaptic vesicle pools , and in neurotransmitter release during intense neuronal activity .
we discuss how calcium handling , atp production and other mitochondrial mechanisms may influence synaptic vesicle pool organization and synaptic function .
given the link between synaptic mitochondrial function and neuronal communication , efforts toward better understanding mitochondrial biology may lead to novel therapeutic approaches of neurological disorders including alzheimer 's disease , parkinson 's disease , amyotrophic lateral sclerosis and psychiatric disorders that are at least in part caused by mitochondrial deficits . | Introduction
Calcium Buffering by Presynaptic Mitochondria Influences Exo- and Endocytosis of SVs
Mitochondrial ATP Production is Required for SV Mobilization and Recycling Under Intense Stimulation
Mitochondria and Disease
Conflict of Interest Statement | they are strikingly abundant in neurons and in particular at chemical synapses ( palay , 1956 ) where neuronal communication takes place . it has long been recognized that mitochondria are important for the maintenance of neuronal integrity and function , as diseases caused by a mitochondrial failure often display a neurodegenerative component affecting the central or peripheral nervous system ( dimauro and schon , 2008 ) . these observations suggest an intimate involvement of mitochondria in maintaining neuronal integrity , possibly in part by controlling neurotransmission , yet the exact function of these organelles in synaptic activity has long remained elusive . disorders are listed in this table along with their most characteristic phenotype , when known , their causative gene(s ) and the mitochondrial features reported to be defective . besides their predominant role in energy metabolism ( figure 1 ) , they are involved in many other cellular processes that are also relevant for neuronal function , including the regulation of calcium homeostasis and a form of programmed cell death ( or apoptosis ) . defects in all these functions as well as in mitochondrial fusion or fission are predicted to affect various aspects of neuronal physiology ( dimauro and schon , 2008 ; chen and chan , 2009 ) . however , unraveling the link between a mitochondrial defect and its consequences on neuronal function or survival has proven a complex task , mostly because different mitochondrial functions are interdependent ( see for example niemann et al . ( 6 ) mitochondria are also involved in calcium homeostasis , and ca ions can be sequestered in the matrix under the form of a reversible phosphate complex . this review focuses on the role of presynaptic mitochondria in the control of synaptic vesicle ( sv ) function . together , the readily releasable pool and the recycling pool , sometimes referred to as the exo - endo cycling pool ( ecp ) , typically represent about 1020% of the svs and are released under moderate or intense neuronal activity , whereas vesicles from the rp are recruited only upon high frequency stimulation ( figure 2 ) ( kuromi and kidokoro , 2005 ; rizzoli and betz , 2005 ) . because the synaptic vesicle cycle involves numerous atp - consuming steps and is tightly controlled by the cytosolic calcium concentration ( [ ca]cyto ) , vesicle cycling appears to be highly dependent on mitochondrial function . in the following sections we will review recent reports indicating how mitochondria can affect the distribution in pools and release properties of svs . in particular , we reexamine experimental evidence which together indicate that these organelles are essential for synaptic vesicle release under conditions of intense neuronal activity through vesicle mobilization from the rp . current studies dedicated to this link between mitochondria and the sv cycle will hopefully lead to new therapeutic strategies for the handling of neurological disorders characterized by a mitochondrial defect and which have a tremendous social impact , like alzheimer 's , huntington 's and parkinson 's diseases . ( 1 ) synaptic vesicles ( svs ) from the exo - endo cycling pool ( ecp ) participate in neurotransmitter release at active zones ( in red ) . ( 3 ) the atp produced by the mitochondria also plays an important role for preynaptic ca buffering in the endoplasmic reticulum ( er ) . in neurons ,
calcium signaling is involved in neurotransmitter release from the presynaptic terminals , in vesicle recycling via endocytosis and in long - term neuronal responses like neurite growth and degeneration , or modulation of synaptic strength ( ghosh and greenberg , 1995 ; zucker , 1999 ; jonas , 2006 ; yao et al . the mcu shows a very low affinity for calcium ions ( km estimated between 1 and 189 m in different mitochondrial preparations ) ( gunter and sheu , 2009 ) , that can be overcome by the proximity between mitochondria and the ca source , i.e. , 2004 )
( see also below ) , and in drosophila ca ions were found to disrupt the interaction between the motor protein kinesin and the miro gtpase , which is required for the transport of these organelles along microtubules ( figure 1 ) ( macaskill et al . in neurons , [ ca]cyto peaks stimulate sv exocytosis and hence neurotransmitter release . this role of mitochondria in ca buffering is prominent in chromaffin cells ( montero et al . stimulation of sv exocytosis by cytosolic calcium ions is also at the heart of the so - called residual calcium hypothesis , according to which residual calcium ions present in the cytoplasm during the decay of the [ ca]cyto peak after each action potential increase the probability of neurotransmitter release ( katz and miledi , 1968 ) . this has been well described at the crayfish nmj , where inhibition of mitochondrial ca uptake leads to a higher [ ca]cyto during tetanic stimulation , as in the work from the groups of forsythe and barrett described above , and increases short - term augmentation ( tang and zucker , 1997 ) . recently , the importance of mitochondria in controlling presynaptic [ ca]cyto and hence in synaptic plasticity has been illustrated in mammals as well . defective calcium buffering at mouse hippocampal presynaptic terminals , resulting from a lack of the microtubule - binding protein syntaphilin required for axonal docking of mitochondria ( figure 1 ) , was shown to cause an elevation of [ ca]cyto during intense stimulation , prolonging the facilitation phenomenon observed in control neurons ( kang et al . in short , a second consequence of calcium buffering by mitochondria is that neurotransmitter release can be prolonged after the stimulus end as ca ions are progressively unloaded from the mitochondrial matrix into the cytosol . this apparent discrepancy is most likely due to the use of different stimulation protocols in various model systems , and to the different approaches used to observe calcium buffering , either via mobile calcium buffers ( dittman and regehr , 1998 ; wang and kaczmarek , 1998 ) or specifically by the mitochondria ( billups and forsythe , 2002 ; david and barrett , 2003 ) . more importantly , this controversy also stresses the versatile role of calcium in the sv cycle . the molecular mechanism of this regulation is only starting to be elucidated , but it appears that the calcium - binding protein calmodulin initiates endocytosis at the synapse and facilitates both plasma membrane invagination and fission ( wu et al . the overall effect of ca ions on neurotransmitter release should thus be regarded as a balance between the stimulation of sv exocytosis , which can promote either recovery from depression or sv depletion depending on the experimental conditions , and a positive effect on sv recycling . yet a reduced ca - buffering capacity of synaptic mitochondria , caused under pathogenic conditions or by aging , may presumably increase their susceptibility to mpt and subsequent release of cell - death molecules ( see below ) , leading to synapse degeneration as observed e.g. , in alzheimer 's disease ( selkoe , 2002 ) , schizophrenia ( glantz et al . , 2010
, the drosophila nmj mitochondrial contribution to ca buffering appears to be only minor and/or easily compensated by alternative mechanisms , at least during mild stimulation ( guo et al . , 2006 ) ,
calcium storage in the er lumen contributes to evoked neurotransmitter release at the drosophila nmj , supposedly by allowing the [ ca]cyto to remain elevated after the stimulus end ( sanyal et al . in this context , it is worth noting that a very close physical connection between the er and mitochondria can be observed in many cell types , including neurons ( mironov and symonchuk , 2006 ) . in mouse respiratory neurons , where both the er and mitochondria contribute to calcium - dependent modulation of synaptic activity , some of the ca ions transiently accumulated within er vesicles in response to a stimulus are subsequently transferred to the mitochondrial matrix ( mironov and symonchuk , 2006 ) . given the low affinity of the mitochondrial calcium uptake system , mams may thus play a critical role in the capacity of mitochondria to promptly respond to a change in [ ca]cyto and thereby affect many aspects of mitochondrial biology ( hayashi et al . three enzymes of the krebs cycle residing in the mitochondrial matrix are indeed activated in a calcium - dependent manner ( duchen , 1992 ) , so that mitochondrial calcium uptake increases the flux of electrons through the electron transport chain ( etc ) and hence atp production ( figure 1 ) ( graier et al . as a consequence
, drugs preventing mitochondrial calcium uptake also negatively affect atp production , thereby complicating the interpretation of experimental data . the most well - known function of mitochondria is to produce energy , and in neurons they generate the majority of atp ( about 90 % ) via aerobic metabolism ( figure 1 ) , the rest being provided by glycolysis in the cytosol ( mathews and van holde , 1995 ) . this energy supply is critical since proper neuronal function requires atp in order to fuel ion pumps , to organize cytoskeletal components , to support numerous phosphorylation reactions ( attwell and laughlin , 2001 ) and for many steps in the sv cycle including myosin - dependent axonal transport of svs , their snare - mediated fusion with the plasma membrane , membrane scission , vesicle uncoating and refilling of vesicles with neurotransmitter ( murthy and camilli , 2003 ) . because of the highly specialized architecture of neurons , with their long processes contacting cells located far away from the cell body , atp diffusion from a central location to the distal synapses is thought to be too slow to meet these energy requirements ( kuiper et al . accordingly , in neurons mitochondrial biogenesis occurs mostly in the soma ( davis and clayton , 1996 ) but these organelles can subsequently be transported along cytoskeleton tracks in the neuronal processes ( figure 1 ) ( hollenbeck and saxton , 2005 ) , where they show saltatory bidirectional movement , with frequent pauses and changes of direction ( cai and sheng , 2009 ) . mitochondria are then specifically retained in regions of the axon where the energy demand is high ( hollenbeck , 2005 ) , including synapses ( palay , 1956 ; nguyen et al . moreover , there is a positive correlation between anterograde axonal transport of mitochondria and the electrical potential across their inner membrane ( miller and sheetz , 2004 ) which drives atp production ( alfonzo and racker , 1979 ) ( figure 1 ) , and mitochondria with a high potential tend to accumulate at synapses ( lee and peng , 2006 ) . finally , there appears to be a direct relationship between the frequency and/or the intensity of activity in a given synapse and the number and metabolic activity of presynaptic mitochondria , both under physiological and pathological conditions ( atwood et al . all these observations point to a central role of mitochondrial atp production in neuronal transmission , but the exact molecular mechanisms involved in this regulation are only beginning to be deciphered . high ca leads to calcineurin activation ( guerini , 1997 ) and in turn to dephosphorylation and activation of drp1 and thus mitochondrial fission , which has been associated with the transport of mitochondria into the synapse ( verstreken et al . ,
, these data support a model where dynamic mitochondria distribution in neurons is adjusted to meet the energy requirement for proper neuronal communication . at drosophila
nmj synapses , svs can be assigned to different , but spatially mixed functional vesicle pools , the ecp and rp ( kuromi and kidokoro , 2005 ) . because both the sesb and drp1 mutations enhance the phenotype of the paralytic mutant shibire , where endocytosis is known to be blocked at the restrictive temperature ( hinshaw and schmid , 1995 ) , one possibility proposed by the authors is that reduced atp levels could affect endocytosis , thereby preventing efficient sv recycling ( rikhy et al . remarkably , in the absence of presynaptic mitochondria in drp1 or miro ( mitochondrial rho - gtpase ) loss - of - function mutant flies basal synaptic activity at the drosophila neuromuscular junction ( nmj ) appears not to be significantly altered . ,
2005 ) , suggesting that energy production by presynaptic mitochondria is essential only during intense neuronal activity . failure of the drp1 mutant to sustain neuronal activity during intense stimulation is due to a defect in rp mobilization , while basal exocytosis and endocytosis of ecp vesicles is unaffected ( verstreken et al . similar effects on rp vesicle mobilization are observed at wild - type nmjs treated with oligomycin , an inhibitor of the mitochondrial atp synthase ( figure 1 ) , and forward - filling drp1 motor neurons with atp restores the vesicle trafficking defects in the rp ( verstreken et al . in particular , atp appears to be needed for rp vesicle propulsion along actin filaments mediated by the myosin atpase complex ( verstreken et al . a very similar dependence of neurotransmission on mitochondrial atp production has recently been observed in mammalian neurons , where anterograde transport of mitochondria along the axon relies on the adaptor protein syntabulin , linking these organelles to the kinesin heavy chain ( figure 1 ) ( cai et al . interestingly , in rat hippocampal neurons the gtpase activity of drp1 appears to be positively regulated by the antiapoptotic protein bcl - xl ( li et al . this is likely to be attributed to increased atp release from mitochondria since , at least at the squid giant synapse , the positive effects of bcl - xl overexpression on neurotransmission can be mimicked by atp injection ( jonas et al . compared to other mitochondria in the same neuron , mac mitochondria display a higher density of crista - membranes , suggesting increased metabolic activity ( perkins et al . in both types of calyces , this particular distribution of mitochondria relative to synaptic vesicles might contribute to trap atp and glutamate which is generated in mitochondria from precursors emanating from the krebs cycle or from glutamine produced by glial cells ( figure 1 ) in restricted areas , thereby facilitating vesicle refilling and mobilization , ensuring sustained synaptic transmission during intense neuronal activity . in conclusion , although most processes involved in the sv cycle are known to be atp - dependent , experimental evidence point to a critical role of mitochondrial atp production mostly in the establishment of large sv pools and in sustained neurotransmitter release during intense neuronal activity , i.e. in contrast , low atp levels produced by glycolysis in the cytosol appear to be sufficient to fuel other steps of the sv cycle ( calupca et al . as described above , energy generation by mitochondria is of major importance for the sv cycle in neurons , and one can expect that disturbed atp production may have deleterious consequences for neurons in health and disease . defects in most of the complexes of the etc ( figure 1 ) are indeed associated with different neurodegenerative diseases and numerous mutations in mitochondrial - associated proteins have been linked to a variety of neurological diseases ( ly and verstreken , 2006 ) . the first evidence that mitochondria are involved in parkinson 's disease ( pd ) came from the observation that mitochondrial drugs like mptp and rotenone that specifically affect complex i of the etc ( figure 1 ) cause pd - like symptoms , including degeneration of dopaminergic neurons ( german et al . it was later shown that mutations in the parkinson related gene pink1 similarly lead to a reduced complex i activity ( gautier et al . the product of two other parkinson related genes , parkin and dj-1 , also appear to be involved in the regulation of mitochondrial functions ( bonifati et al . , 2003 ;
furthermore , amyotrophic lateral sclerosis ( als ) , leigh 's syndrome and multiple sclerosis can also be caused by defects in several complexes of the etc ( kalman et al . aside from direct etc dysfunction that leads to reduced atp production , disturbed mitochondrial transport to sites of high energy demand , including synapses , may also lead to local atp depletion and thus synaptic transmission deficits . as discussed before , drp1 , a regulator of mitochondrial fission appears to be a key regulatory switch controlling mitochondrial transport and thus synaptic atp production , possibly in response to calcium influx and neuronal activity ( lee et al . while to date no inherited disease resulting directly from mutations in genes regulating fission are known ; increased levels of drp1 , and thus presumably also increased mitochondrial fission and synaptic transport has been implicated in alleviating parkinson disease - like phenotypes in flies , yet it is still unclear whether these effects are direct or not ( morais et al . , 2000 ;
it might seem that mutations in two genes encoding proteins involved in mitochondrial fusion cause two diseases with differential phenotypes . linking these mitochondrial fusion related diseases to defects in neuronal function , the reduced atp levels as a consequence of less mitochondrial fusion may be responsible for defects in sv release and/or recycling under intense stimulation . taken together , these data support the hypothesis that atp production may play a critical role in neuronal communication during intense stimulation and that mitochondria play a specific role in regulating synaptic strength , suggesting that neurological disorders with defects in presynaptic mitochondrial function or mitochondrial transport also display abnormalities in the sv cycle . first of all , the particular mode of mitochondrial inheritance might contribute to the appearance of clinical manifestations only in a specific cell population , since stochastic distribution of mitochondria between daughter cells can lead to very different ratio of mutant to wild - type organelles after cytokinesis ( chan , 2006 ) . this is expected to be even more critical for neurons possessing a very long axon , like those innervating distal muscles that are affected in patients with cmt disease ( misko et al . a defect in mitochondrial calcium handling capacity and/or bioenergetics would in this model predispose dopaminergic neurons to excitotoxic damage and be particularly detrimental for them compared to other cell types ( greene and greenamyre , 1996 ; nicholls et al . we thus surmise that dysfunction or degeneration of specific neuronal populations in specific neurological diseases may be a consequence of both cell autonomous effects that inherently regulate mitochondrial function and cell non - autonomous effects , where the particular cellular context a neuron finds itself in may ( over- ) stress mitochondrial function . in conclusion
, there is now accumulating evidence that the capacity of presynaptic mitochondria to produce atp and to buffer ca is of major importance to maintain neuronal communication , at least in part by controlling svs mobilization and recycling . mitochondrial dysfunctions linked to neurodegenerative diseases are thus likely to exert their effect at least in part through an alteration of the sv cycle . in the light of the literature
detailed above , it seems reasonable to speculate that disease - related mutations that eventually cause a reduction of mitochondrial atp production and calcium buffering capacity in the nerve terminals would negatively affect neurotransmission by preventing replenishment of the ecp and vesicle mobilization from the rp , as indeed observed in pink1 mutant drosophila where expression of a clinical mutant fails to rescue rp mobilization ( morais et al . further work is now needed to shed light on the exact implications of altered mitochondrial metabolism and dynamics in the pathogenesis of neuronal disorders , and hopefully to generate more efficient treatments for these diseases . | [
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fatigue is a common complaint of 33% patients presenting in the ambulatory primary care offices in the usa .
the prevalence of fatigue in the usa workforce has been estimated as 37.9% in a 2-week period .
although fatigue is defined as a state of inability to maintain or sustain a force , nevertheless it is interchangeably used by the patients to describe a state of tiredness or low energy .
it has significant negative impact on social life , family life , and work performance .
excluding advanced systemic illness ( e.g. , malignancy ) , major organ dysfunction ( e.g. , stroke , congestive heart failure , chronic obstructive airway disorder , end stage liver disease , severe anemia , myxedema , end stage renal disease , advanced rheumatologic disease , etc . ) , or acute illness ( e.g. , infection , psychiatric illness ) ; most patients with fatigue have no obvious cause .
a detailed diagnostic work up of fatigue can find a specific cause in only 5% .
studies have shown an association of low vitamin d levels and fatigue in cancer patients and in patients with myasthenia gravis ; and improvement in fatigue symptom scores after normalization of vitamin d levels .
there are many reports which indicate high prevalence of low vitamin d in the general population .
low vitamin d leads to bone abnormalities ( e.g. , osteomalacia , osteopenia , and osteoporosis ) and worsens muscle strength . in otherwise healthy individuals ,
fatigue can be a manifestation of low vitamin d levels and its impact on reduced maximum functioning of skeletal muscles via vitamin d receptors .
we designed this study to observe the effect of correction of low vitamin d in patients with fatigue and stable comorbid medical condition(s ) , if any .
this study was a prospective non - randomized therapeutic study that observed the effect of correction of low vitamin d in fatigue scores of the patients who presented with fatigue , stable comorbid medical condition(s ) if any , and low serum 25-hydroxyvitamin d ( 25-ohd ) levels .
patient enrolment started in may 2012 and the follow - up completed in november 2013 .
the study was reviewed and approved by the institutional review board of the cooper health system , camden , new jersey , usa .
adult patients between the ages of 18 and 75 who presented in our internal medicine office with fatigue as their chief complaint volunteered to enroll in the study .
we excluded patients over age 75 due to several factors that are known to contribute fatigue in this age group such as , decline in the functional ability , physical health , sensorycapacity , cognitive and mental health , and socio - environmental limitations .
furthermore , studies have shown a strong association between age greater than 75 and functional decline , and fatigue .
the inclusion criteria were fatigue present for greater than 4 weeks and stable chronic medical condition(s ) , if any .
the exclusion criteria were normal level of serum 25-ohd , active or advanced cancer , advanced neurological condition ( e.g. , cerebrovascular disease , degenerative neurological disease , multiple sclerosis , etc . ) , advanced cardiac condition with poor performance state ( e.g. , nyha ii - iv congestive heart failure , left ventricular ejection fraction of less than 40% ) , advanced pulmonary disorder ( e.g. , chronic obstructive or restrictive airway disease requiring ambulatory oxygen therapy ) , chronic kidney disease ( e.g. , end stage renal disease requiting hemodialysis ) , end stage liver disease ( e.g. , cirrhosis ) , hematological disorders leading to severe anemia ( hemoglobin less than 9.0 g / dl ) , uncontrolled endocrine disorder ( e.g. , uncontrolled hypothyroidism or hyperthyroidism , diabetes mellitus with glycosylated hemoglobin greater than 9% , etc . ) , advanced or uncontrolled rheumatologic disorder ( e.g. , rheumatoid arthritis , systemic lupus erythematosus , osteoarthritis , etc . ) , chronic fatigue syndrome , fibromyalgia , advanced multisystem disorder ( e.g. , sarcoidosis ) , pregnancy , nursing mother , inability to adhere with the instructions of the intervention ( e.g. , developmental disorder , mentally challenged , severe dementia , non - compliance , etc . ) , contraindication to vitamin d therapy ( e.g. , hypercalcemia ) , and established sleep disorder . after obtaining informed consent
the study physicians asked patients to complete a fatigue scale assessment questionnaire ( multidimensional fatigue symptom inventory- short form , mfsi - sf ) . patients
all patients with low serum 25-ohd levels ( less than 30 ng / ml ) received oral vitamin d ( ergocalciferol 50,000 usp international unit ; manufactured by sigmapharm laboratories , llc , bensalem , pennsylvania ) three times per week for 5 weeks , if not contraindicated .
after completion of ergocalciferol therapy all patients were asked to complete the mfsi - sf and their serum 25-ohd were retested to ensure normalization .
we collected the following data for each patient : age , gender , race , associated stable medical condition(s ) , use of over the counter supplemental vitamin d if any , pulse rate , blood pressure , mfsi - sf score before treatment , serum 25-ohd level ( ng / ml ) before treatment , mfsi - sf score after treatment , serum 25-ohd level ( ng / ml ) after treatment , and adverse effect(s ) of ergocalciferol therapy .
we entered the patient data in microsoft excel 2007 spreadsheet , and analyzed using statistical package for the social sciences ( spss ) ( version 15.01 ) .
a total of 116 patients were planned for sequential sampling who presented with fatigue and low serum 25-ohd levels in order to achieve the largest sample that would provide 80% power to the study hypotheses .
we used wilcoxon paired test ( nonparametric analog of the paired t test ) to calculate the median difference on pre and post treatment serum 25-ohd levels assuming the distribution of difference of scores is not normal .
al - sunduqchi ( 1990 ) showed that sample size and power calculations for the wilcoxon test can be made using the standard t test results with a simple adjustment to the sample size .
a sample size of 116 achieved 84% power to detect a 50% relative increase in serum 25-ohd levels over an average baseline of 10 ng / ml ( + 5 ng / ml ) with a standard deviation of 10 ng / ml using a two - sided wilcoxon test with p 0.05 defining statistical significance and assuming that the actual distribution of post - pre differences is uniformly distributed over subjects .
it should be noted that once the data was collected , we found that it was normally distributed and so for analysis , the paired t test was used to compare the mean of vitamin d levels and fatigue scores pre and post - intervention except for the comparison between pre and post - intervention physical score and total score .
paired t test was used to compare the mean pre- and post - intervention vitamin d levels , and fatigue scores except the physical scale .
wilcoxon ranked sum test was used to compare the median pre- and post - intervention physical scale scores and total scores .
independent t test was used to compare continuous variables between the normal and abnormal ( low ) vitamin d group .
pearson chi square and fisher exact tests were used to compare categorical variables between the normal and abnormal ( low ) vitamin d group .
correlations were used to assess the relationship between pre - intervention vitamin d levels with the pre - intervention fatigue scores , and the relationship between post - intervention vitamin d levels with the post - intervention fatigue scores .
multivariable logistic regression was applied to assess the relationship between having a normal post - intervention serum 25-ohd level and change on total mfsi - sf adjusting for the association for the effects of demographics and stable medical condition(s ) that were significant at the p 0.2 level .
the variables that were placed in the model were race , depression , and use of over - the - counter supplemental vitamin d.
this study was a prospective non - randomized therapeutic study that observed the effect of correction of low vitamin d in fatigue scores of the patients who presented with fatigue , stable comorbid medical condition(s ) if any , and low serum 25-hydroxyvitamin d ( 25-ohd ) levels .
patient enrolment started in may 2012 and the follow - up completed in november 2013 .
the study was reviewed and approved by the institutional review board of the cooper health system , camden , new jersey , usa .
adult patients between the ages of 18 and 75 who presented in our internal medicine office with fatigue as their chief complaint volunteered to enroll in the study .
we excluded patients over age 75 due to several factors that are known to contribute fatigue in this age group such as , decline in the functional ability , physical health , sensorycapacity , cognitive and mental health , and socio - environmental limitations .
furthermore , studies have shown a strong association between age greater than 75 and functional decline , and fatigue .
the inclusion criteria were fatigue present for greater than 4 weeks and stable chronic medical condition(s ) , if any .
the exclusion criteria were normal level of serum 25-ohd , active or advanced cancer , advanced neurological condition ( e.g. , cerebrovascular disease , degenerative neurological disease , multiple sclerosis , etc . ) , advanced cardiac condition with poor performance state ( e.g. , nyha ii - iv congestive heart failure , left ventricular ejection fraction of less than 40% ) , advanced pulmonary disorder ( e.g. , chronic obstructive or restrictive airway disease requiring ambulatory oxygen therapy ) , chronic kidney disease ( e.g. , end stage renal disease requiting hemodialysis ) , end stage liver disease ( e.g. , cirrhosis ) , hematological disorders leading to severe anemia ( hemoglobin less than 9.0 g / dl ) , uncontrolled endocrine disorder ( e.g. , uncontrolled hypothyroidism or hyperthyroidism , diabetes mellitus with glycosylated hemoglobin greater than 9% , etc . ) , advanced or uncontrolled rheumatologic disorder ( e.g. , rheumatoid arthritis , systemic lupus erythematosus , osteoarthritis , etc . ) , chronic fatigue syndrome , fibromyalgia , advanced multisystem disorder ( e.g. , sarcoidosis ) , pregnancy , nursing mother , inability to adhere with the instructions of the intervention ( e.g. , developmental disorder , mentally challenged , severe dementia , non - compliance , etc . ) , contraindication to vitamin d therapy ( e.g. , hypercalcemia ) , and established sleep disorder .
after obtaining informed consent the study physicians asked patients to complete a fatigue scale assessment questionnaire ( multidimensional fatigue symptom inventory- short form , mfsi - sf ) . patients
all patients with low serum 25-ohd levels ( less than 30 ng / ml ) received oral vitamin d ( ergocalciferol 50,000 usp international unit ; manufactured by sigmapharm laboratories , llc , bensalem , pennsylvania ) three times per week for 5 weeks , if not contraindicated .
after completion of ergocalciferol therapy all patients were asked to complete the mfsi - sf and their serum 25-ohd were retested to ensure normalization .
we collected the following data for each patient : age , gender , race , associated stable medical condition(s ) , use of over the counter supplemental vitamin d if any , pulse rate , blood pressure , mfsi - sf score before treatment , serum 25-ohd level ( ng / ml ) before treatment , mfsi - sf score after treatment , serum 25-ohd level ( ng / ml ) after treatment , and adverse effect(s ) of ergocalciferol therapy .
we entered the patient data in microsoft excel 2007 spreadsheet , and analyzed using statistical package for the social sciences ( spss ) ( version 15.01 ) .
a total of 116 patients were planned for sequential sampling who presented with fatigue and low serum 25-ohd levels in order to achieve the largest sample that would provide 80% power to the study hypotheses .
we used wilcoxon paired test ( nonparametric analog of the paired t test ) to calculate the median difference on pre and post treatment serum 25-ohd levels assuming the distribution of difference of scores is not normal .
al - sunduqchi ( 1990 ) showed that sample size and power calculations for the wilcoxon test can be made using the standard t test results with a simple adjustment to the sample size .
a sample size of 116 achieved 84% power to detect a 50% relative increase in serum 25-ohd levels over an average baseline of 10 ng / ml ( + 5 ng / ml ) with a standard deviation of 10 ng / ml using a two - sided wilcoxon test with p 0.05 defining statistical significance and assuming that the actual distribution of post - pre differences is uniformly distributed over subjects .
it should be noted that once the data was collected , we found that it was normally distributed and so for analysis , the paired t test was used to compare the mean of vitamin d levels and fatigue scores pre and post - intervention except for the comparison between pre and post - intervention physical score and total score .
paired t test was used to compare the mean pre- and post - intervention vitamin d levels , and fatigue scores except the physical scale .
wilcoxon ranked sum test was used to compare the median pre- and post - intervention physical scale scores and total scores .
independent t test was used to compare continuous variables between the normal and abnormal ( low ) vitamin d group .
pearson chi square and fisher exact tests were used to compare categorical variables between the normal and abnormal ( low ) vitamin d group .
correlations were used to assess the relationship between pre - intervention vitamin d levels with the pre - intervention fatigue scores , and the relationship between post - intervention vitamin d levels with the post - intervention fatigue scores .
multivariable logistic regression was applied to assess the relationship between having a normal post - intervention serum 25-ohd level and change on total mfsi - sf adjusting for the association for the effects of demographics and stable medical condition(s ) that were significant at the p 0.2 level .
the variables that were placed in the model were race , depression , and use of over - the - counter supplemental vitamin d.
a total of 174 patients with fatigue completed mfsi - sf fatigue assessment questionnaires . of them , 171 ( 98.3% ) patients underwent serum 25-ohd level tests while 3 ( 1.7% ) did not .
thirty - nine ( 22.8% ) patients were excluded due to normal levels of serum 25-ohd , and the rest 132 ( 77.2% ) patients received vitamin d therapy [ figure 1 ] .
the prevalence of low vitamin d was 77.2% in all 171 patients , despite the fact that 51.5% patients with low vitamin d levels were regularly taking over - the - counter vitamin d-3 between 1000 and 2000 international units [ table 1 ] .
majority of the patients were in the age range of 36 and 65 [ figure 2 ] , female ( 72.4% ) , and non - hispanic caucasians ( 59.6% ) [ table 1 ] .
demographics of 171 patients showed a higher prevalence of low vitamin d in the african - american patients ( 88.6% ) and in the other race category ( asian and hispanic patients ) ( 85.3% ) , compared to 70.6% in the non - hispanic caucasian patients .
there was no significant difference between the number of patients taking over - the - counter vitamin d-3 in low vitamin d group ( 51.5% ) and normal vitamin d group ( 64.1% ) ( p = 0.166 ) .
other factors , such as associated stable comorbid conditions , pulse and blood pressure between low vitamin d group and normal vitamin d group were comparable , except for the history of depression .
patients with history of depression were twice likely to have a normal vitamin d level than a low vitamin d level [ table 1 ] .
after completion of vitamin d therapy 16 ( 12.1% ) patients did not go for serum 25-ohd test ; hence , they were excluded from the study [ figure 1 ] .
frequency of fatigue through age ranges , gender , and vitamin d levels ( ldf = low serum 25-ohd female , ldm = low serum 25-ohd male , ndf = normal serum 25-ohd female , ndm = normal serum 25-ohd male , nrf = did not go for serum 25-ohd test female , nrm = did not go for serum 25-ohd test male ) the mfsi - sf is a 30-item form that provides scores in the empirically derived 5 subscale categories .
the assessment of fatigue is based on scores on the items that are rated on a 5-point scale indicating patients agreement to the truthfulness of each statement asking about their experience during the previous week ( 0 = not at all , to 4 = extremely ) .
the mfsi - sf has excellent psychometric properties , hence it is an effective substitute for a more time consuming 83-item self - report measure ( multidimensional fatigue symptom inventory ) .
the questions are randomly arranged to assess five different subscales , which include general scale , physical scale , emotional scale , mental scale , and vigor scale .
each scale has six questions , hence a possible maximum score for each subscale is 24 .
a high level of fatigue is indicated by high scores in general , physical , emotional , and mental scales , and low score in vigor scale .
the total score is calculated by subtracting the score of vigor scale from the sum of the scores of all other scales .
. there was no statistically significant difference in the mean mfsi - sf scores in the general scale , emotional scale , mental scale and vigor scale ; and the median mfsi - sf scores in the physical scale and total score between all 171 patients and 116 patients with low vitamin d levels at initial encounter [ table 2 ] .
there was no significant correlation between the baseline vitamin d levels and baseline mfsi - sf scores in 171 patients ; and post vitamin d therapy normalized vitamin d levels and mfsi - sf scores in 116 patients .
pearson correlation was used to calculate all except for the vitamin d levels and mfsi - sf total scores , for which spearman rho correlation was used .
after 5 weeks of vitamin d therapy in 116 patients , 111 ( 95.7% ) patients achieved normal levels of serum 25-ohd and 5 ( 4.3% ) patients required a second course of vitamin d therapy to achieve normal levels of serum 25-ohd .
after ergocalciferol therapy mean serum 25-ohd level of all 116 patients normalized and significantly improved from a mean pre - treatment level of 19.71 ( sd , 5.98 ) ng / ml to a mean post - treatment level of 52.29 ( sd , 21.73 ) ng / ml ( p < 0.001 ) [ table 2 ] .
fatigue scores : multidimensional fatigue symptom inventory - short form mean mfsi - sf scores in the general scale , emotional scale , mental scale , and vigor scale ; and the median mfsi - sf scores in the physical scale and total score improved significantly ( p < 0.001 ) after normalization of vitamin d levels [ table 2 ] .
the multivariate analysis showed that history of depression was the only significant factor that impacted the post - intervention vitamin d level .
patients who had a history of depression had 12 times greater odds of having a normal vitamin d level than those who did not have a history of depression ( 95% ci , 1.7 to 88 times ) .
the reasons of discontinuation and consequent exclusion from the study were non - willingness for continued participation in 3 ( 1.7% ) patients , inability to go for serum 25-ohd test due to loss of medical insurance coverage in 7 ( 6.0% ) patients , and protocol violation due to a significant delay in getting serum 25-ohd test in 5 ( 3.8% ) patients .
majority of the patients were in the age range of 36 and 65 [ figure 2 ] , female ( 72.4% ) , and non - hispanic caucasians ( 59.6% ) [ table 1 ] .
demographics of 171 patients showed a higher prevalence of low vitamin d in the african - american patients ( 88.6% ) and in the other race category ( asian and hispanic patients ) ( 85.3% ) , compared to 70.6% in the non - hispanic caucasian patients .
there was no significant difference between the number of patients taking over - the - counter vitamin d-3 in low vitamin d group ( 51.5% ) and normal vitamin d group ( 64.1% ) ( p = 0.166 ) .
other factors , such as associated stable comorbid conditions , pulse and blood pressure between low vitamin d group and normal vitamin d group were comparable , except for the history of depression .
patients with history of depression were twice likely to have a normal vitamin d level than a low vitamin d level [ table 1 ] .
after completion of vitamin d therapy 16 ( 12.1% ) patients did not go for serum 25-ohd test ; hence , they were excluded from the study [ figure 1 ] .
frequency of fatigue through age ranges , gender , and vitamin d levels ( ldf = low serum 25-ohd female , ldm = low serum 25-ohd male , ndf = normal serum 25-ohd female , ndm = normal serum 25-ohd male , nrf = did not go for serum 25-ohd test female , nrm = did not go for serum 25-ohd test male )
the mfsi - sf is a 30-item form that provides scores in the empirically derived 5 subscale categories .
the assessment of fatigue is based on scores on the items that are rated on a 5-point scale indicating patients agreement to the truthfulness of each statement asking about their experience during the previous week ( 0 = not at all , to 4 = extremely ) .
the mfsi - sf has excellent psychometric properties , hence it is an effective substitute for a more time consuming 83-item self - report measure ( multidimensional fatigue symptom inventory ) .
the questions are randomly arranged to assess five different subscales , which include general scale , physical scale , emotional scale , mental scale , and vigor scale .
each scale has six questions , hence a possible maximum score for each subscale is 24 .
a high level of fatigue is indicated by high scores in general , physical , emotional , and mental scales , and low score in vigor scale .
the total score is calculated by subtracting the score of vigor scale from the sum of the scores of all other scales .
a high total score indicates high level of fatigue . there was no statistically significant difference in the mean mfsi - sf scores in the general scale , emotional scale , mental scale and vigor scale ; and the median mfsi - sf scores in the physical scale and total score between all 171 patients and 116 patients with low vitamin d levels at initial encounter [ table 2 ] .
there was no significant correlation between the baseline vitamin d levels and baseline mfsi - sf scores in 171 patients ; and post vitamin d therapy normalized vitamin d levels and mfsi - sf scores in 116 patients .
pearson correlation was used to calculate all except for the vitamin d levels and mfsi - sf total scores , for which spearman rho correlation was used .
after 5 weeks of vitamin d therapy in 116 patients , 111 ( 95.7% ) patients achieved normal levels of serum 25-ohd and 5 ( 4.3% ) patients required a second course of vitamin d therapy to achieve normal levels of serum 25-ohd .
after ergocalciferol therapy mean serum 25-ohd level of all 116 patients normalized and significantly improved from a mean pre - treatment level of 19.71 ( sd , 5.98 ) ng / ml to a mean post - treatment level of 52.29 ( sd , 21.73 ) ng / ml ( p < 0.001 ) [ table 2 ] .
mean mfsi - sf scores in the general scale , emotional scale , mental scale , and vigor scale ; and the median mfsi - sf scores in the physical scale and total score improved significantly ( p < 0.001 ) after normalization of vitamin d levels [ table 2 ] .
the multivariate analysis showed that history of depression was the only significant factor that impacted the post - intervention vitamin d level .
patients who had a history of depression had 12 times greater odds of having a normal vitamin d level than those who did not have a history of depression ( 95% ci , 1.7 to 88 times ) .
the reasons of discontinuation and consequent exclusion from the study were non - willingness for continued participation in 3 ( 1.7% ) patients , inability to go for serum 25-ohd test due to loss of medical insurance coverage in 7 ( 6.0% ) patients , and protocol violation due to a significant delay in getting serum 25-ohd test in 5 ( 3.8% ) patients .
this prospective study of low vitamin d and fatigue demonstrated two major observations : first , a very high prevalence of low vitamin d ( serum 25-ohd ) in patients who complained of fatigue .
second , a significant reduction in the severity of fatigue ( fatigue symptom scores ) after normalization of vitamin d levels .
our strict exclusion criteria supported the observation that improvement in fatigue symptoms were primarily due to normalization of low vitamin d levels and not secondary to amelioration of the chronic medical condition(s ) in patients who had one or more associated stable comorbid condition(s ) .
patients who suffer from fatigue have higher mortality , morbidity , and disability especially after middle age .
it facilitates the absorption of calcium from the intestine , reabsorption of calcium from the kidneys , and release of calcium from the skeleton .
a low vitamin d level reduces serum total and ionized calcium which results into increase in parathormone level , decrease in bone density , increase in bone turnover , decrease in urinary calcium excretion , increase in urinary phosphate excretion , and decrease in serum phosphate level , ultimately resulting into skeletal demineralization and muscle weakness .
fatigue is the end result of the underlying muscle fatigue , which is more commonly encountered than muscle weakness .
low vitamin d is also associated with inappropriate activation of the renin - angiotensin system , which fails to inhibit abnormal cell proliferation .
additionally , vitamin d influences our innate immune system by enhancing the production of human cathelicidin antimicrobial peptide , and modulates the adaptive immune system by controlling cytokine responses and t helper cell balance . the overall impact of these mechanisms results into further lowering of vitamin d levels , which have been associated with fatigue in patients with cancer , and myasthenia gravis .
low vitamin d levels are also associated with cardiovascular diseases , hypertension , congestive heart failure , cancer , diabetes mellitus , neuromuscular disorders , pneumonia , upper respiratory tract infections , chronic obstructive pulmonary disease , and all - cause mortality .
a meta - analysis of 18 randomized clinical trials concluded that vitamin d supplementation was associated with lower all - cause mortality . a survey of over 18,000 subjects in the united states general population showed 6.7% current and 24.4% lifetime prevalence of fatigue .
although fatigue is twice as common in women as in men , it is not strongly associated with age or occupation .
more recent analysis of 74 million adults in the united states aged 51 and above showed 31.2% prevalence of fatigue , being more in women ( 33.3% ) than men ( 28.6% ) and more in minorities ( 47.5% ) than in caucasians ( 27.5% ) . in our study
we found that fatigue was more common in non - hispanic caucasians ( 59.6% ) , and it affected about three - quarter ( 72.4% ) of women than men . in the usa , 41% of men and 53% of women
we observed a higher prevalence of low vitamin d ( 77.2% ) in our patients who presented with fatigue , despite the fact that more than half of the patients were taking over - the - counter vitamin d-3 regularly .
low vitamin d may be caused by less exposure to sunlight , application of sunscreen , elevated melanin in skin , or fully covered skin .
a higher prevalence of low vitamin d in the african american patients ( 88.6% ) in our study can be explained by the racial differences in vitamin d metabolism .
the african american population has an increased activity of 25-hydroxyvitamin d-1--hydroxylase ( cyp27b1 ) , either driven by increased parathormone activity or by racial differences in cyp27b1 affinity for substrate , which results into lower serum 25-ohd levels .
racial difference in variable vitamin d receptor affinity for vitamin d metabolites may explain similar variation in the prevalence of low vitamin d across other races with the observation of at least one vitamin d receptor polymorphism ( foki ) which is known to differ by race and ethnicity .
a critical review of methods and instruments that have been developed to measure fatigue found good psychometric properties in only 3 comprehensive instruments : fatigue symptom inventory ( fsi ) , multidimensional assessment of fatigue ( maf ) , and mfsi - sf . mfsi - sf is a valid , reliable and least time consuming instrument in the outpatient setting .
functional neuroimaging offers a more objective assessment of fatigue by measuring fatigue during brain scan and correlating it with cerebral activation .
a comparison of our study results with other relevant studies showed similar observations in different settings .
correction of low vitamin d in breast cancer patients with fatigue showed improvement in fatigue and other musculoskeletal symptoms .
similarly , improvement in fatigue scores were observed in patients with myasthenia gravis after vitamin d replacement .
our findings provide a consistent body of evidence that normalization of low vitamin d levels in patients with fatigue and stable chronic medical conditions , if any , significantly improves their fatigue symptom scores . among various therapeutic regimens of oral ergocalciferol
the 50,000 international unit 3 times weekly for 5 weeks regimen has shown the highest efficacy ( 82% ) , without adverse events , or toxicity .
a double - blind randomized controlled trial with ergocalciferol and placebo interventions could have provided a comparative analysis , nevertheless correction of low serum 25-ohd was mandatory in all patients based on the current guidelines .
normalization of low vitamin d level with ergocalciferol therapy significantly improves the severity of fatigue symptoms in adult patients who present in the primary care office with fatigue and stable chronic medical conditions , if any . with fatigue being a major presenting complaint in the primary care offices ,
serum 25-ohd level should be tested and low serum 25-ohd levels should be corrected with an effective ergocalciferol therapy regimen in order to improve their symptoms . | background : fatigue is a common presenting complaint of patients in the primary care offices
. low levels of vitamin d have been associated with fatigue in cancer patients .
normalization of vitamin d level improves their fatigue . whether low vitamin d plays a role in fatigue in medically stable patients is not known.aims:this prospective non - randomized therapeutic study observed the prevalence of low vitamin d in fatigue and the effect of normalization of vitamin d on fatigue.material and methods : one hundred and seventy four adult patients , who presented in our primary care office with fatigue and stable chronic medical conditions , completed fatigue assessment questionnaires .
patients with low vitamin d levels received ergocalciferol therapy for 5 weeks .
scores of pre- and post - treatment fatigue assessment questionnaires were compared.results:prevalence of low vitamin d was 77.2% in patients who presented with fatigue .
after normalization of vitamin d levels fatigue symptom scores improved significantly ( p < 0.001 ) in all five subscale categories of fatigue assessment questionnaires.conclusion:the prevalence of low vitamin d is high in patients who present with fatigue and stable chronic medical conditions , if any .
normalization of vitamin d levels with ergocalciferol therapy significantly improves the severity of their fatigue symptoms . | Introduction
Materials and Methods
Study selection
Data collection
Data synthesis and analysis
Results
Baseline characteristics
Fatigue assessment score
Symptom scores after normalization of vitamin D levels
Discussion
Conclusion | fatigue is a common complaint of 33% patients presenting in the ambulatory primary care offices in the usa . the prevalence of fatigue in the usa workforce has been estimated as 37.9% in a 2-week period . studies have shown an association of low vitamin d levels and fatigue in cancer patients and in patients with myasthenia gravis ; and improvement in fatigue symptom scores after normalization of vitamin d levels . there are many reports which indicate high prevalence of low vitamin d in the general population . low vitamin d leads to bone abnormalities ( e.g. in otherwise healthy individuals ,
fatigue can be a manifestation of low vitamin d levels and its impact on reduced maximum functioning of skeletal muscles via vitamin d receptors . we designed this study to observe the effect of correction of low vitamin d in patients with fatigue and stable comorbid medical condition(s ) , if any . this study was a prospective non - randomized therapeutic study that observed the effect of correction of low vitamin d in fatigue scores of the patients who presented with fatigue , stable comorbid medical condition(s ) if any , and low serum 25-hydroxyvitamin d ( 25-ohd ) levels . adult patients between the ages of 18 and 75 who presented in our internal medicine office with fatigue as their chief complaint volunteered to enroll in the study . the inclusion criteria were fatigue present for greater than 4 weeks and stable chronic medical condition(s ) , if any . patients
all patients with low serum 25-ohd levels ( less than 30 ng / ml ) received oral vitamin d ( ergocalciferol 50,000 usp international unit ; manufactured by sigmapharm laboratories , llc , bensalem , pennsylvania ) three times per week for 5 weeks , if not contraindicated . we collected the following data for each patient : age , gender , race , associated stable medical condition(s ) , use of over the counter supplemental vitamin d if any , pulse rate , blood pressure , mfsi - sf score before treatment , serum 25-ohd level ( ng / ml ) before treatment , mfsi - sf score after treatment , serum 25-ohd level ( ng / ml ) after treatment , and adverse effect(s ) of ergocalciferol therapy . a total of 116 patients were planned for sequential sampling who presented with fatigue and low serum 25-ohd levels in order to achieve the largest sample that would provide 80% power to the study hypotheses . we used wilcoxon paired test ( nonparametric analog of the paired t test ) to calculate the median difference on pre and post treatment serum 25-ohd levels assuming the distribution of difference of scores is not normal . it should be noted that once the data was collected , we found that it was normally distributed and so for analysis , the paired t test was used to compare the mean of vitamin d levels and fatigue scores pre and post - intervention except for the comparison between pre and post - intervention physical score and total score . paired t test was used to compare the mean pre- and post - intervention vitamin d levels , and fatigue scores except the physical scale . wilcoxon ranked sum test was used to compare the median pre- and post - intervention physical scale scores and total scores . correlations were used to assess the relationship between pre - intervention vitamin d levels with the pre - intervention fatigue scores , and the relationship between post - intervention vitamin d levels with the post - intervention fatigue scores . multivariable logistic regression was applied to assess the relationship between having a normal post - intervention serum 25-ohd level and change on total mfsi - sf adjusting for the association for the effects of demographics and stable medical condition(s ) that were significant at the p 0.2 level . the variables that were placed in the model were race , depression , and use of over - the - counter supplemental vitamin d.
this study was a prospective non - randomized therapeutic study that observed the effect of correction of low vitamin d in fatigue scores of the patients who presented with fatigue , stable comorbid medical condition(s ) if any , and low serum 25-hydroxyvitamin d ( 25-ohd ) levels . adult patients between the ages of 18 and 75 who presented in our internal medicine office with fatigue as their chief complaint volunteered to enroll in the study . we excluded patients over age 75 due to several factors that are known to contribute fatigue in this age group such as , decline in the functional ability , physical health , sensorycapacity , cognitive and mental health , and socio - environmental limitations . the inclusion criteria were fatigue present for greater than 4 weeks and stable chronic medical condition(s ) , if any . patients
all patients with low serum 25-ohd levels ( less than 30 ng / ml ) received oral vitamin d ( ergocalciferol 50,000 usp international unit ; manufactured by sigmapharm laboratories , llc , bensalem , pennsylvania ) three times per week for 5 weeks , if not contraindicated . we collected the following data for each patient : age , gender , race , associated stable medical condition(s ) , use of over the counter supplemental vitamin d if any , pulse rate , blood pressure , mfsi - sf score before treatment , serum 25-ohd level ( ng / ml ) before treatment , mfsi - sf score after treatment , serum 25-ohd level ( ng / ml ) after treatment , and adverse effect(s ) of ergocalciferol therapy . a total of 116 patients were planned for sequential sampling who presented with fatigue and low serum 25-ohd levels in order to achieve the largest sample that would provide 80% power to the study hypotheses . it should be noted that once the data was collected , we found that it was normally distributed and so for analysis , the paired t test was used to compare the mean of vitamin d levels and fatigue scores pre and post - intervention except for the comparison between pre and post - intervention physical score and total score . paired t test was used to compare the mean pre- and post - intervention vitamin d levels , and fatigue scores except the physical scale . wilcoxon ranked sum test was used to compare the median pre- and post - intervention physical scale scores and total scores . correlations were used to assess the relationship between pre - intervention vitamin d levels with the pre - intervention fatigue scores , and the relationship between post - intervention vitamin d levels with the post - intervention fatigue scores . multivariable logistic regression was applied to assess the relationship between having a normal post - intervention serum 25-ohd level and change on total mfsi - sf adjusting for the association for the effects of demographics and stable medical condition(s ) that were significant at the p 0.2 level . the variables that were placed in the model were race , depression , and use of over - the - counter supplemental vitamin d.
a total of 174 patients with fatigue completed mfsi - sf fatigue assessment questionnaires . thirty - nine ( 22.8% ) patients were excluded due to normal levels of serum 25-ohd , and the rest 132 ( 77.2% ) patients received vitamin d therapy [ figure 1 ] . the prevalence of low vitamin d was 77.2% in all 171 patients , despite the fact that 51.5% patients with low vitamin d levels were regularly taking over - the - counter vitamin d-3 between 1000 and 2000 international units [ table 1 ] . majority of the patients were in the age range of 36 and 65 [ figure 2 ] , female ( 72.4% ) , and non - hispanic caucasians ( 59.6% ) [ table 1 ] . demographics of 171 patients showed a higher prevalence of low vitamin d in the african - american patients ( 88.6% ) and in the other race category ( asian and hispanic patients ) ( 85.3% ) , compared to 70.6% in the non - hispanic caucasian patients . there was no significant difference between the number of patients taking over - the - counter vitamin d-3 in low vitamin d group ( 51.5% ) and normal vitamin d group ( 64.1% ) ( p = 0.166 ) . other factors , such as associated stable comorbid conditions , pulse and blood pressure between low vitamin d group and normal vitamin d group were comparable , except for the history of depression . patients with history of depression were twice likely to have a normal vitamin d level than a low vitamin d level [ table 1 ] . frequency of fatigue through age ranges , gender , and vitamin d levels ( ldf = low serum 25-ohd female , ldm = low serum 25-ohd male , ndf = normal serum 25-ohd female , ndm = normal serum 25-ohd male , nrf = did not go for serum 25-ohd test female , nrm = did not go for serum 25-ohd test male ) the mfsi - sf is a 30-item form that provides scores in the empirically derived 5 subscale categories . the assessment of fatigue is based on scores on the items that are rated on a 5-point scale indicating patients agreement to the truthfulness of each statement asking about their experience during the previous week ( 0 = not at all , to 4 = extremely ) . there was no statistically significant difference in the mean mfsi - sf scores in the general scale , emotional scale , mental scale and vigor scale ; and the median mfsi - sf scores in the physical scale and total score between all 171 patients and 116 patients with low vitamin d levels at initial encounter [ table 2 ] . there was no significant correlation between the baseline vitamin d levels and baseline mfsi - sf scores in 171 patients ; and post vitamin d therapy normalized vitamin d levels and mfsi - sf scores in 116 patients . after 5 weeks of vitamin d therapy in 116 patients , 111 ( 95.7% ) patients achieved normal levels of serum 25-ohd and 5 ( 4.3% ) patients required a second course of vitamin d therapy to achieve normal levels of serum 25-ohd . after ergocalciferol therapy mean serum 25-ohd level of all 116 patients normalized and significantly improved from a mean pre - treatment level of 19.71 ( sd , 5.98 ) ng / ml to a mean post - treatment level of 52.29 ( sd , 21.73 ) ng / ml ( p < 0.001 ) [ table 2 ] . fatigue scores : multidimensional fatigue symptom inventory - short form mean mfsi - sf scores in the general scale , emotional scale , mental scale , and vigor scale ; and the median mfsi - sf scores in the physical scale and total score improved significantly ( p < 0.001 ) after normalization of vitamin d levels [ table 2 ] . the multivariate analysis showed that history of depression was the only significant factor that impacted the post - intervention vitamin d level . patients who had a history of depression had 12 times greater odds of having a normal vitamin d level than those who did not have a history of depression ( 95% ci , 1.7 to 88 times ) . the reasons of discontinuation and consequent exclusion from the study were non - willingness for continued participation in 3 ( 1.7% ) patients , inability to go for serum 25-ohd test due to loss of medical insurance coverage in 7 ( 6.0% ) patients , and protocol violation due to a significant delay in getting serum 25-ohd test in 5 ( 3.8% ) patients . majority of the patients were in the age range of 36 and 65 [ figure 2 ] , female ( 72.4% ) , and non - hispanic caucasians ( 59.6% ) [ table 1 ] . demographics of 171 patients showed a higher prevalence of low vitamin d in the african - american patients ( 88.6% ) and in the other race category ( asian and hispanic patients ) ( 85.3% ) , compared to 70.6% in the non - hispanic caucasian patients . there was no significant difference between the number of patients taking over - the - counter vitamin d-3 in low vitamin d group ( 51.5% ) and normal vitamin d group ( 64.1% ) ( p = 0.166 ) . other factors , such as associated stable comorbid conditions , pulse and blood pressure between low vitamin d group and normal vitamin d group were comparable , except for the history of depression . patients with history of depression were twice likely to have a normal vitamin d level than a low vitamin d level [ table 1 ] . frequency of fatigue through age ranges , gender , and vitamin d levels ( ldf = low serum 25-ohd female , ldm = low serum 25-ohd male , ndf = normal serum 25-ohd female , ndm = normal serum 25-ohd male , nrf = did not go for serum 25-ohd test female , nrm = did not go for serum 25-ohd test male )
the mfsi - sf is a 30-item form that provides scores in the empirically derived 5 subscale categories . the assessment of fatigue is based on scores on the items that are rated on a 5-point scale indicating patients agreement to the truthfulness of each statement asking about their experience during the previous week ( 0 = not at all , to 4 = extremely ) . a high level of fatigue is indicated by high scores in general , physical , emotional , and mental scales , and low score in vigor scale . there was no statistically significant difference in the mean mfsi - sf scores in the general scale , emotional scale , mental scale and vigor scale ; and the median mfsi - sf scores in the physical scale and total score between all 171 patients and 116 patients with low vitamin d levels at initial encounter [ table 2 ] . there was no significant correlation between the baseline vitamin d levels and baseline mfsi - sf scores in 171 patients ; and post vitamin d therapy normalized vitamin d levels and mfsi - sf scores in 116 patients . pearson correlation was used to calculate all except for the vitamin d levels and mfsi - sf total scores , for which spearman rho correlation was used . after 5 weeks of vitamin d therapy in 116 patients , 111 ( 95.7% ) patients achieved normal levels of serum 25-ohd and 5 ( 4.3% ) patients required a second course of vitamin d therapy to achieve normal levels of serum 25-ohd . after ergocalciferol therapy mean serum 25-ohd level of all 116 patients normalized and significantly improved from a mean pre - treatment level of 19.71 ( sd , 5.98 ) ng / ml to a mean post - treatment level of 52.29 ( sd , 21.73 ) ng / ml ( p < 0.001 ) [ table 2 ] . mean mfsi - sf scores in the general scale , emotional scale , mental scale , and vigor scale ; and the median mfsi - sf scores in the physical scale and total score improved significantly ( p < 0.001 ) after normalization of vitamin d levels [ table 2 ] . the multivariate analysis showed that history of depression was the only significant factor that impacted the post - intervention vitamin d level . patients who had a history of depression had 12 times greater odds of having a normal vitamin d level than those who did not have a history of depression ( 95% ci , 1.7 to 88 times ) . this prospective study of low vitamin d and fatigue demonstrated two major observations : first , a very high prevalence of low vitamin d ( serum 25-ohd ) in patients who complained of fatigue . second , a significant reduction in the severity of fatigue ( fatigue symptom scores ) after normalization of vitamin d levels . our strict exclusion criteria supported the observation that improvement in fatigue symptoms were primarily due to normalization of low vitamin d levels and not secondary to amelioration of the chronic medical condition(s ) in patients who had one or more associated stable comorbid condition(s ) . a low vitamin d level reduces serum total and ionized calcium which results into increase in parathormone level , decrease in bone density , increase in bone turnover , decrease in urinary calcium excretion , increase in urinary phosphate excretion , and decrease in serum phosphate level , ultimately resulting into skeletal demineralization and muscle weakness . low vitamin d is also associated with inappropriate activation of the renin - angiotensin system , which fails to inhibit abnormal cell proliferation . the overall impact of these mechanisms results into further lowering of vitamin d levels , which have been associated with fatigue in patients with cancer , and myasthenia gravis . low vitamin d levels are also associated with cardiovascular diseases , hypertension , congestive heart failure , cancer , diabetes mellitus , neuromuscular disorders , pneumonia , upper respiratory tract infections , chronic obstructive pulmonary disease , and all - cause mortality . a meta - analysis of 18 randomized clinical trials concluded that vitamin d supplementation was associated with lower all - cause mortality . a survey of over 18,000 subjects in the united states general population showed 6.7% current and 24.4% lifetime prevalence of fatigue . although fatigue is twice as common in women as in men , it is not strongly associated with age or occupation . more recent analysis of 74 million adults in the united states aged 51 and above showed 31.2% prevalence of fatigue , being more in women ( 33.3% ) than men ( 28.6% ) and more in minorities ( 47.5% ) than in caucasians ( 27.5% ) . in our study
we found that fatigue was more common in non - hispanic caucasians ( 59.6% ) , and it affected about three - quarter ( 72.4% ) of women than men . in the usa , 41% of men and 53% of women
we observed a higher prevalence of low vitamin d ( 77.2% ) in our patients who presented with fatigue , despite the fact that more than half of the patients were taking over - the - counter vitamin d-3 regularly . a higher prevalence of low vitamin d in the african american patients ( 88.6% ) in our study can be explained by the racial differences in vitamin d metabolism . racial difference in variable vitamin d receptor affinity for vitamin d metabolites may explain similar variation in the prevalence of low vitamin d across other races with the observation of at least one vitamin d receptor polymorphism ( foki ) which is known to differ by race and ethnicity . a critical review of methods and instruments that have been developed to measure fatigue found good psychometric properties in only 3 comprehensive instruments : fatigue symptom inventory ( fsi ) , multidimensional assessment of fatigue ( maf ) , and mfsi - sf . mfsi - sf is a valid , reliable and least time consuming instrument in the outpatient setting . correction of low vitamin d in breast cancer patients with fatigue showed improvement in fatigue and other musculoskeletal symptoms . similarly , improvement in fatigue scores were observed in patients with myasthenia gravis after vitamin d replacement . our findings provide a consistent body of evidence that normalization of low vitamin d levels in patients with fatigue and stable chronic medical conditions , if any , significantly improves their fatigue symptom scores . a double - blind randomized controlled trial with ergocalciferol and placebo interventions could have provided a comparative analysis , nevertheless correction of low serum 25-ohd was mandatory in all patients based on the current guidelines . normalization of low vitamin d level with ergocalciferol therapy significantly improves the severity of fatigue symptoms in adult patients who present in the primary care office with fatigue and stable chronic medical conditions , if any . with fatigue being a major presenting complaint in the primary care offices ,
serum 25-ohd level should be tested and low serum 25-ohd levels should be corrected with an effective ergocalciferol therapy regimen in order to improve their symptoms . | [
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a plausible prebiotic synthesis of the canonical nucleotides has long been a major goal in origins of life research .
the oligomerization of hydrogen cyanide one of the major products of spark discharge in atmospheres containing methane and nitrogen to furnish the hcn tetramer diaminomaleonitrile ( 1 ) , its near - quantitative intramolecular photochemical rearrangement to 5-aminoimidazole-4-carbonitrile ( aicn , 2 ) , and the subsequent hydrolysis of aicn 2 to 5-aminoimidazole-4-carboxamide ( aica , 3 ) were demonstrated over 50 years ago.(4 ) however , the further elaboration of aminoimidazoles 2 and 3 to give the purine nucleobases and , after ribosylation and phosphorylation , the purine nucleotides is both very low yielding and , most importantly , not selective for the canonical nucleotides over isomers thereof .
for example , adenine and hypoxanthine have been condensed under dry conditions at high temperatures with an excess of ribose in the presence of a high concentration of magnesium salts to yield 3 and 15% of the respective canonical n9-linked -d - ribofuranonucleosides in complex mixtures with many other isomers.(5 ) thus , irrespective of the prebiotic availability of ribose , the formation of adenosine ( and inosine ) is in low yield , alongside a multitude of regioisomers and pyranosyl isomers .
furthermore , guanine , cytosine , and uracil do not give any of their respective nucleosides under similar conditions.(6 ) recently , a stepwise route from simple abiotic molecules considered to be prebiotically available to activated pyrimidine nucleotides was demonstrated ( scheme 1b).(1 ) however , this route does not solve the problem of purine synthesis .
ideally , further development of this chemistry would also lead to a stepwise synthetic route for purine nucleotide assembly , thus providing a plausible pathway by which the four nucleotides required for an information - rich rna coding system would be prebiotically available .
the key to effective pyrimidine synthesis was the stepwise increase in molecular complexity from simple aldehydes and cyanamide 4 through in situ formation of 2-aminooxazole ( 2ao , 5 ) as a hybrid sugar and nucleobase synthon .
therefore , we considered ways in which 5 could additionally participate in the synthesis of purines .
the diversity of chemical species likely to have been present under prebiotic conditions and the proven utility of multicomponent reactions ( mcrs ) to yield biologically relevant structures suggested that the development of novel mcrs could lead to the rapid buildup of molecular complexity and , in particular , to the structures that are central to molecular biology .
the formation of 1 is catalyzed by simple aldehydes via the formation of cyanohydrins during hydrogen cyanide oligomerization ( scheme 1a).(9 ) it therefore seemed important to assess the chemistry of hydrogen cyanide tetramers with simple aldehydes in aqueous solution toward the prebiotic synthesis of purine ribo - nucleotides.(10 ) specifically , we proposed that the addition of 2 or 3 to the reaction of 5 with aldehydes might yield regiospecifically tethered aminoimidazole nucleotide intermediates .
the close relationship of aminoimidazole tethering and pyrimidine synthesis is depicted in scheme 1b , wherein we propose that divergent access to purines and pyrimidines may be achieved by the addition of 5 to an imine or an aldehyde , respectively .
as the five carbon atoms of ribose are contiguous , the formation of the canonical purine ribonucleotides from simple aldehydes should be considered both in the context of one c2 and one c3 fragment , and of one c1 and two c2 fragments .
we first assessed the efficiency of forming an aminoimidazole - tethered masked c3-aldehyde upon reaction of 2ao 5 with formaldehyde and aicn 2 or aica 3 , which could ultimately translate into a c5-tethered aminoimidazole . upon mixing formaldehyde , 5 , and 2 or 3 in water at room temperature and near neutral ph , the rapid , efficient synthesis of rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines 6 or 7 was observed ( scheme 2 ) .
synthesis of 6 and 7 demonstrates the selective sequestration of 2 and 3 through sequential formation of iminium ion 8 at equilibrium , followed by intermolecular carboncarbon bond formation to furnish 9 and 6-exo - trig intramolecular imidazoleiminium trapping ( scheme 3 ) .
these reactions were found to be scalable to multigram levels , and the products crystallized from concentrated aqueous solution , giving 6 and 7 in 89 and 80% isolated yield , respectively .
the -fused tricyclic structures were proven by x - ray diffraction analysis of a single crystal of 7 crystallized at ph 6.0 and a single crystal of the hemi - hydrochloride salt of 6 crystallized at ph 5.0 .
pyrimidine nucleotide precursors result from two - component chemistry ( 2cr),(1 ) while three - component chemistry ( 3cr ) leads to potential purine nucleotide precursors . to further explore the generality of the mcr
the reaction was shown to tolerate acetaldehyde to yield 11 and 12 as a 2:1 ( erythro : threo ( e : t ) ) mixture of diastereomers .
the minor rac-[2s,3r]-isomer 12 was then isolated by precipitation and crystallization , which allowed the structure to be proven unambiguously by single - crystal x - ray diffraction . to explore this reaction outside of the prebiotic context
, an aromatic aldehyde , 13 , was shown to undergo reaction in the presence of dmso as a co - solvent , giving a 3:2 e : t ratio of products 14 and 15 ( scheme 4 ) . to continue our investigation into the potential of this mcr for prebiotic nucleotide assembly
the reaction of these -hydroxyaldehydes with aicn 2 or aica 3 and 2ao 5 was very clean and high yielding for the expected c4 ( c2 + c2 ) and c5 ( c2 + c3 ) products.(11 ) ph - dependent reactivity was observed , with three - component chemistry dominating at lower ph values and two - component chemistry dominating a higher ph values ( vide infra ) .
notably , the products were uncontaminated with homo - aldolization or homo - mannich byproducts , and the three - component products exhibited a high diastereoselectivity for rac-[3r,4r]-products important for realization of a -ribo - nucleotide synthesis by c3-stereochemical inversion ( schemes 5 and 6 ) .
proposed routes for rna synthesis must be continually evaluated for their potential to yield alternative structures capable of watsoncrick base - paring .
glycolaldehyde , as a c2 sugar synthon , has the potential to furnish precursors of threose nucleic acid ( tna ) upon reaction with 5.(12 ) at pd 5.0 , the reaction of glycolaldehyde , aicn 2 , and 5 gave a 4:1 mixture of diastereoisomers 16 and 17 , alongside 10% two - component tetrose aminooxazoline products 18 and 19 . at pd 4.0 ,
the reaction gave a near - quantitative conversion to three - component products ; however , 21% of these products were 20 and 21 , which result from 5-exo - trig ring closure of intermediate 9.(13 ) an improvement in diastereoselectivity accrued upon reaction with aica 3 in lieu of aicn 2 .
at pd 5.0 , a 7:1 mixture of diastereoisomers 22 and 23 ( 95% yield as determined by h nmr integration ) was observed , with complete 6-exo - trig selectivity .
compound 22 was isolated in 74% yield and crystallized to allow the [ 2r,3r]-stereochemical relationship to be proven by x - ray crystallography .
interestingly , 22 and 16 are related to tna via c3-stereochemical inversion.(12 ) glyceraldehyde is a c3 sugar synthon ; therefore , its reaction with the c2 sugar synthon 2ao 5 can yield precursors of pentose nucleic acids ( e.g. , rna).(1 ) however , a high - yielding synthesis of ribo - purinosides via c3-tethered aminoimidazoles would require high [ 3r,4r]-diastereoselectivity ( lyxo / xylo ) , a reversal of the facial selectivity of glyceraldehyde upon bimolecular reaction with 5.(14 ) at pd 5.0 , the reaction of glyceraldehyde , 5 , and aica 3 furnished lyxo - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine ( 24 ) in 60% yield ( by h nmr analysis ; 46% isolated yield ) , alongside three other diastereoisomers , 2527 , in 11 , 5 , and 8% yields , respectively ( by h nmr analysis ) .
at pd 4.0 , the reaction of aicn 2 , 5 , and glyceraldehyde was highly selective for three - component products ( 89% by h nmr analysis ) . however , likely due to an increased thorpeingold effect in 9 ( where r = ch(oh)ch2oh relative to r = ch2oh ) and the lower ph required for selective three - component reaction of 2 relative to 3 , competitive 6-exo - trig imidazole cyclization and 5-exo - trig hydroxyl cyclization resulted in a 1:1 mixture of rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines 2831 and rac-3-aminoimidazole-2-aminooxazolines 3235 .
the three - component reaction of glyceraldehyde with 2 or 3 and 5 is highly lyxo - selective , yielding 66% of 28/32 or 60% of 24 , respectively , whereas the bimolecular reaction of glyceraldehyde and 5 is highly ribo / arabino - selective ( ribo36 = 44% and arabino37 = 30%).(14 ) the high lyxo - selectivity of the three - component reaction establishes the required c1 , c3 , and c4 stereochemistry for the proposed ribo - nucleotide synthesis by c3 stereochemical inversion , while the ribo / arabino - selectivity of the two - component reaction establishes the required c3 and c4 stereochemistry for pyrimidine synthesis .
the stereochemical preference for lyxo / xylo three - component products or ribo / arabino two - component products likely derives from the control of facial selectivity of the imine or aldehyde component , respectively,(14 ) coupled with the obligatory z - geometry of the nucleophilic c = c bond of 5 ( see supporting information , figure s36 ) , though calculations to support this conjecture have not been carried out .
interestingly , with regard to the furanosyl selectivity of ribo - nucleotide synthesis , isolated 32 is observed to equilibrate with 28 , but not with detectable amounts of the pyranosyl isomer 40,(16 ) though lyxo - furanosyl aminooxazoline 39-f equilibrates to give significant amounts of the pyranosyl isomer ( 39-p/39-f , 5:1 ) ( scheme 7).(17 ) equilibration of 28 and 32 is proposed to occur via intermediate 9 and to be controlled by imidazole protonation ; therefore , at low ph , protonation of the imidazole moiety results in complete furanosyl selectivity ( see supporting information , figure s27 , for h nmr spectra of 28/32 at pd 112 ) . although efficient three - component reactivity is observed at ph values lower than 6.0 , the reaction profile of -hydroxyaldehydes with aicn 2 or aica 3 and 2ao 5 was observed to shift to furnish only bimolecular aminooxazoline products at higher ph s , even in the presence of a large excess of 2 or 3 .
the reaction of glycolaldehyde and 5 with 2 or 3 , above pd 6.5 and 7.0 , respectively , was found to predominantly yield tetrose aminooxazolines 18 and 19 in a 1.2:1 [ 2r,3r]:[2s,3r ] ratio.(14 ) similarly , the reaction of glyceraldehyde , 2 or 3 , and 5 at pd 7.0 yields a mixture of pentose aminooxazolines 3639 in a 1.5:1 [ 2r,3r]:[2s,3r ] ratio.(14 ) the more or less complete switch between two- and three - component reactivity is observed to occur over 2 ph units at an aminoimidazole pka - dependent ph . however , surprisingly , at pd 7.0 , formaldehyde and 2 or 3 were observed to react selectively with 5 as imine / iminium 8 , resulting in the synthesis of 6 or 7 ( schemes 2 and 3 ) . even at elevated ph , and in the absence of 2 and 3 ,
no hydroxymethylation of 5 to give 41 was observed a standard sample of this latter compound being prepared by the reaction of glyceraldehyde with cyanamide 4 ( scheme 8).(18 ) it is possible that , due to the stability of the hemiaminal 42 formed between the amino group of 5 and formaldehyde , there is insufficient free aldehyde form of formaldehyde to react with free 5 at an observable rate.(19 ) the selective mcr of formaldehyde , and the absence of hydroxymethylation of 5 , may have interesting implications for the selectivity of prebiotic nucleotide synthesis . in effect , glycolaldehyde may be sequestered as 5 by the action of cyanamide 4 even in the presence of other aldehydes , such as formaldehyde , that can not undergo oxazole or aminooxazoline formation . as an additional point ,
aldehydes are known to accelerate the rate of hydrolysis of syn - disposed -aminonitriles at elevated ph values , but no concurrent nitrile hydrolysis was observed under the conditions of tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine formation .
as the nitrile of derivatives of 2 retains the potential to give divergent access to adenine and guanine nucleotides , it is of note that no significant nitrile hydrolysis was observed in our experiments .
selective cyanovinylation of the aminooxazoline moiety of the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines is one of several routes that may allow for the subsequent removal of those atoms derived from cyanamide 4 ( scheme 3 ) through photoactivated hydrolytic ( or otherwise ) removal of the resultant cytosine moiety , thereby affording products constitutionally closer to purine nucleotides . to investigate this potential for pyrimidine nucleobase elaboration and potential subsequent loss , a series of cyanovinylation experiments was undertaken to show that the aminooxazoline moiety of the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine structure could be selectively cyanovinylated . because there are several potentially nucleophilic heteroatoms in the generic tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine structure that could react with an alkylating agent , we were initially unsure as to the likely regioselectivity of cyanovinylation upon treatment with aqueous cyanoacetylene . nonetheless , with regard to the nucleophilicty of n1 , we were encouraged by the observation that 6 crystallized as the hemihydrochloride salt at ph 5.0 , wherein two molecules of 6 were hydrogen - bonded via a common proton between n1 ( see supporting information , figure s3 ) . furthermore , we thought that the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines were likely to be intrinsically protected from c3nh cyanovinylation by delocalization of the nitrogen lone pair into the imidazole ring . interestingly , and very pleasingly ,
highly selective cyanovinylation was observed upon treatment of 6 , 17 , 21 , 24 , and 30 with cyanoacetylene . even in the presence of 5 equiv of cyanoacetylene , clean conversion of 6 , 17 , 21 , 24 , and 30 to 4-amino-1-(3-hydroxy-1,2,3,4-tetrahydroimidazo[1,5-a]-pyrimidin-4-yl)pyrimidin-2-(h)-ones 4347
was observed over the period of 13 days at 60 c in unbuffered aqueous solution in 8095% yield.(21 ) this range of products demonstrates the efficacy this cyanovinyl in both tetrose and pentose series for both 2,3-cis- and 2,3-trans - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines .
additionally , compounds 43 and 44 were isolated and crystallized from d2o , providing samples for x - ray diffraction to unambiguously prove the novel structural motif ( scheme 9 ) .
experiments to investigate the propensity for cytosine loss from these compounds under a variety of conditions are now underway .
we have described a concise and high - yielding route to the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines . on the basis of these key intermediates
cytidines are known to undergo both pyrimidine loss and c2-epimerization upon uv - irradiation , possibly due to the photochemical generation of nucleobaseiminium ions.(22 ) these intermediates can undergo dissociative hydrolytic processes that are similar to those observed during the acid - catalyzed hydrolysis of -d - uridine,(23 ) but at near - neutrality .
it is of note that the mcr described provides a high yield of [ 3r,4r]-products ( 70% lyxo / xylo ) , reversing the facial selectivity of glyceraldehyde observed in the biomolecular reaction with 2ao 5 ( 70% ribo / arabino ) , with the lyxo and xylo products being stereochemically related via c2-epimerization.(24 ) upon cyanovinylation and dissociative cytosine cleavage , compounds 27 , 31 , and 34 should , via intramolecular ring closure , furnish 5,3-anhydro - aicn - riboside 49 ( where r = ch2oh ) , which has an obvious relationship to aica - riboside 50 , an intermediate in the de novo biosynthesis of purines ( scheme 10 ) .
furthermore , those compounds with trans-2-hydroxyl-3-anhydronucleoside relationships , such as 49 , have the clear potential to allow 2,3-cyclic phosphate formation under urea - mediated phosphoryl - transfer conditions,(1 ) thus generating 51 .
access to 49 should allow further study of the selectivity issues involved in synthesizing the canonical nucleotides as opposed to closely related chemical structures capable of watsoncrick base - pairing interactions .
it is of note that the proposed phosphorylation of the 2-hydroxyl group of 49 could lead to purines via a c3 stereochemical inversion , which would be directly analogous to the c2 inversion previously described to give access to the pyrimidine nucleotides .
center : comparison to aica - riboside 50 , an intermediate in the de novo stepwise biosynthesis of canonical nucleotides . in summary , we have described additional predisposed complex structures that can be accessed through the participation of the key intermediate 2-aminooxazole 5 in a novel aqueous multicomponent reaction system . this mcr has proved to be scalable and high - yielding , and it generates a ph - dependent product distribution .
further , the intrinsically controlled cyanovinylation of the low - ph mcr products generates potential precursors of the purine nucleotides .
these reactions help to define the chemical structural space and chemical distribution likely to be accessible under plausible prebiotic chemical scenarios .
in particular , we have demonstrated the concurrent synthesis of 37 and the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines in the ph range in which activated pyrimidine synthesis can occur ( ph < 6.5 , where hydrolysis of 53 is prevented),(1 ) suggesting that both the pyrimidine and the purine ribonucelotides could be made together at the same time , in the same place , and under the same conditions.(29 ) the reactions may therefore be of direct relevance to the problem of abiogenesis of a full set of canonical ribonucleotides , and hence the chemical origins of molecular biology .
reagents and solvents were purchased from sigma - aldrich , tci america , frontier scientific , or cambridge isotope laboratories .
flash column chromatography was carried out using merck 9385 silica gel 60 ( 230400 mesh ) .
nmr spectroscopy was carried out on a varian nmr spectrometer ( oxford as-400 ) operating at 20 c probe temperature ( unless specified ) .
where possible , the chemical shift of the corresponding solvent was used as a reference .
high - resolution mass spectrometry was carried out on a waters q - tof micro lc / ms / ms system .
infrared spectra were recorded as manually pressed potassium bromide ( kbr ) discs on a perkinelmer spectrum 100 series ft - ir spectrometer .
single - crystal x - ray crystallography was carried out with a bruker apex ii ccd diffractometer ( mo k radiation , = 0.71073 ) , equipped with an oxford cryosystems nitrogen flow apparatus , at 100 k. data integration down to 0.76 resolution was carried out using saint v7.46 a ( bruker diffractometer , 2009 ) with reflection spot size optimization .
the structure was solved by the direct methods procedure and refined by least - squares methods against f using shelxs-97 and shelxl-97 ( sheldrick , 2008 ) .
non - hydrogen atoms were refined anisotropically . method a : a solution of aminoimidazole 2 or 3 ( 1 equiv ) and 2-aminooxazole 5 ( 1 equiv ) in d2o was adjusted to the desired pd .
a solution of aldehyde ( 1 equiv ) in d2o and , if required , dmso - d6 was added .
the pd was adjusted as necessary by addition of dcl or naod , and the reaction was volumetrically adjusted to the required concentration and then incubated at the specified temperature .
method b : a solution of aminoimidazole 2 or 3 ( 1 equiv ) and 2-aminooxazole 5 ( 1 equiv ) in h2o was adjusted to the desired ph . a solution of aldehyde ( 1 equiv ) in h2o was added .
the ph was adjusted as necessary by addition of hcl or naoh , and the reaction was volumetrically adjusted to the required concentration and then incubated at the specified temperature .
method c : an aqueous solution of cyanoacetylene ( 0.98 m , 35 equiv ) was added to tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines ( 20 mm ) , and the solution was then heated at 60 c for 13 d with stirring .
method a : 5-aminoimidazole-4-carbonitrile 2 ( 24.8 mg , 0.23 mmol ) and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 1.0 ml ) at pd 5.0 , 6.0 , or 7.0 .
formaldehyde ( 37% , 18.6 mg , 0.23 mmol ) was added in d2o ( 0.5 ml ) , the pd was rechecked , and the reaction volume was adjusted to 2 ml by the addition of d2o .
the reactions were incubated at room temperature , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s4 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) .
6 was then crystallized by cooling the reaction at pd 5.0 to 4 c for 2 d. 6 was isolated by filtration and washed with ice - cold water , and a single crystal was removed for x - ray diffraction .
method b : 5-aminoimidazole-4-carbonitrile 2 ( 1.29 g , 11.9 mmol ) and 2-aminooxazole 5 ( 1.00 g , 11.9 mmol ) were dissolved in h2o ( 10 ml ) at ph 5.0 .
formaldehyde ( 37% , 0.96 g , 11.9 mmol ) was added , the ph was rechecked , and the reaction volume was adjusted to 20 ml with h2o . after 20 min a white precipitate had formed in the reaction .
the reaction was mechanically stirred for a further 15 h. the solids present were isolated by filtration , and the filtrate was lyophilized .
slowly cooling to room temperature gave 1.68 g ( 69% ) of 6 as a white solid .
recrystallization of the lyophilized filtrate afforded a further 0.5 g ( 20% ) of 6 .
ir ( kbr , cm ) : 3467 ( nh ) , 3380 ( oh ) , 3115 , 2986 , 2938 ( ch ) , 2212 ( cn ) 1698 , 1610 ( n = c ) .
h nmr ( 400 mhz , d2o ) : h 7.39 ( s , 1h , h-(c2 ) ) ; 6.08 ( d , j = 7.8 hz , 1h , h-(c1 ) ) ; 5.30 ( ddd , j = 7.8 , 2.0 , 1.8 hz , 1h , h-(c2 ) ) ; 3.54 ( abx , j = 14.2 , 1.8 hz , 1h , ha-(c3 ) ) ; 3.37 ( abx , j = 14.2 , 2.0 hz , 1h , hb-(c3 ) ) [ peak partially obscured by hod peak in dmso ; see supporting information , figure s2 ] .
c nmr ( 101 mhz , dmso - d6 ) : c 166.6 ( c = o ) ; 164.0 ( c2 ) ; 142.1 ( c5 ) ; 129.6 ( c2 ) ; 112.7 ( c4 ) ; 76.4 ( c2 ) ; 72.8 ( c1 ) ; 42.3 ( c3 ) .
m / z ) : 205 ( 100% , [ m + h ] ) , 227 ( 10% , [ m + na ] ) .
hrms ( m / z ) : [ m ] calcd for c8h9n6o , 205.0832 ; found , 205.0831 .
method a : 5-aminoimidazole-4-carboxamide 3 ( 29.0 mg , 0.23 mmol ) and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2.0 ml ) at pd 5.0 , 6.0 , or 7.0 .
formaldehyde ( 37% , 18.6 mg , 0.23 mmol ) was added in d2o ( 0.5 ml ) , the pd was rechecked , and the reaction volume was adjusted to 2 ml with d2o .
the reactions were incubated for 15 h , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s4 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) .
7 was then crystallized by cooling the reaction solution to 4 c for 1 d. 7 was isolated by filtration and washed with ice - cold water .
method b : 5-aminoimidazole-4-carboxamide 3 ( 1.50 g , 11.9 mmol ) and 2-aminooxazole 5 ( 1.00 g , 11.9 mmol ) were dissolved in h2o ( 10 ml ) at ph 5.0 .
formaldehyde ( 37% , 0.96 g , 11.9 mmol ) was added , the ph was rechecked , and the reaction volume was adjusted to 20 ml with h2o .
the reaction was mechanically stirred for 15 h. the solids present were isolated by filtration , and the filtrate was lyophilized .
the combined solids were redissolved in water by heating , agitated , and then allowed to cool to give 1.95 g of 7 as a yellow solid .
the solids were dissolved in hot water ( 4 ml ) , and the product was allowed to crystallize upon cooling .
7 was isolated by filtration , washed with methanol ( 10 ml ) , and air - dried to give 1.30 g ( 49% ) of 7 as a white solid .
a further portion of methanol was added to the filtrate , and the solution was then cooled to give a second batch ( 0.82 g , 31% ) of 7 as an off - white powder .
ir ( kbr , cm ) : 3365 ( nh ) , 3096 , 2979 , 2765 , 2660 ( ch ) , 1676 ( n = c ) , 1640 ( c = o ) .
h nmr ( 400 mhz , d2o ) : h 7.21 ( s , 1h , h-(c2 ) ) ; 5.83 ( d , j = 7.8 hz , 1h , h-(c1 ) ) ; 5.03 ( dt , j = 7.8 , 1.8 hz , 1h , h-(c2 ) ) ; 3.53 ( abx , j = 13.8 , 1.8 hz , 1h , ha-(c3 ) ) ; 3.18 ( abx , j = 13.8 , 1.8 hz , 1h , hb-(c3 ) ) .
c nmr ( 101 mhz , d2o ) : c 166.2 ( c = o ) ; 164.1 ( c2 ) ; 144.3 ( c5 ) ; 130.0 ( c2 ) ; 110.4 ( c4 ) ; 78.4 ( c2 ) ; 66.1 ( c1 ) ; 43.4 ( c3 ) .
m / z ) : 223 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ]
x - ray diffraction structure solved ; deposited as ccdc 784244 . method a : acetaldehyde ( 10 mg , 0.23 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 86.9 mg , 0.69 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 6.0 .
the reaction was incubated for 15 h , and crystals ( colorless needles ) were observed to form .
the supernatant was removed , and two products were observed by nmr spectroscopy ( see supporting information , figure s7 , for h nmr spectrum ) .
the crystals were dissolved in the supernatant by warming , and nmr spectroscopy showed a 2:1 ratio of 11:12 .
the crystals were isolated by filtration ( 18 mg ) , and an analytical sample was dissolved in dmso - d6 ( 0.75 ml ) for nmr spectroscopy .
the remaining crystals were recrystallized from slowly cooling h2o to provide a sample for x - ray diffraction .
h nmr ( 400 mhz , d2o ) : h 7.18 ( s , 1h , har ) ) ; 5.81 ( d , j = 7.6 hz , 1h , h-(c1 ) ) ; 4.74 ( dd , j = 7.6 , 2.9 hz , 1h , h-(c2 ) ) ; 3.65 ( dq , j = 6.9 , 2.9 hz , 1h , h-(c3 ) ) ; 0.90 ( d , j = 6.9 hz , 3h , me ) .
m / z ) : 237 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c9h13n6o2 , 237.1095 ; found , 237.1099 . 12 .
ir ( kbr , cm ) : 3413 ( nh ) , 3178 , 3087 , 2960 ( ch ) , 1684 , 1663 ( n = c ) , 1616 ( c = o ) .
h nmr ( 400 mhz , d2o ) : h 7.15 ( s , 1h , h-(c2 ) ) ; 5.81 ( d , j = 7.7 hz , 1h , h-(c1 ) ) ; 4.83 ( dd , j = 7.7 , 2.0 hz , 1h , h-(c2 ) ) ; 3.32 ( dq , j = 6.6 , 2.0 hz , 1h , h-(c3 ) ) ; 1.16 ( d , j = 6.6 hz , 3h , me ) .
h nmr ( 400 mhz , dmso - d6 ) : h 7.16 ( s , 1h , h - ar ) ; 6.68 ( br d , 2h , nh2 ) ; 6.40 ( br s , 2h , nh2 ) ; 5.79 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 5.59 ( br s , 1h , nh ) ; 4.70 ( dd , j = 7.4 , 0.5 hz , 1h , h-(c2 ) ) ; 3.41 ( dd , j = 6.0 , 0.5 hz , 1h , h-(c3 ) ) ; 1.29 ( d , j = 6.0 hz , 3h , me ) .
c nmr ( 101 mhz , dmso - d6 ) : c 166.7 ( c = o ) ; 166.7 ( c2 ) ; 142.0 ( c5 ) ; 129.5 ( c2 ) ; 112.5 ( c4 ) ; 79.7 ( c2 ) ; 73.6 ( c1 ) ; 47.9 ( c3 ) ; 17.9 ( me ) . es - ms ( pos .
m / z ) : 237 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c9h13n6o2 , 237.1095 ; found , 237.1099 .
x - ray diffraction structure solved ; deposited as ccdc 784245 . method a : 2-pyridine carboxaldehyde 13 ( 24.6 mg , 0.23 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 58 mg , 0.42 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o : dmso - d6 ( 10:1 , 2 ml ) at pd 6.0 .
the reaction was incubated for 15 h , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s9 , for h nmr spectrum ) . the formation of two products , 14/15 , was observed in a 3:2 ratio by h nmr spectroscopy .
compound 14 was observed to crystallize from the solution over 3 d and was isolated by centrifugation , washed with methanol , and dried to give 15 mg ( 20% ) of 14 .
an analytical sample of crystalline 14 was then recrystallized from ethanol three times for single - crystal x - ray diffraction .
ir ( kbr , cm ) : 3412 ( nh ) , 2937 , 2528 , 2458 ( ch ) , 1693 ( n = c ) , 1669 , 1637 ( c = o ) 1591 .
h nmr ( 400 mhz , dmso - d6 ) : h 9.1 ( s , nh ) ; 8.43 ( dd , j = 4.8 , 0.8 hz , 1h , ar ) ; 7.75 ( td , j = 7.8 , 1.8 hz , 1h , ar ) ; 7.36 ( d , j = 7.8 hz , 1h , ar ) ; 7.24 ( ddd , j = 7.8 , 4.8 , 1.8 hz , 1h , ar ) ; 5.78 ( d , j = 7.8 hz , 1h , h-(c1 ) ) ; 5.40 ( dd , j = 7.6 , 2.5 hz , 1h , h-(c2 ) ) ; 4.88 ( d , j = 2.5 hz , 1h , h-(c3 ) ) .
c nmr ( 101 mhz , dmso - d6 ) : c 166.6 ( c = o ) ; 163.7 ( c2 ) ; 159.0 ( ar ) ; 149.6 ( c5 ) ; 139.9 ( arh ) ; 137.9 ( arh ) ; 129.8 ( c2 ) ; 123.6 ( arh ) ; 122.2 ( arh ) ; 112.7 ( c4 ) ; 78.6 ( c2 ) ; 72.3 ( c1 ) ; 56.2 ( c3 ) .
m / z ) : 300 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c13h14n7o2 , 300.1203 ; found , 300.1205 .
x - ray diffraction structure solved ; deposited as ccdc 784252 . method a : glycolaldehyde ( 14.2 mg , 0.23 mmol ) , 5-aminoimidazole-4-carbonitrile 2 ( 24.8 mg , 0.23 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 4.0 , 5.0 , 5.5 , or 6.0 .
the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see table 1 and supporting information , figure s11 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) .
the formation of 16 ( 60% ) , 17 ( 16% ) , 20 ( 14% ) , and 21 ( 7% ) was observed at pd 4.0 ( see supporting information , figure s16 , for h , hh cosy , and 2d noesy spectra ) .
17 was isolated by precipitation from warm methanol and recrystallized four times from ethanol and once by vapor diffusion ( chloroform into methanol ) to provide a sample for x - ray crystallography .
17 was studied by 2d homo- and heteronuclear nmr spectroscopy ( see supporting information , figures s13s15 ) .
the formation of 18 ( 60% ) and 19 ( 40% ) was observed at pd 7.0 .
noe intensity volumes are given as strong ( s ) , medium ( m ) , weak ( w ) , or not observed ( ) relative to the volume of h(c1)h(c2 ) of the same compound which is defined as strong .
h nmr ( 400 mhz , d2o ) : h 7.22 ( s , 1h , h-(c2 ) ) ; 5.81 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 4.99 ( dd , j = 7.4 , 2.7 hz , 1h , h-(c2 ) ) ; 3.74 ( dt , j = 5.7 , 2.7 hz , 1h , h-(c3 ) ) ; 3.50 ( t , j = 5.7 hz , 2h , h-(c4 ) ) .
m / z ) : 235 ( 100% , [ m + h ] ) , 257 ( 10% , [ m + na ] ) , 273 ( 3% , [ m + k ] ) . hrms ( m / z ) : [ m ] calcd for c9h11n6o2 , 235.0938 ; found , 235.0944 .
ir ( kbr , cm ) : 3467 ( nh ) , 3380 ( oh ) , 3115 , 2986 , 2938 ( ch ) , 2212 ( cn ) 1698 , 1610 ( n = c ) .
h nmr ( 400 mhz , d2o ) : h 7.16 ( s , 1h , h - ar ) ) ; 5.78 ( d , j = 7.7 hz , 1h , h-(c1 ) ) ; 4.8 ( dd , j = 7.7 , 1.9 hz , 1h , h-(c2 ) ) ; 3.63 ( abx , j = 11.4 , 6.6 hz , 1h , ha-(c4 ) ) ; 3.56 ( abx , j = 11.4 , 6.8 hz , 1h , hb-(c4 ) ) ; 3.56 ( app td , j = 6.7 , 1.9 hz , 1h , h-(c3 ) ) .
h nmr ( 400 mhz , dmso - d6 , 25 c ) : h 7.35 ( s , 1h , h - ar ) ; 6.83 ( s , 1h , hn ) ; 6.55 ( s , 2h , nh2 ) ; 5.87 ( d , j = 7.5 hz , 1h , h-(c1 ) ) ; 5.08 ( t , j = 5.85 hz , 1h , oh ) ; 4.92 ( d , j = 7.5 hz , 1h , h-(c2 ) ) ; 3.74 ( abx , j = 10.5 , 6.0 hz , 1h , ha-(c4 ) ) ; 3.60 ( abx , j = 10.5 , 6.7 hz , 1h , hb-(c4 ) ) ; 3.42 ( m , 1h , h-(c3 ) ) .
c nmr ( 101 mhz , dmso - d6 , 25 c ) : c 163.6 ( c2 ) ; 145.4 ( c5 ) ; 131.9 ( c2 ) ; 116.9 ( cn ) ; 88.8 ( c4 ) ; 75.5 ( c2 ) ; 73.5 ( c1 ) ; 60.3 ( c4 ) ; 53.6 ( c3 ) .
m / z ) : 235 ( 100% , [ m + h ] ) , 257 ( 10% , [ m + na ] ) .
m / z ) : 233 ( 70% , [ m h ] ) .
hrms ( m / z ) : [ m ] calcd for c9h11n6o2 , 235.0938 ; found , 235.0944 .
x - ray diffraction structure solved ; deposited as ccdc 784246 . method a : glycolaldehyde ( 14.3 mg , 0.23 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 29.0 mg , 0.23 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 5.0 , 6.0 , or 7.0 . the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s17 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . the formation of 22 ( 78% ) and 23 ( 11% ) was observed at pd 5.0 .
22 was isolated by precipitation with methanol and studied by 2d homo- and heteronuclear nmr spectroscopy ( see supporting information , figures s19s21 ) .
the formation of 18 ( 60% ) and 19 ( 40% ) was observed at pd 7.0 .
method b : glycolaldehyde ( 357 mg , 5.9 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 743 mg , 5.9 mmol ) , and 2-aminooxazole 5 ( 500 mg , 5.9 mmol ) were dissolved in h2o ( 20 ml ) at ph 6.0 .
the reaction was incubated for 15 h , after which a white precipitate had formed .
the precipitate was isolated by filtration and washed with ice - cold water to give 1.1 g ( 74% ) of 22 .
an analytical sample of 22 was then recrystallized five times from aqueous ethanol for x - ray diffraction analysis .
ir ( kbr , cm ) : 3315 ( oh ) , 3188 ( nh ) , 2922 , 2850 ( ch ) , 1711 ( n = c ) , 1659 ( c = o ) , 1614 , 1575 ( n = c ) .
h nmr ( 400 mhz , dmso - d6 ) : h 7.14 ( s , 1h , h-(c2 ) ) ; 6.65 ( bd , 2h , nh2 ) ) ; 6.45 ( s , 2h , nh2 ) ) ; 6.18 ( s , 1h , nh2 ) ) ; 5.74 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 5.04 ( t , j = 5.12 hz , 1h , ho ) ) ; 4.78 ( dd , j = 7.4 , 2.9 hz , 1h , h-(c2 ) ) ; 3.49 ( m , 1h , h-(c3 ) ) ; 3.44 ( abx , j = 11.0 , 5.0 hz , 1h , ha-(c4 ) ) ; 3.38 ( abx , j = 11.0 , 5.3 hz , 1h , hb-(c4 ) ) .
c nmr ( 101 mhz , dmso - d6 ) : c 166.7 ( c = o ) ; 163.4 ( c = n ) ; 140.4 ( c5 ) ; 129.2 ( c2 ) ; 112.2 ( c4 ) ; 76.3 ( c1 ) ; 72.5 ( c2 ) ; 62.4 ( c3 ) ; 54.2 ( c4 ) .
m / z ) : 253 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c9h13n6o3 , 253.1044 ; found , 253.1049 .
glyceraldehyde ( 20.7 mg , 0.23 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 29.0 mg , 0.23 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 5.0 , 6.0 , 6.5 , or 7.0 .
the reaction was incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s23 , for h nmr spectra at pd 5.0 , 6.0 , 6.5 , and 7.0 ) . the predominant formation of four tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines ( 84% ) 24 ( 60% ) , 25 ( 11% ) , 26 ( 5% ) and 27 ( 8% ) was observed at pd 5.0 . the predominant formation of pentose aminoxaolines 36 ( 44% ) , 37 ( 30% ) , 38 ( 8% ) , 35-f ( 2% ) , and 35-p ( 10% ) was observed at pd 7.0 .
the reaction initiated at pd 5.0 was lyophilized and dissolved in methanol ( 5 ml ) .
24 was isolated by precipitation with anhydrous diethyl ether ( 10 ml ) and crystallized from methanol to give 29 mg ( 46% ) of 24 as colorless crystals .
an analytical sample of 24 was crystallized three times from aqueous ethanol and then recrystallized from methanol : ch2cl2 ( 1.5:1 ) by evaporation .
ir ( kbr , cm ) : 3443 ( nh ) , 3305 ( oh ) , 2866 , 2807 , 2662 ( ch ) , 1678 ( c = o ) 1652 , 1577 ( n = c ) .
h nmr ( 400 mhz , d2o ) : h 7.16 ( s , 1h , har ) ; 5.72 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 4.93 ( dd , j = 7.4 , 2.4 hz , 1h , h-(c2 ) ) ; 3.72 ( dd , j = 4.2 , 2.4 hz , 1h , h-(c3 ) ) ; 3.63 ( app q , j = 5.2 hz , 1h , h-(c4 ) ) ; 3.50 ( abx , j = 11.7 , 5.0 hz , 1h , ha-(c5 ) ) ; 3.42 ( abx , j = 11.7 , 6.2 hz , 1h , hb-(c5 ) ) .
c nmr ( 101 mhz , d2o ) : c 168.1 ( c = o ) ; 165.1 ( c2 ) ; 141.3 ( c5 ) ; 131.0 ( c2 ) ; 110.4 ( c4 ) ; 78.4 ( c2 ) ; 71.3 ( c1 ) ; 62.5 ( c4 ) ; 53.3 ( c5 ) ; 48.9 ( c3 ) .
m / z ) : 283 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c10h15n6o4 , 283.1149 ; found , 283.1154 .
x - ray diffraction structure solved ; deposited as ccdc 784247 . method a : glyceraldehyde ( 20.7 mg , 0.23 mmol ) , 5-aminoimidazole-4-carbonitrile 2 ( 49.6 mg , 0.46 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 4.0 , 4.5 , 5.0 , 6.0 , or 7.0 . the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see table 2 and supporting information , figure s26 , for h nmr spectra at pd 4.0 , 4.5 , 5.0 , 6.0 , and 7.0 ) . at pd 4.0 ,
the formation of four rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines , 2831 ( 46% , 13:2.2:1 ratio ) , and four 3-imidazoloaminooxazolines , 3235 ( 43% , 16:2.2:1 ratio ) , was observed by h nmr spectroscopy ( see supporting information , page s17 , for partial nmr characterization of compounds 28 , 29 , 31 , 33 , 34 , and 35 ) .
the predominant formation of pentose aminoxaolines 36 ( 44% ) , 37 ( 30% ) , 38 ( 8% ) , 35-f ( 2% ) , and 35-p ( 10% ) was observed at pd 7.0 .
noe intensity volumes are given as strong ( s ) , medium ( m ) , weak ( w ) , or not observed ( ) relative to the volume of h(c1)h(c2 ) of the same compound which is defined as strong .
method b : glyceraldehyde ( 207 mg , 2.3 mmol ) , 5-aminoimidazole-4-carboxamide 2 ( 496 mg , 2.3 mmol ) , and 2-aminooxazole 5 ( 200 mg , 2.3 mmol ) were dissolved in h2o ( 10 ml ) at ph 4.0 .
the reaction was incubated for 2 d , lyophilized , and dissolved in meoh ( 10 ml ) .
sio2 ( 2 g ) was added , the mixture was concentrated to a fine , free - flowing powder in vacuo , and the compounds were eluted with 1:4 to 1:3 methanol : ch2cl2 .
the solids were dissolved in aqueous methanol ( h2o : meoh 0.5:10 ml ) , and etoac ( 6 ml ) was added to partially precipitate the mixture .
the solids were filtered and washed with methanol to give 98 mg of 32 ( 16% ) .
those fractions eluted from silica gel that predominately contained 2831 were concentrated in vacuo to a white foam .
the foam was dissolved in d2o and studied by h , hh cosy , and hh noesy nmr .
the solution was lyophilized , and the solid was dissolved in warm aqueous ethanol . upon cooling ,
white crystalline solids formed ; these were isolated by centrifugation and then dissolved in d2o .
repeated recrystallization of the crystalline solids from h2o afforded 20 mg of 30 as fine , clear needles ( see supporting information , figure s25 ) .
ir ( kbr , cm ) : 3440 ( nh ) , 3323 ( oh ) , 2873 , 2800 , 2658 ( ch ) , 2216 ( cn ) , 1579 ( n = c ) .
h nmr ( 400 mhz , d2o ) : h 7.40 ( s , 1h , h-(c2 ) ) ; 6.00 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 5.22 ( dd , j = 7.4 , 1.9 hz , 1h , h-(c2 ) ) ; 3.93 ( ddd , j = 8.1 , 5.6 , 3.9 hz , 1h , h-(c4 ) ) ; 3.83 ( abx , j = 12.1 , 3.9 hz , 1h , ha-(c5 ) ) ; 3.73 ( abx , j = 12.1 , 5.6 hz , 1h , hb-(c5 ) ) ; 3.55 ( dd , j = 8.1 , 1.9 hz , 1h , h-(c3 ) ) . es - ms ( pos .
m / z ) : 265 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c10h12n6o3 , 265.144 ; found , 265.1050 .
h nmr ( 400 mhz , d2o , pd 1.0 ) : 8.21 ( s , 1h , h-(c2 ) ) ; 5.97 ( d , j = 5.4 hz , 1h , h-(c1 ) ) ; 5.45 ( t , j = 5.4 hz , 1h , h-(c2 ) ) ; 4.17 ( dd , j = 10.0 , 5.4 hz , 1h , h-(c3 ) ) ; 3.87 ( ddd , j = 10.0 , 3.9 , 2.0 hz , 1h , h-(c4 ) ) ; 3.77 ( abx , j = 12.5 , 2.0 hz , 1h , ha-(c5 ) ) ; 3.55 ( abx , j = 12.5 , 3.9 hz , 1h , hb-(c5 ) ) .
c nmr ( 400 mhz , d2o ) : 162.7 ( c2 ) ; 151.4 ( c5 ) ; 135.5 ( c2 ) ; 114.9 ( cn ) ; 88.2 ( c1 ) ; 84.9 ( c2 ) ; 84.0 ( c4 ) ; 78.0 ( c4 ) ; 59.1 ( c5 ) ; 55.5 ( c3 ) .
m / z ) : 265 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c10h12n6o3 , 265.144 ; found , 265.1051 .
glyceraldehyde ( 1.8 g , 20 mmol ) and cyanamide 4 ( 840 mg , 20 mmol ) were dissolved in h2o ( 10 ml ) at ph 10 and heated at 60 c for 2 h. the solution was then cooled , diluted with h2o ( 10 ml ) , and extracted in etoac ( 5 20 ml ) .
the residue was dissolved in etoac and recrystallized by the addition of heptane to give 1.5 g ( 66% ) of 5-(hydroxymethyl)-2-aminooxazole 41 as a white solid .
h nmr ( 400 mhz , meod ) : h 6.58 ( s , 1h , h-(c4 ) ) ; 4.40 ( s , 2h , ch2 ) .
c nmr ( 400 mhz , meod ) : c 163.9 ( c2 ) ; 146.2 ( c5 ) ; 124.5 ( c4 ) ; 55.2 ( ch2 ) .
m / z ) : 115 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c4h7n2o2 , 115.0502 ; found , 115.0507 .
method c:7 ( 100 mg , 0.49 mmol ) and cyanoacetylene ( 0.98 m in h2o , 1.5 ml ) were incubated at 60 c for 72 h. the product was then purified by silica gel chromatography ( eluting with 1:5 to 1:1 ch2cl2:meoh ) , and the fractions containing the product ( rf 0.3 ; 1:5 ch2cl2:meoh ) were concentrated in vacuo .
the product was crystallized from aqueous ethanol to give 120 mg of 43 ( 85% ) as clear , colorless crystals .
h nmr ( 400 mhz , d2o ) : h 7.25 ( d , j = 7.6 hz , 1h , h-(c6 ) ) ; 7.08 ( s , 1h , h - ar ) ; 6.75 ( d , j = 3.8 hz , 1h , h-(c1 ) ) ; 5.93 ( d , j = 3.8 hz , 1h , h-(c5 ) ) ; 4.37 ( ddd , j = 5.3 , 3.8 , 2.2 hz , 1h , h-(c2 ) ) ; 3.53 ( abx , j = 13.0 , 2.2 hz , 1h , h-(c3 ) ) ; 3.41 ( abx , j = 11.7 , 5.0 hz , 1h , h-(c3 ) ) .
c nmr ( 101 mhz , d2o ) : c 168.2 ( c = o ) ; 166.3 ( c2 ) ; 157.8 ( c4 ) ; 144.0 ( c6 ) ; 142.7 ( c5 ) ; 130.6 ( c2 ) ; 109.5 ( c5 ) ; 96.6 ( c4 ) ; 64.8 ( c2 ) ; 62.4 ( c1 ) ; 43.5 ( c3 ) .
m / z ) : 292 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c11h12n7o4 , 292.1153 ; found , 292.1146 .
method c:17 ( 117 mg , 0.50 mmol ) and cyanoacetylene ( 0.98 m in h2o , 2.0 ml ) were incubated at 60 c for 48 h. ( 2r,3s,4s)-3-hydroxy-2-(hydroxymethyl)-4-(2-imino-6-oxo-2h - pyrimido[1,6-a]-pyrimidin-7(6h)-yl)-1,2,3,4-tetrahydroimidazo[1,5-a]-pyrimidine-8-carbonitrile ( 48 ) was observed to precipitate from solution over 4 h and was removed by filtration to give 2 mg of 48 as a yellow solid .
( 2r,3s,4s)-4-(4-amino-2-oxopyrimidin-1(2h)-yl)-3-hydroxy-2-(hydroxymethyl)-1,2,3,4-tetrahydroimidazo[1,5-a]-pyrimidine-8-carbonitrile ( 44 ) was observed to crystallize from aqueous solution over 48 h. the large colorless crystals were used directly for x - ray diffraction .
h nmr ( 400 mhz , d2o ) : h 7.28 ( d , j = 7.4 hz , 1h , h-(c6 ) ) ; 7.13 ( s , 1h , h-(c2 ) ) ; 6.76 ( d , j = 2.7 hz , 1h , h-(c1 ) ) ; 5.96 ( d , j = 7.4 hz , 1h , h-(c5 ) ) ; 4.27 ( d , j = 2.7 hz , 1h , h-(c2 ) ) ; 3.773.87 ( m , 1h , ha-(c4 ) ) ; 3.673.77 ( m , 2h , h-(c3 ) , hb-(c4 ) ) .
c nmr ( 101 mhz , d2o ) : c 166.2 ( c2 ) ; 157.4 ( c4 ) ; 147.2 ( c5 ) ; 142.7 ( c6 ) ; 131.1 ( c2 ) ; 116.5 ( cn ) ; 96.3 ( c5 ) ; 88.6 ( c4 ) ; 66.0 ( c2 ) ; 62.2 ( c1 ) ; 60.4 ( c4 ) ; 55.2 ( c3 ) .
m / z ) : 304 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c12h14n7o3 , 304.1152 ; found , 304.1119 .
h nmr ( 400 mhz , d2o ) : h 8.34 ( s , 1h , h-(c2 ) ) ; 7.62 ( d , j = 7.5 hz , 1h , h-(c6 ) ) ; 7.33 ( d , j = 9.4 hz , 1h , h-(c7 ) ) ; 6.10 ( d , j = 3.5 hz , 1h , h-(c1 ) ) ; 5.96 ( d , j = 7.5 hz , 1h , h-(c5 ) ) ; 5.55 ( d , j = 9.4 hz , 1h , h-(c8 ) ) ; 4.48 ( dd , j = 3.5 , 2.0 hz , 1h , h-(c2 ) ) ; 4.43 ( dd , j = 9.8 , 4.0 hz , 1h , ha-(c4 ) ) ; 4.26 ( app dt , j = 3.0 , 1.9 hz , 1h , h-(c3 ) ) ; 4.0 ( dd , j = 9.8 , 1.9 hz , 1h , hb-(c4 ) ) .
uvvis : max 252 nm ( 9760 ) , 320 ( 6400 ) . es - ms ( pos .
m / z ) : 355 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c15h15n8o3 , 355.1261 ; found , 355.1261 .
method c:21 ( 50 mg , 0.20 mmol ) and cyanoacetylene ( 0.98 m in h2o , 0.5 ml ) were incubated at 60 c for 24 h. the formation of 45 was observed in 80% yield by h nmr integration .
h nmr ( 400 mhz , d2o ) : h 7.28 ( d , j = 7.4 hz , 1h , h-(c6 ) ) ; 7.13 ( s , 1h , h-(c2 ) ) ; 6.76 ( d , j = 2.7 hz , 1h , h-(c1 ) ) ; 5.96 ( d , j = 7.4 hz , 1h , h-(c5 ) ) ; 4.27 ( d , j = 2.7 hz , 1h , h-(c2 ) ) ; 3.773.87 ( m , 1h , h-(c4 ) ) ; 3.673.77 ( m , 2h , h-(c3 ) , h-(c4 ) ) .
c nmr ( 101 mhz , d2o ) : c 167.6 ( c = o ) ; 165.6 ( c2 ) ; 157.3 ( c4 ) ; 142.4 ( c5 ) ; 142.0 ( c6 ) ; 129.5 ( c2 ) ; 108.4 ( c4 ) ; 96.2 ( c5 ) ; 63.3 ( c1 ) ; 62.9 ( c2 ) ; 60.9 ( c4 ) ; 54.1 ( c3 ) .
m / z ) : 304 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c12h16n7o4 , 322.1258 ; found , 322.1284 .
method c:30 ( 5 mg , 0.019 mmol ) and cyanoacetylene ( 0.98 m in h2o , 50 l ) were incubated at 60 c for 24 h. the formation of 46 was observed in 82% yield by h nmr integration ( see supporting information , figure s34 , for crude h nmr ) .
h nmr ( 400 mhz , d2o ) : 7.16 ( d , j = 7.6 hz , 1h , h-(c6 ) ) ; 7.03 ( s , 1h , h-(c2 ) ) ; 6.64 ( d , j = 3.1 hz , 1h , h-(c1 ) ) ; 5.84 ( d , j = 7.6 hz , 1h , h-(c5 ) ) ; 4.30 ( d , j = 3.1 , 1.0 hz , 1h , h-(c2 ) ) ; 3.663.77 ( m , 2h , h-(c4 ) , ha-(c5 ) ) ; 3.58 ( abx , j = 12.1 , 5.8 hz , 1h , hb-(c5 ) ) ; 3.51 ( app d , j = 8.6 hz , 1h , h-(c3 ) ) .
c nmr ( 101 mhz , d2o ) : c 165.6 ( c2 ) ; 157.5 ( c4 ) ; 147.2 ( c5 ) ; 142.0 ( c6 ) ; 130.8 ( c2 ) ; 116 ( cn ) ; 96.0 ( c4 ) ; 96.0 ( c5 ) ; 65.9 ( c1 ) ; 61.7 ( c2 ) ; 69.0 ( c4 ) ; 62.3 ( c5 ) ; 55.2 ( c3 ) .
m / z ) : 334 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c13h16n7o4 , 334.1258 ; found , 334.1251 .
method c:24 ( 20 mg , 0.07 mmol ) and cyanoacetylene ( 0.98 m in h2o , 0.35 ml ) were incubated at 60 c for 24 h. the formation of 47 was observed in 87% yield by h nmr integration ( see supporting information , figure s34 , for crude h nmr ) .
h nmr ( 400 mhz , d2o ) : h 7.16 ( d , j = 7.5 hz , 1h , h-(c6 ) ) ; 7.00 ( s , 1h , h-(c2 ) ) ; 6.74 ( d , j = 4.3 hz , 1h , h-(c1 ) ) ; 5.81 ( d , j = 7.5 hz , 1h , h-(c5 ) ) ; 4.22 ( dd , j = 5.8 , 4.3 hz , 1h , h-(c2 ) ) ; 3.68 ( ddd , j = 5.6 , 4.8 , 2.7 hz , 1h , h-(c4 ) ) ; 3.60 ( abx , j = 11.8 , 4.8 hz , 1h , h-(c5 ) ) ; 3.53 ( abx , j = 11.8 , 5.6 hz , 1h , h-(c5 ) ) ; 3.50 ( dd , j = 5.8 , 2.7 hz , 1h , h-(c3 ) ) .
c nmr ( 101 mhz , d2o ) : c 168.3 ( c = o ) ; 166.1 ( c4 ) ; 157.8 ( c2 ) ; 143.2 ( c5 ) ; 142.6 ( c6 ) ; 129.8 ( c2 ) ; 109.0 ( c4 ) ; 96.7 ( c5 ) ; 69.7 ( c4 ) ; 64.4 ( c2 ) ; 63.7 ( c1 ) ; 63.6 ( c5 ) ; 55.5 ( c3 ) .
m / z ) : 352 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c13h16n7o4 , 352.1364 ; found , 352.1363 | the recent development of a sequential , high - yielding route to activated pyrimidine nucleotides , under conditions thought to be prebiotic , is an encouraging step toward the greater goal of a plausible prebiotic pathway to rna and the potential for an rna world .
however , this synthesis has led to a disparity in the methodology available for stepwise construction of the canonical pyrimidine and purine nucleotides . to address this problem , and further explore prebiotically accessible chemical systems ,
we have developed a high - yielding , aqueous , one - pot , multicomponent reaction that tethers masked - sugar moieties to prebiotically plausible purine precursors .
a ph - dependent three - component reaction system has been discovered that utilizes key nucleotide synthons 2-aminooxazole and 5-aminoimidazoles , which allows the first divergent purine / pyrimidine synthesis to be proposed .
due to regiospecific aminoimidazole tethering , the pathway allows n9 purination only , thus suggesting the first prebiotically plausible mechanism for regiospecific n9 purination . | Introduction
Results and Discussion
Outlook and Summary
Experimental Section | a plausible prebiotic synthesis of the canonical nucleotides has long been a major goal in origins of life research . the oligomerization of hydrogen cyanide one of the major products of spark discharge in atmospheres containing methane and nitrogen to furnish the hcn tetramer diaminomaleonitrile ( 1 ) , its near - quantitative intramolecular photochemical rearrangement to 5-aminoimidazole-4-carbonitrile ( aicn , 2 ) , and the subsequent hydrolysis of aicn 2 to 5-aminoimidazole-4-carboxamide ( aica , 3 ) were demonstrated over 50 years ago. (4 ) however , the further elaboration of aminoimidazoles 2 and 3 to give the purine nucleobases and , after ribosylation and phosphorylation , the purine nucleotides is both very low yielding and , most importantly , not selective for the canonical nucleotides over isomers thereof . for example , adenine and hypoxanthine have been condensed under dry conditions at high temperatures with an excess of ribose in the presence of a high concentration of magnesium salts to yield 3 and 15% of the respective canonical n9-linked -d - ribofuranonucleosides in complex mixtures with many other isomers. (5 ) thus , irrespective of the prebiotic availability of ribose , the formation of adenosine ( and inosine ) is in low yield , alongside a multitude of regioisomers and pyranosyl isomers . (6 ) recently , a stepwise route from simple abiotic molecules considered to be prebiotically available to activated pyrimidine nucleotides was demonstrated ( scheme 1b). ideally , further development of this chemistry would also lead to a stepwise synthetic route for purine nucleotide assembly , thus providing a plausible pathway by which the four nucleotides required for an information - rich rna coding system would be prebiotically available . therefore , we considered ways in which 5 could additionally participate in the synthesis of purines . the diversity of chemical species likely to have been present under prebiotic conditions and the proven utility of multicomponent reactions ( mcrs ) to yield biologically relevant structures suggested that the development of novel mcrs could lead to the rapid buildup of molecular complexity and , in particular , to the structures that are central to molecular biology . as the five carbon atoms of ribose are contiguous , the formation of the canonical purine ribonucleotides from simple aldehydes should be considered both in the context of one c2 and one c3 fragment , and of one c1 and two c2 fragments . upon mixing formaldehyde , 5 , and 2 or 3 in water at room temperature and near neutral ph , the rapid , efficient synthesis of rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines 6 or 7 was observed ( scheme 2 ) . these reactions were found to be scalable to multigram levels , and the products crystallized from concentrated aqueous solution , giving 6 and 7 in 89 and 80% isolated yield , respectively . the -fused tricyclic structures were proven by x - ray diffraction analysis of a single crystal of 7 crystallized at ph 6.0 and a single crystal of the hemi - hydrochloride salt of 6 crystallized at ph 5.0 . pyrimidine nucleotide precursors result from two - component chemistry ( 2cr),(1 ) while three - component chemistry ( 3cr ) leads to potential purine nucleotide precursors . to further explore the generality of the mcr
the reaction was shown to tolerate acetaldehyde to yield 11 and 12 as a 2:1 ( erythro : threo ( e : t ) ) mixture of diastereomers . the minor rac-[2s,3r]-isomer 12 was then isolated by precipitation and crystallization , which allowed the structure to be proven unambiguously by single - crystal x - ray diffraction . (11 ) ph - dependent reactivity was observed , with three - component chemistry dominating at lower ph values and two - component chemistry dominating a higher ph values ( vide infra ) . notably , the products were uncontaminated with homo - aldolization or homo - mannich byproducts , and the three - component products exhibited a high diastereoselectivity for rac-[3r,4r]-products important for realization of a -ribo - nucleotide synthesis by c3-stereochemical inversion ( schemes 5 and 6 ) . (12 ) at pd 5.0 , the reaction of glycolaldehyde , aicn 2 , and 5 gave a 4:1 mixture of diastereoisomers 16 and 17 , alongside 10% two - component tetrose aminooxazoline products 18 and 19 . at pd 4.0 ,
the reaction gave a near - quantitative conversion to three - component products ; however , 21% of these products were 20 and 21 , which result from 5-exo - trig ring closure of intermediate 9. (1 ) however , a high - yielding synthesis of ribo - purinosides via c3-tethered aminoimidazoles would require high [ 3r,4r]-diastereoselectivity ( lyxo / xylo ) , a reversal of the facial selectivity of glyceraldehyde upon bimolecular reaction with 5. (14 ) at pd 5.0 , the reaction of glyceraldehyde , 5 , and aica 3 furnished lyxo - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine ( 24 ) in 60% yield ( by h nmr analysis ; 46% isolated yield ) , alongside three other diastereoisomers , 2527 , in 11 , 5 , and 8% yields , respectively ( by h nmr analysis ) . at pd 4.0 , the reaction of aicn 2 , 5 , and glyceraldehyde was highly selective for three - component products ( 89% by h nmr analysis ) . however , likely due to an increased thorpeingold effect in 9 ( where r = ch(oh)ch2oh relative to r = ch2oh ) and the lower ph required for selective three - component reaction of 2 relative to 3 , competitive 6-exo - trig imidazole cyclization and 5-exo - trig hydroxyl cyclization resulted in a 1:1 mixture of rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines 2831 and rac-3-aminoimidazole-2-aminooxazolines 3235 . the three - component reaction of glyceraldehyde with 2 or 3 and 5 is highly lyxo - selective , yielding 66% of 28/32 or 60% of 24 , respectively , whereas the bimolecular reaction of glyceraldehyde and 5 is highly ribo / arabino - selective ( ribo36 = 44% and arabino37 = 30%). (14 ) the high lyxo - selectivity of the three - component reaction establishes the required c1 , c3 , and c4 stereochemistry for the proposed ribo - nucleotide synthesis by c3 stereochemical inversion , while the ribo / arabino - selectivity of the two - component reaction establishes the required c3 and c4 stereochemistry for pyrimidine synthesis . the stereochemical preference for lyxo / xylo three - component products or ribo / arabino two - component products likely derives from the control of facial selectivity of the imine or aldehyde component , respectively,(14 ) coupled with the obligatory z - geometry of the nucleophilic c = c bond of 5 ( see supporting information , figure s36 ) , though calculations to support this conjecture have not been carried out . (17 ) equilibration of 28 and 32 is proposed to occur via intermediate 9 and to be controlled by imidazole protonation ; therefore , at low ph , protonation of the imidazole moiety results in complete furanosyl selectivity ( see supporting information , figure s27 , for h nmr spectra of 28/32 at pd 112 ) . although efficient three - component reactivity is observed at ph values lower than 6.0 , the reaction profile of -hydroxyaldehydes with aicn 2 or aica 3 and 2ao 5 was observed to shift to furnish only bimolecular aminooxazoline products at higher ph s , even in the presence of a large excess of 2 or 3 . (14 ) similarly , the reaction of glyceraldehyde , 2 or 3 , and 5 at pd 7.0 yields a mixture of pentose aminooxazolines 3639 in a 1.5:1 [ 2r,3r]:[2s,3r ] ratio. (14 ) the more or less complete switch between two- and three - component reactivity is observed to occur over 2 ph units at an aminoimidazole pka - dependent ph . however , surprisingly , at pd 7.0 , formaldehyde and 2 or 3 were observed to react selectively with 5 as imine / iminium 8 , resulting in the synthesis of 6 or 7 ( schemes 2 and 3 ) . (18 ) it is possible that , due to the stability of the hemiaminal 42 formed between the amino group of 5 and formaldehyde , there is insufficient free aldehyde form of formaldehyde to react with free 5 at an observable rate. as the nitrile of derivatives of 2 retains the potential to give divergent access to adenine and guanine nucleotides , it is of note that no significant nitrile hydrolysis was observed in our experiments . selective cyanovinylation of the aminooxazoline moiety of the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines is one of several routes that may allow for the subsequent removal of those atoms derived from cyanamide 4 ( scheme 3 ) through photoactivated hydrolytic ( or otherwise ) removal of the resultant cytosine moiety , thereby affording products constitutionally closer to purine nucleotides . to investigate this potential for pyrimidine nucleobase elaboration and potential subsequent loss , a series of cyanovinylation experiments was undertaken to show that the aminooxazoline moiety of the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine structure could be selectively cyanovinylated . furthermore , we thought that the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines were likely to be intrinsically protected from c3nh cyanovinylation by delocalization of the nitrogen lone pair into the imidazole ring . even in the presence of 5 equiv of cyanoacetylene , clean conversion of 6 , 17 , 21 , 24 , and 30 to 4-amino-1-(3-hydroxy-1,2,3,4-tetrahydroimidazo[1,5-a]-pyrimidin-4-yl)pyrimidin-2-(h)-ones 4347
was observed over the period of 13 days at 60 c in unbuffered aqueous solution in 8095% yield. we have described a concise and high - yielding route to the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines . it is of note that the mcr described provides a high yield of [ 3r,4r]-products ( 70% lyxo / xylo ) , reversing the facial selectivity of glyceraldehyde observed in the biomolecular reaction with 2ao 5 ( 70% ribo / arabino ) , with the lyxo and xylo products being stereochemically related via c2-epimerization. (24 ) upon cyanovinylation and dissociative cytosine cleavage , compounds 27 , 31 , and 34 should , via intramolecular ring closure , furnish 5,3-anhydro - aicn - riboside 49 ( where r = ch2oh ) , which has an obvious relationship to aica - riboside 50 , an intermediate in the de novo biosynthesis of purines ( scheme 10 ) . access to 49 should allow further study of the selectivity issues involved in synthesizing the canonical nucleotides as opposed to closely related chemical structures capable of watsoncrick base - pairing interactions . it is of note that the proposed phosphorylation of the 2-hydroxyl group of 49 could lead to purines via a c3 stereochemical inversion , which would be directly analogous to the c2 inversion previously described to give access to the pyrimidine nucleotides . in summary , we have described additional predisposed complex structures that can be accessed through the participation of the key intermediate 2-aminooxazole 5 in a novel aqueous multicomponent reaction system . this mcr has proved to be scalable and high - yielding , and it generates a ph - dependent product distribution . further , the intrinsically controlled cyanovinylation of the low - ph mcr products generates potential precursors of the purine nucleotides . these reactions help to define the chemical structural space and chemical distribution likely to be accessible under plausible prebiotic chemical scenarios . in particular , we have demonstrated the concurrent synthesis of 37 and the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines in the ph range in which activated pyrimidine synthesis can occur ( ph < 6.5 , where hydrolysis of 53 is prevented),(1 ) suggesting that both the pyrimidine and the purine ribonucelotides could be made together at the same time , in the same place , and under the same conditions. (29 ) the reactions may therefore be of direct relevance to the problem of abiogenesis of a full set of canonical ribonucleotides , and hence the chemical origins of molecular biology . where possible , the chemical shift of the corresponding solvent was used as a reference . method c : an aqueous solution of cyanoacetylene ( 0.98 m , 35 equiv ) was added to tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines ( 20 mm ) , and the solution was then heated at 60 c for 13 d with stirring . formaldehyde ( 37% , 18.6 mg , 0.23 mmol ) was added in d2o ( 0.5 ml ) , the pd was rechecked , and the reaction volume was adjusted to 2 ml by the addition of d2o . the reactions were incubated at room temperature , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s4 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . formaldehyde ( 37% , 0.96 g , 11.9 mmol ) was added , the ph was rechecked , and the reaction volume was adjusted to 20 ml with h2o . the reaction was mechanically stirred for a further 15 h. the solids present were isolated by filtration , and the filtrate was lyophilized . formaldehyde ( 37% , 18.6 mg , 0.23 mmol ) was added in d2o ( 0.5 ml ) , the pd was rechecked , and the reaction volume was adjusted to 2 ml with d2o . the reactions were incubated for 15 h , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s4 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . formaldehyde ( 37% , 0.96 g , 11.9 mmol ) was added , the ph was rechecked , and the reaction volume was adjusted to 20 ml with h2o . the reaction was mechanically stirred for 15 h. the solids present were isolated by filtration , and the filtrate was lyophilized . the solids were dissolved in hot water ( 4 ml ) , and the product was allowed to crystallize upon cooling . the reaction was incubated for 15 h , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s9 , for h nmr spectrum ) . the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see table 1 and supporting information , figure s11 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s17 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . the reaction was incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s23 , for h nmr spectra at pd 5.0 , 6.0 , 6.5 , and 7.0 ) . the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see table 2 and supporting information , figure s26 , for h nmr spectra at pd 4.0 , 4.5 , 5.0 , 6.0 , and 7.0 ) . at pd 4.0 ,
the formation of four rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines , 2831 ( 46% , 13:2.2:1 ratio ) , and four 3-imidazoloaminooxazolines , 3235 ( 43% , 16:2.2:1 ratio ) , was observed by h nmr spectroscopy ( see supporting information , page s17 , for partial nmr characterization of compounds 28 , 29 , 31 , 33 , 34 , and 35 ) . sio2 ( 2 g ) was added , the mixture was concentrated to a fine , free - flowing powder in vacuo , and the compounds were eluted with 1:4 to 1:3 methanol : ch2cl2 . the solution was lyophilized , and the solid was dissolved in warm aqueous ethanol . method c:7 ( 100 mg , 0.49 mmol ) and cyanoacetylene ( 0.98 m in h2o , 1.5 ml ) were incubated at 60 c for 72 h. the product was then purified by silica gel chromatography ( eluting with 1:5 to 1:1 ch2cl2:meoh ) , and the fractions containing the product ( rf 0.3 ; 1:5 ch2cl2:meoh ) were concentrated in vacuo . | [
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] |
root canal therapy is known as the main etiologic factor for iatrogenic tooth discoloration ( entombment of pulp tissue remnants or metal - including root canal sealers ) . as a result
, nonvital bleaching has gained so much attention , due to the growing demand for beautiful smiles and white teeth . walking or internal bleaching technique
effective bleaching agents used to lighten the tooth color in this technique include hydrogen peroxide ( h2o2 ) and sodium perborate , the latter being effective by releasing hydrogen peroxide . given the appropriate application , the walking bleaching is a relatively low - risk and minimally invasive intervention and bears only slight risks if performed correctly .
nevertheless , concerns exist regarding decreasing the likelihood of cervical root resorption as an important complication .
it is stated that the oxidative action of bleaching agent and releasing of nascent oxygen which is later transferred to the cervical periodontal ligament ( pdl ) through the dentinal tubules and cementum defects can act as a stimulus for inflammatory changes of the region and subsequent up - regulation of odontoclastic cells responsible for invasive cervical root resorption .
this phenomenon is probably rendered by the high concentration of intra - coronal oxidizing agents reaching the root surface .
effective sealing of the canal orifice can eliminate the risk for this tubular transferring of the pdl irritants and thus can reduce the risk of this phenomenon . due to decreased ph at the root surface , cementum necrosis happens , the periodontium is inflamed , and eventually root resorption emerges .
the incidence of cervical root resorption has been reported rather high , from 1% to 13% .
the general recommendation today is :
not to heat the bleaching agent in the access cavity , because heat can damage periodontal tissue and lead to an increased resorption rate at the root surface , andto cover the root canal filling material with a base in order to eliminate or decrease the diffusion of oxidizing agents from the access cavity toward root surface .
not to heat the bleaching agent in the access cavity , because heat can damage periodontal tissue and lead to an increased resorption rate at the root surface , and to cover the root canal filling material with a base in order to eliminate or decrease the diffusion of oxidizing agents from the access cavity toward root surface .
the importance of this cervical sealing has been extensively documented , for instance , over a 20-year period of follow - up of nonvital teeth bleached with 30% hydrogen peroxide with adequate cervical sealing , no root resorption was observed . a variety of dental materials such as hydrophilic filling materials ( cavit , coltosol ) , intermediate restorative material ( irm ) , zinc oxide - eugenol cement , zinc phosphate cement , photo - activated temporary resin materials , and conventional or resin - modified glass - ionomer ( gi )
have been suggested as sealing agents to be laid on root - canal filling material during intra - coronal bleaching .
if temporary filling materials are used , their removal prior to final restoration of the bleached tooth is essential .
therefore , a 2-mm layer of gi cement has been recommended as a standard protective intra - orifice barrier during bleaching , which can be left in place to be a base for the final restoration .
microleakage tests can well demonstrate the sealing ability of root filling materials . in endodontics , this index is measured principally based on the penetration of trace agents through the filled canal ; which include radioisotopes , dyes , bacterium and their products such as proteins . comparing with the other methods available for leakage assessment in the field of endodontic research , from our standpoint , protein leakage test is superior , particularly because it has more clinical relevancy ,
furthermore , there is no liability for sample destruction , and hence the leakage might be evaluated repeatedly , if needed . in case of mineral trioxide aggregate ( mta ) , numerous studies have shown its suitability for a wide variety of applications such as successful treatment of invasive cervical resorption .
an outstanding specification of mta is its superior leakage resistance , which may be explained by its marginal adaptation .
mta has biological benefits due to high concentration of calcium hydroxide , in its formulation after mixing with water . upon high alkalinity
, it is hypothesized that mta may prevent or arrest tooth resorption . on the other hand ,
the effect of alkaline and acidic ph values on physical properties of wmta ( white mta ) has been well - documented .
the potential tooth discoloration can be the only reason that prevents mta to be used as an effective intra - orifice barrier during tooth bleaching .
recently , calcium enriched mixture ( cem ) cement has been introduced as a new root - end filling material .
it is dominantly composed of calcium compounds . due to being biocompatible , well - tolerable by oral tissues such as pulp and pdl and providing comparable seal to mta this new biomaterial
is proved to be applicable for other purposes in the field of endodontics , such as perforations repair , coronal seal , root canal filling , and vital pulp therapies as well .
besides all the aforementioned criteria , cem cement offers the advantage of not causing tooth discoloration that is favorable in cases of intra - coronal bleaching .
this interventional study was designated and conducted to compare the sealing ability of gi , mta and cem cement when used as intra - orifice barrier in nonvital bleaching .
ethical committee of mazandaran university of medical sciences approved ( code : 91 - 175 ) to use the extracted teeth after the informed consent form would have been filled out by the volunteers .
seventy freshly extracted human maxillary incisors with standard root length ( 10 - 13 mm ) were selected for this study . the teeth were extracted because of periodontal reasons . after cleaning the teeth , radiographies were taken in buccolingual and mesiodistal directions .
teeth with calcification , internal or external resorption , cracks ( detectable under light stereomicroscope ) , severe coronal or root caries , large coronal restoration , root fractures , dilacerations , deep depression on root surfaces , and those with apical foramen larger than file size # 40 were excluded , and if necessary , they were replaced with intact ones .
all teeth had mature apices and straight patent canals . to control the cross infection and minimize soft tissues and periodontal remnants ,
all teeth were stored in normal saline then in a 5.25% solution of sodium hypochlorite ( naocl ) ( golrang , tehran , iran ) for 6 h and were finally stored in 0.5% chloramine solution until experiment commencement .
the access cavity was prepared using a high - speed handpiece and # 2 round diamond bur ( jota , ruthi , switzerland ) under copious water irrigation .
then , the working length was determined by # 15 k - file ( mailer , ballaigues , switzerland ) inserted into the canal until the file tip got visible at the apex .
the coronal and mid segments of the canals were prepared with sizes 3 - 1 gates - glidden drills ( mailer , ballaigues , switzerland ) and then complete preparation of the canals was conducted using mtwo rotary system ( vdw , munich , germany ) in standard preparation technique up to file 40/0.04 as the master apical preparation . the canal was alternatively irrigated with 2 ml of 2.6% naocl during instrumentation between each file size .
, chongchong buch do , korea ) for removal of the smear layer , followed by 5 ml of normal saline .
the canals were then dried with absorbent paper points ( ariadent , tehran , iran ) and obturated using gutta - percha ( diadent inc .
, chongchong buch do , korea ) and ah-26 sealer ( dentsply , tulsa dental , tulsa , ok , usa ) with cold lateral condensation technique
. excess sealer of the pulp chamber and dentinal tubules was removed with alcohol - soaked cotton pellets and the final obturation radiography was taken [ figure 1a ] .
thereafter , a peeso reamer # 3 ( maillefer , ballaigues , switzerland ) was used to remove the intra canal gutta - percha , 3 mm below the cementoenamel junction ( cej ) in palatal aspect .
the cavity depth was confirmed by a periodontal probe and radiographed as well [ figure 1b ] .
the teeth were randomly divided into three experimental groups ( n = 20 ) and two positive and negative control groups , of five each using simple randomization allocation .
the canal obturating material was covered as follows : group 1-rmgic - lc fuji ii ( fuji corp .
hyogo , japan ) , group 2-wmta ( tooth - colored formula , anglus , londrina , brazil ) , and group 3-cem cement ( bioniquedent , tehran , iran ) .
all the materials were prepared according to the manufacturer 's instruction [ table 1 ] , and were packed into the unfilled cervical portion of the canals up to the palatal and facial aspects of cej to provide a 3-mm thick barrier . at this step ,
all samples were radiographically evaluated for the length and density of the intra - orifice barrier [ figure 1c ] .
wet cotton pellets were placed over wmta and cem cement to provide their setting hydration .
all the teeth were temporized with cavit ( espe - premier , norristown , pa , usa ) and incubated at 37c for 24 h at a 90% relative humidity .
the bleaching agent used in the study was a mixture of 0.15 g sodium perborate ( merck , darmstadt , germany and 0.05 ml distilled water .
it was placed in the pulp chamber , and the teeth were restored again with cavit and then incubated composition and usage instruction for material used in the study the bleaching period was arranged for 9 days . the bleaching agent was being refreshed every 3 days . at the end of the 9 day , cavit was removed again , and pulp chamber was rinsed with distilled water and dried and was then filled with cavit for the last time . in positive control group , teeth received neither coronal barrier nor temporal restoration after canal obturation .
external surfaces of teeth except for the 2-mm apical part were covered using two layers of nail polish . in negative control group ,
all surfaces even the temporal restoration were covered and sealed with two layers of nail polish after filling the canals with sticky wax . in case of specimens in three experimental groups ,
all surfaces were covered with two layers of nail polish except for access cavity margins and apical 2-mm portion .
all the samples were mounted in a dual - chamber leakage apparatus as shown in schematic view [ figure 2 ] .
first , the teeth were inserted from the cap end of a 3-ml plastic eppendorf cylinder ( elkay , shrewbury , ma , usa ) .
the ending 3 mm of cylinders were cut previously , so the root tips passed through this part and were visible .
the glass vials had been filled previously with 9 ml of distilled water . the junction line between microtube and vial
was tightly covered and sealed with parafilm ( supa co. , tehran , iran ) .
the whole system was sterilized with ethylene oxide gas for 12 h. the plastic cylinders were filled with 1 ml of 22% bovine serum albumin ( bsa , sigma - aldrich , st .
the assembly was incubated again at 37c and a relative humidity of 90% for 30 days ( test period ) .
bradford indicator was used to measure the concentration of leaked albumin form the upper chamber into the lower one at the end of the 30 day .
bradford protein reagent is an aqueous solution of coomassie brilliant blue g ( sigma - aldrich , st .
the procedure is based on the formation of a complex between the dye , brilliant blue g , and proteins in solution .
according to the manufacturer , albumin leakage into the solution and subsequent formation of the protein - dye complex would shift the wavelength of maximum absorption of coomassie brilliant blue g from 465 to 596 nm .
then 100 l of test solution of the vials was pipetted into a new eppendorf tube and 1 ml of bradford protein reagent was added to the tube , and the contents were mixed .
statistical analysis of the data was conducted using the kruskal - wallis followed by the mann - whitney tests .
the level of significance was set at 0.05 ( 17.0 spss for windows , chicago , il , usa ) .
ethical committee of mazandaran university of medical sciences approved ( code : 91 - 175 ) to use the extracted teeth after the informed consent form would have been filled out by the volunteers .
seventy freshly extracted human maxillary incisors with standard root length ( 10 - 13 mm ) were selected for this study . the teeth were extracted because of periodontal reasons . after cleaning the teeth , radiographies were taken in buccolingual and mesiodistal directions .
teeth with calcification , internal or external resorption , cracks ( detectable under light stereomicroscope ) , severe coronal or root caries , large coronal restoration , root fractures , dilacerations , deep depression on root surfaces , and those with apical foramen larger than file size # 40 were excluded , and if necessary , they were replaced with intact ones .
all teeth had mature apices and straight patent canals . to control the cross infection and minimize soft tissues and periodontal remnants ,
all teeth were stored in normal saline then in a 5.25% solution of sodium hypochlorite ( naocl ) ( golrang , tehran , iran ) for 6 h and were finally stored in 0.5% chloramine solution until experiment commencement .
the access cavity was prepared using a high - speed handpiece and # 2 round diamond bur ( jota , ruthi , switzerland ) under copious water irrigation .
then , the working length was determined by # 15 k - file ( mailer , ballaigues , switzerland ) inserted into the canal until the file tip got visible at the apex .
the coronal and mid segments of the canals were prepared with sizes 3 - 1 gates - glidden drills ( mailer , ballaigues , switzerland ) and then complete preparation of the canals was conducted using mtwo rotary system ( vdw , munich , germany ) in standard preparation technique up to file 40/0.04 as the master apical preparation . the canal was alternatively irrigated with 2 ml of 2.6% naocl during instrumentation between each file size .
, chongchong buch do , korea ) for removal of the smear layer , followed by 5 ml of normal saline .
the canals were then dried with absorbent paper points ( ariadent , tehran , iran ) and obturated using gutta - percha ( diadent inc .
, chongchong buch do , korea ) and ah-26 sealer ( dentsply , tulsa dental , tulsa , ok , usa ) with cold lateral condensation technique
. excess sealer of the pulp chamber and dentinal tubules was removed with alcohol - soaked cotton pellets and the final obturation radiography was taken [ figure 1a ] .
thereafter , a peeso reamer # 3 ( maillefer , ballaigues , switzerland ) was used to remove the intra canal gutta - percha , 3 mm below the cementoenamel junction ( cej ) in palatal aspect .
the cavity depth was confirmed by a periodontal probe and radiographed as well [ figure 1b ] .
the teeth were randomly divided into three experimental groups ( n = 20 ) and two positive and negative control groups , of five each using simple randomization allocation .
in the experimental groups , the canal obturating material was covered as follows : group 1-rmgic - lc fuji ii ( fuji corp .
hyogo , japan ) , group 2-wmta ( tooth - colored formula , anglus , londrina , brazil ) , and group 3-cem cement ( bioniquedent , tehran , iran ) .
all the materials were prepared according to the manufacturer 's instruction [ table 1 ] , and were packed into the unfilled cervical portion of the canals up to the palatal and facial aspects of cej to provide a 3-mm thick barrier . at this step ,
all samples were radiographically evaluated for the length and density of the intra - orifice barrier [ figure 1c ] .
wet cotton pellets were placed over wmta and cem cement to provide their setting hydration .
all the teeth were temporized with cavit ( espe - premier , norristown , pa , usa ) and incubated at 37c for 24 h at a 90% relative humidity . the bleaching agent used in the study was a mixture of 0.15 g sodium perborate ( merck , darmstadt , germany and 0.05 ml distilled water .
it was placed in the pulp chamber , and the teeth were restored again with cavit and then incubated composition and usage instruction for material used in the study the bleaching period was arranged for 9 days .
the bleaching agent was being refreshed every 3 days . at the end of the 9 day
, cavit was removed again , and pulp chamber was rinsed with distilled water and dried and was then filled with cavit for the last time .
in positive control group , teeth received neither coronal barrier nor temporal restoration after canal obturation .
external surfaces of teeth except for the 2-mm apical part were covered using two layers of nail polish . in negative control group ,
all surfaces even the temporal restoration were covered and sealed with two layers of nail polish after filling the canals with sticky wax . in case of specimens in three experimental groups , all surfaces were covered with two layers of nail polish except for access cavity margins and apical 2-mm portion .
all the samples were mounted in a dual - chamber leakage apparatus as shown in schematic view [ figure 2 ] .
first , the teeth were inserted from the cap end of a 3-ml plastic eppendorf cylinder ( elkay , shrewbury , ma , usa ) .
the ending 3 mm of cylinders were cut previously , so the root tips passed through this part and were visible .
the glass vials had been filled previously with 9 ml of distilled water . the junction line between microtube and vial was tightly covered and sealed with parafilm ( supa co. , tehran , iran ) .
the whole system was sterilized with ethylene oxide gas for 12 h. the plastic cylinders were filled with 1 ml of 22% bovine serum albumin ( bsa , sigma - aldrich , st .
the assembly was incubated again at 37c and a relative humidity of 90% for 30 days ( test period ) .
bradford indicator was used to measure the concentration of leaked albumin form the upper chamber into the lower one at the end of the 30 day .
bradford protein reagent is an aqueous solution of coomassie brilliant blue g ( sigma - aldrich , st .
the procedure is based on the formation of a complex between the dye , brilliant blue g , and proteins in solution .
according to the manufacturer , albumin leakage into the solution and subsequent formation of the protein - dye complex would shift the wavelength of maximum absorption of coomassie brilliant blue g from 465 to 596 nm .
then 100 l of test solution of the vials was pipetted into a new eppendorf tube and 1 ml of bradford protein reagent was added to the tube , and the contents were mixed .
schematic view of dual - chamber technique used for protein leakage analysis . using spectrophotometry ,
statistical analysis of the data was conducted using the kruskal - wallis followed by the mann - whitney tests .
the level of significance was set at 0.05 ( 17.0 spss for windows , chicago , il , usa ) .
the mean standard deviation leakage of samples from negative control , positive control , gi , mta , and cem cement groups were 0.0 , 8.9 0.03 , 0.47 0.02 , 0.48 0.02 , and 0.49 0.02 mg / ml , respectively .
the decrease of acidity on external surfaces of teeth during internal bleaching has been considered as an important causative factor for cervical root resorption . for the very same reason , it is recommended to cover the canal filling materials with a barrier to occlude the dentinal tubules as a way of diffusion between pulp cavity and root surface .
considering the fact that root fillings per se are not leak proof , and that no sealer cement or obturation technique consistently prevents percolation through the canal , it is critical to take most care in creating bests of the seal and consider the quality of intra - orifice barrier in nonvital bleaching .
the success of walking bleaching rests somehow upon the sealing quality of the cervical barrier , especially when the remaining dentin walls are very thin .
it is strongly suggested to apply only low concentration of bleaching agents , or to mix sodium perborate with distilled water instead of hydrogen peroxide , in order to govern the detrimental effects of the bleaching agents , although the importance of cervical barrier is not allowed to be ignored .
attempts are underway to introduce more qualified materials with the potential to provide a long - term seal .
recently mta has been implemented to seal the root canal . to date , no study evaluated the intra - orifice sealing ability of cem cement in bleaching .
its formulation is composed of a different mixture of calcium compounds to provide a bioactive calcium - phosphate enriched material .
. studies on cem cement showed that it can release phosphorous and calcium ions which promotes the alkalinity and also leads to mineralization process , implying its hard tissue inductivity .
hydroxyapatite formation is demonstrated to be resulted via the reaction between calcium and phosphorous ions released with endogenous and exogenous sources . to date
, cem cement has not been studied as an intra - orifice plug to resist the microleakage in nonvital bleaching process while it has been implemented for treatment of furcal perforation , vital pulp therapies in permanent and primary teeth , root end fillings , management of root resorption and pathologic / iatrogenic perforations , periradicular surgery , and revascularization for necrotic immature permanent molars .
compared to mta , cem cement has a shorter setting time and also significantly superior results in film thickness and flow , easier handling , and enhanced antibacterial effect as well as better abilities to form hydroxyapatite in normal saline .
in addition to the mentioned properties , it has been shown that this material provides favorable apical / coronal sealing property similar to that of commercial types of mta and superior to intermediate restorative material ( irm ) .
it is shown that cem cement has less microleakage compared to different types of mta although not significant .
the sealing ability of endodontic materials has been evaluated using dye or bacterial penetration , electrical method , fluid filtration technique , human saliva leakage model , glucose leakage model , radioisotope tracing , and marginal adaptation . some advantages and also disadvantages
have been reported for each of these techniques , for instance , bacterial leakage assesses the sealing ability of all portions while in dye leakage only the walls adjacent to material are observed .
bacterial leakage studies focus on the main pathogenic factors , but even this method can not mirror the real clinical condition as special species or a limited number of bacteria are used and so , bacterial synergistic effect , influence of environment , thermal changes , salivary enzymes , buffering materials and antibodies are neglected . of these techniques ,
some of them provide higher biological and clinical relevance . in this study , protein leakage approach was adopted since
is met our answers more convincingly than the others . to show the importance of cervical barrier in leakage prevention , valadares et al . stated that the use of cervical barrier prevents the microleakage of enterococcus faecalis . according to yavari et al . , cem and mta are more effective than amalgam and composite resin , in case of coronal sealing in endodontically treated teeth .
roghanizad and jones revealed that amalgam , as an orifice plug , is more efficacious than cavit in preventing coronal microleakage .
tselnik et al . reported no difference between gray mta , white mta , or a resin modified gi , in terms of bacterial penetration with human saliva model .
based on the findings of a recent study , using the glucose penetration model , bailn - snchez et al .
cavit , and proroot mta has similar abilities to resist leakage when used as intra - orifice barriers .
barrieshi - nusair and hammad compared gi and mta as orifice plugs and reported that gi has more microleakage which is not concurring with our findings . in their study
, each group included 30 samples , and the leakage assessment method was dye leakage for 48 h , but we had 20 teeth in each experimental group which were analyzed in protein leakage approach for 30 days .
the mentioned facts might be the reason of the different results obtained . in another study that was conducted by vosoughhosseini et al . to compare mta and gi as coronal barrier in nonvital bleaching ,
the results of the present study revealed that cem cement has sealing potential as an intra - orifice barrier against protein penetration that is comparable with that of mta and gi . as mentioned before ,
this noticeable sealing ability can be probably the result of the reaction between calcium and phosphorous ions , although , in most part , can be related to hydrophilic nature , good antibacterial / fungal potential , high ph and formation of hydroxyapatite crystals as well .
the obtained results opened perspectives for use of this newly - introduced biomaterial in bleaching interventions on ground of sealing ability .
other experimental settings , particularly for leakage assessment are needed to simulate the clinical reality . among the limitations ,
technique sensitivity of protein leakage assessment method , and the cost of the materials were more prominent .
considering the results of the present study , on ground of the leakage resistance , it is concluded that all materials used ; gi , mta , and cem cement are able to convincingly seal the coronal portion of the canal during nonvital bleaching in order to prevent the cervical root resorption .
the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article .
the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . | background : this study was designed to assess the microleakage of glass - ionomer ( gi ) , mineral trioxide aggregate ( mta ) , and calcium - enriched mixture ( cem ) cement as coronal orifice barrier during walking bleaching.materials and methods : in this experimental study , endodontic treatment was done for 70 extracted human incisors without canal calcification , caries , restoration , resorption , or cracks .
the teeth were then divided into three experimental using simple randomization allocation ( n = 20 ) and two control groups ( n = 5 ) .
the three cements were applied as 3-mm intra - orifice barrier in test groups , and bleaching process was then conducted using a mixture of sodium perborate powder and distilled water , for 9 days . for leakage evaluation ,
bovine serum albumin marker was traced in a dual - chamber technique with bradford indicator .
the kruskal - wallis and mann - whitney tests were used for statistical analysis.results:the mean standard deviation leakage of samples from negative control , positive control , gi , mta , and cem cement groups were 0.0 , 8.9 0.03 , 0.47 0.02 , 0.48 0.02 , and 0.49 0.02 mg / ml , respectively .
statistical analysis showed no significant difference between three experimental groups ( p > 0.05).conclusion : it is concluded that gi , mta , and cem cements are considered as suitable intra - orifice barrier to provide coronal seal during walking bleaching . | INTRODUCTION
MATERIALS AND METHODS
Preparation of specimens
Experimental groups
Control groups
Protein leakage test
RESULTS
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest | walking or internal bleaching technique
effective bleaching agents used to lighten the tooth color in this technique include hydrogen peroxide ( h2o2 ) and sodium perborate , the latter being effective by releasing hydrogen peroxide . given the appropriate application , the walking bleaching is a relatively low - risk and minimally invasive intervention and bears only slight risks if performed correctly . it is stated that the oxidative action of bleaching agent and releasing of nascent oxygen which is later transferred to the cervical periodontal ligament ( pdl ) through the dentinal tubules and cementum defects can act as a stimulus for inflammatory changes of the region and subsequent up - regulation of odontoclastic cells responsible for invasive cervical root resorption . this phenomenon is probably rendered by the high concentration of intra - coronal oxidizing agents reaching the root surface . due to decreased ph at the root surface , cementum necrosis happens , the periodontium is inflamed , and eventually root resorption emerges . not to heat the bleaching agent in the access cavity , because heat can damage periodontal tissue and lead to an increased resorption rate at the root surface , and to cover the root canal filling material with a base in order to eliminate or decrease the diffusion of oxidizing agents from the access cavity toward root surface . the importance of this cervical sealing has been extensively documented , for instance , over a 20-year period of follow - up of nonvital teeth bleached with 30% hydrogen peroxide with adequate cervical sealing , no root resorption was observed . a variety of dental materials such as hydrophilic filling materials ( cavit , coltosol ) , intermediate restorative material ( irm ) , zinc oxide - eugenol cement , zinc phosphate cement , photo - activated temporary resin materials , and conventional or resin - modified glass - ionomer ( gi )
have been suggested as sealing agents to be laid on root - canal filling material during intra - coronal bleaching . therefore , a 2-mm layer of gi cement has been recommended as a standard protective intra - orifice barrier during bleaching , which can be left in place to be a base for the final restoration . comparing with the other methods available for leakage assessment in the field of endodontic research , from our standpoint , protein leakage test is superior , particularly because it has more clinical relevancy ,
furthermore , there is no liability for sample destruction , and hence the leakage might be evaluated repeatedly , if needed . in case of mineral trioxide aggregate ( mta ) , numerous studies have shown its suitability for a wide variety of applications such as successful treatment of invasive cervical resorption . upon high alkalinity
, it is hypothesized that mta may prevent or arrest tooth resorption . on the other hand ,
the effect of alkaline and acidic ph values on physical properties of wmta ( white mta ) has been well - documented . the potential tooth discoloration can be the only reason that prevents mta to be used as an effective intra - orifice barrier during tooth bleaching . recently , calcium enriched mixture ( cem ) cement has been introduced as a new root - end filling material . it is dominantly composed of calcium compounds . due to being biocompatible , well - tolerable by oral tissues such as pulp and pdl and providing comparable seal to mta this new biomaterial
is proved to be applicable for other purposes in the field of endodontics , such as perforations repair , coronal seal , root canal filling , and vital pulp therapies as well . besides all the aforementioned criteria , cem cement offers the advantage of not causing tooth discoloration that is favorable in cases of intra - coronal bleaching . this interventional study was designated and conducted to compare the sealing ability of gi , mta and cem cement when used as intra - orifice barrier in nonvital bleaching . seventy freshly extracted human maxillary incisors with standard root length ( 10 - 13 mm ) were selected for this study . the teeth were extracted because of periodontal reasons . after cleaning the teeth , radiographies were taken in buccolingual and mesiodistal directions . teeth with calcification , internal or external resorption , cracks ( detectable under light stereomicroscope ) , severe coronal or root caries , large coronal restoration , root fractures , dilacerations , deep depression on root surfaces , and those with apical foramen larger than file size # 40 were excluded , and if necessary , they were replaced with intact ones . to control the cross infection and minimize soft tissues and periodontal remnants ,
all teeth were stored in normal saline then in a 5.25% solution of sodium hypochlorite ( naocl ) ( golrang , tehran , iran ) for 6 h and were finally stored in 0.5% chloramine solution until experiment commencement . the access cavity was prepared using a high - speed handpiece and # 2 round diamond bur ( jota , ruthi , switzerland ) under copious water irrigation . the coronal and mid segments of the canals were prepared with sizes 3 - 1 gates - glidden drills ( mailer , ballaigues , switzerland ) and then complete preparation of the canals was conducted using mtwo rotary system ( vdw , munich , germany ) in standard preparation technique up to file 40/0.04 as the master apical preparation . the canals were then dried with absorbent paper points ( ariadent , tehran , iran ) and obturated using gutta - percha ( diadent inc . , chongchong buch do , korea ) and ah-26 sealer ( dentsply , tulsa dental , tulsa , ok , usa ) with cold lateral condensation technique
. the teeth were randomly divided into three experimental groups ( n = 20 ) and two positive and negative control groups , of five each using simple randomization allocation . hyogo , japan ) , group 2-wmta ( tooth - colored formula , anglus , londrina , brazil ) , and group 3-cem cement ( bioniquedent , tehran , iran ) . all the materials were prepared according to the manufacturer 's instruction [ table 1 ] , and were packed into the unfilled cervical portion of the canals up to the palatal and facial aspects of cej to provide a 3-mm thick barrier . at this step ,
all samples were radiographically evaluated for the length and density of the intra - orifice barrier [ figure 1c ] . wet cotton pellets were placed over wmta and cem cement to provide their setting hydration . all the teeth were temporized with cavit ( espe - premier , norristown , pa , usa ) and incubated at 37c for 24 h at a 90% relative humidity . the bleaching agent used in the study was a mixture of 0.15 g sodium perborate ( merck , darmstadt , germany and 0.05 ml distilled water . it was placed in the pulp chamber , and the teeth were restored again with cavit and then incubated composition and usage instruction for material used in the study the bleaching period was arranged for 9 days . at the end of the 9 day , cavit was removed again , and pulp chamber was rinsed with distilled water and dried and was then filled with cavit for the last time . in positive control group , teeth received neither coronal barrier nor temporal restoration after canal obturation . in negative control group ,
all surfaces even the temporal restoration were covered and sealed with two layers of nail polish after filling the canals with sticky wax . in case of specimens in three experimental groups ,
all surfaces were covered with two layers of nail polish except for access cavity margins and apical 2-mm portion . all the samples were mounted in a dual - chamber leakage apparatus as shown in schematic view [ figure 2 ] . first , the teeth were inserted from the cap end of a 3-ml plastic eppendorf cylinder ( elkay , shrewbury , ma , usa ) . the glass vials had been filled previously with 9 ml of distilled water . the whole system was sterilized with ethylene oxide gas for 12 h. the plastic cylinders were filled with 1 ml of 22% bovine serum albumin ( bsa , sigma - aldrich , st . bradford indicator was used to measure the concentration of leaked albumin form the upper chamber into the lower one at the end of the 30 day . the procedure is based on the formation of a complex between the dye , brilliant blue g , and proteins in solution . then 100 l of test solution of the vials was pipetted into a new eppendorf tube and 1 ml of bradford protein reagent was added to the tube , and the contents were mixed . statistical analysis of the data was conducted using the kruskal - wallis followed by the mann - whitney tests . seventy freshly extracted human maxillary incisors with standard root length ( 10 - 13 mm ) were selected for this study . the teeth were extracted because of periodontal reasons . after cleaning the teeth , radiographies were taken in buccolingual and mesiodistal directions . teeth with calcification , internal or external resorption , cracks ( detectable under light stereomicroscope ) , severe coronal or root caries , large coronal restoration , root fractures , dilacerations , deep depression on root surfaces , and those with apical foramen larger than file size # 40 were excluded , and if necessary , they were replaced with intact ones . to control the cross infection and minimize soft tissues and periodontal remnants ,
all teeth were stored in normal saline then in a 5.25% solution of sodium hypochlorite ( naocl ) ( golrang , tehran , iran ) for 6 h and were finally stored in 0.5% chloramine solution until experiment commencement . the coronal and mid segments of the canals were prepared with sizes 3 - 1 gates - glidden drills ( mailer , ballaigues , switzerland ) and then complete preparation of the canals was conducted using mtwo rotary system ( vdw , munich , germany ) in standard preparation technique up to file 40/0.04 as the master apical preparation . the canals were then dried with absorbent paper points ( ariadent , tehran , iran ) and obturated using gutta - percha ( diadent inc . , chongchong buch do , korea ) and ah-26 sealer ( dentsply , tulsa dental , tulsa , ok , usa ) with cold lateral condensation technique
. the teeth were randomly divided into three experimental groups ( n = 20 ) and two positive and negative control groups , of five each using simple randomization allocation . in the experimental groups , the canal obturating material was covered as follows : group 1-rmgic - lc fuji ii ( fuji corp . hyogo , japan ) , group 2-wmta ( tooth - colored formula , anglus , londrina , brazil ) , and group 3-cem cement ( bioniquedent , tehran , iran ) . all the materials were prepared according to the manufacturer 's instruction [ table 1 ] , and were packed into the unfilled cervical portion of the canals up to the palatal and facial aspects of cej to provide a 3-mm thick barrier . at this step ,
all samples were radiographically evaluated for the length and density of the intra - orifice barrier [ figure 1c ] . wet cotton pellets were placed over wmta and cem cement to provide their setting hydration . all the teeth were temporized with cavit ( espe - premier , norristown , pa , usa ) and incubated at 37c for 24 h at a 90% relative humidity . the bleaching agent used in the study was a mixture of 0.15 g sodium perborate ( merck , darmstadt , germany and 0.05 ml distilled water . it was placed in the pulp chamber , and the teeth were restored again with cavit and then incubated composition and usage instruction for material used in the study the bleaching period was arranged for 9 days . at the end of the 9 day
, cavit was removed again , and pulp chamber was rinsed with distilled water and dried and was then filled with cavit for the last time . in positive control group , teeth received neither coronal barrier nor temporal restoration after canal obturation . in negative control group ,
all surfaces even the temporal restoration were covered and sealed with two layers of nail polish after filling the canals with sticky wax . in case of specimens in three experimental groups , all surfaces were covered with two layers of nail polish except for access cavity margins and apical 2-mm portion . all the samples were mounted in a dual - chamber leakage apparatus as shown in schematic view [ figure 2 ] . first , the teeth were inserted from the cap end of a 3-ml plastic eppendorf cylinder ( elkay , shrewbury , ma , usa ) . the glass vials had been filled previously with 9 ml of distilled water . the whole system was sterilized with ethylene oxide gas for 12 h. the plastic cylinders were filled with 1 ml of 22% bovine serum albumin ( bsa , sigma - aldrich , st . bradford indicator was used to measure the concentration of leaked albumin form the upper chamber into the lower one at the end of the 30 day . the procedure is based on the formation of a complex between the dye , brilliant blue g , and proteins in solution . then 100 l of test solution of the vials was pipetted into a new eppendorf tube and 1 ml of bradford protein reagent was added to the tube , and the contents were mixed . schematic view of dual - chamber technique used for protein leakage analysis . using spectrophotometry ,
statistical analysis of the data was conducted using the kruskal - wallis followed by the mann - whitney tests . the mean standard deviation leakage of samples from negative control , positive control , gi , mta , and cem cement groups were 0.0 , 8.9 0.03 , 0.47 0.02 , 0.48 0.02 , and 0.49 0.02 mg / ml , respectively . the decrease of acidity on external surfaces of teeth during internal bleaching has been considered as an important causative factor for cervical root resorption . for the very same reason , it is recommended to cover the canal filling materials with a barrier to occlude the dentinal tubules as a way of diffusion between pulp cavity and root surface . considering the fact that root fillings per se are not leak proof , and that no sealer cement or obturation technique consistently prevents percolation through the canal , it is critical to take most care in creating bests of the seal and consider the quality of intra - orifice barrier in nonvital bleaching . the success of walking bleaching rests somehow upon the sealing quality of the cervical barrier , especially when the remaining dentin walls are very thin . it is strongly suggested to apply only low concentration of bleaching agents , or to mix sodium perborate with distilled water instead of hydrogen peroxide , in order to govern the detrimental effects of the bleaching agents , although the importance of cervical barrier is not allowed to be ignored . attempts are underway to introduce more qualified materials with the potential to provide a long - term seal . to date , no study evaluated the intra - orifice sealing ability of cem cement in bleaching . its formulation is composed of a different mixture of calcium compounds to provide a bioactive calcium - phosphate enriched material . studies on cem cement showed that it can release phosphorous and calcium ions which promotes the alkalinity and also leads to mineralization process , implying its hard tissue inductivity . to date
, cem cement has not been studied as an intra - orifice plug to resist the microleakage in nonvital bleaching process while it has been implemented for treatment of furcal perforation , vital pulp therapies in permanent and primary teeth , root end fillings , management of root resorption and pathologic / iatrogenic perforations , periradicular surgery , and revascularization for necrotic immature permanent molars . compared to mta , cem cement has a shorter setting time and also significantly superior results in film thickness and flow , easier handling , and enhanced antibacterial effect as well as better abilities to form hydroxyapatite in normal saline . it is shown that cem cement has less microleakage compared to different types of mta although not significant . the sealing ability of endodontic materials has been evaluated using dye or bacterial penetration , electrical method , fluid filtration technique , human saliva leakage model , glucose leakage model , radioisotope tracing , and marginal adaptation . some advantages and also disadvantages
have been reported for each of these techniques , for instance , bacterial leakage assesses the sealing ability of all portions while in dye leakage only the walls adjacent to material are observed . bacterial leakage studies focus on the main pathogenic factors , but even this method can not mirror the real clinical condition as special species or a limited number of bacteria are used and so , bacterial synergistic effect , influence of environment , thermal changes , salivary enzymes , buffering materials and antibodies are neglected . in this study , protein leakage approach was adopted since
is met our answers more convincingly than the others . to show the importance of cervical barrier in leakage prevention , valadares et al . stated that the use of cervical barrier prevents the microleakage of enterococcus faecalis . reported no difference between gray mta , white mta , or a resin modified gi , in terms of bacterial penetration with human saliva model . based on the findings of a recent study , using the glucose penetration model , bailn - snchez et al . cavit , and proroot mta has similar abilities to resist leakage when used as intra - orifice barriers . barrieshi - nusair and hammad compared gi and mta as orifice plugs and reported that gi has more microleakage which is not concurring with our findings . in their study
, each group included 30 samples , and the leakage assessment method was dye leakage for 48 h , but we had 20 teeth in each experimental group which were analyzed in protein leakage approach for 30 days . to compare mta and gi as coronal barrier in nonvital bleaching ,
the results of the present study revealed that cem cement has sealing potential as an intra - orifice barrier against protein penetration that is comparable with that of mta and gi . other experimental settings , particularly for leakage assessment are needed to simulate the clinical reality . among the limitations ,
technique sensitivity of protein leakage assessment method , and the cost of the materials were more prominent . considering the results of the present study , on ground of the leakage resistance , it is concluded that all materials used ; gi , mta , and cem cement are able to convincingly seal the coronal portion of the canal during nonvital bleaching in order to prevent the cervical root resorption . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . | [
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] |
although endometriosis occurs in ~10 % of women of reproductive age and in ~50 % of women with infertility , the etiology is poorly understood .
there is accumulating evidence supporting a concept that endometriosis is a disease associated with a genetic ( albertsen et al .
, 2013 ; xiao et al . , 2010 ) and also an epigenetic disorder ( izawa et al . , 2013 ; nasu et al . ,
2011 ; guo , 2009 ; coln - daz et al . , 2012 ) .
genetic mechanisms have been ascribed important roles in endometriosis ( albertsen et al . , 2013 ; xiao et al . ,
genetic and network - based pathway analysis of endometrial and endometriotic tissues revealed that the unique endometriosis susceptibility genes include genes encoding cell cycle , growth factors , signal transduction , transcription factors , hormones , cytokines , chemokines and ( pro)inflammation , proteases , cell adhesion and motility , stress response and detoxification , immune response and metabolism ( khan et al .
kobayashi et al . ( 2013 ) recently showed the overlapping genetic signatures between endometriosis development and decidualization process , suggesting that insufficient decidualization may underline this disorder .
epigenetic alterations reported to date in endometriosis include the genomic dna methylation of progesterone receptor ( pgr)-b , e - cadherin ( cdh1 ) , homeobox a10 ( hoxa10 ) ( cakmak and taylor , 2011 ) , estrogen receptor - beta ( esr2 ) , steroidogenic factor-1 ( nr5a1 ) , aromatase ( cyp19a1 ) ( nasu et al . , 2011 ) , histone deacetylase inhibition ( hdaci ) ( coln - daz et al
. , 2012 ) , cdkn2a / b ( kawano et al . , 2011 ) , igfbp-1 ( cakmak and taylor , 2011 ) , leukemia inhibitory factor ( lif ) ( cakmak and taylor , 2011 ) and dna - methyltransferase ( dnmts ) ( wu et al . , 2007 ) .
there are no data , however , when and how the disruption of such epigenetic changes occurs .
imprinted genes are expressed mainly from one parental allele due to an epigenetic mechanism while the other allele is inactivated .
the paternally expressed / maternally imprinted genes such as insulin - like growth factor ( igf)-2 were related to promoting cell proliferation , differentiation and metabolism , and are involved in regulating placental size and birth weight ( haggarty et al . , 2013 ) .
maternally expressed / paternally imprinted genes reduce the flow of resources to the fetus and are associated with fetal growth restriction , supporting the " parental conflict hypothesis".the previous studies have not yet provided convincing evidence for any susceptibility genomic imprinting genes of endometriosis . in the present study , from the genomic imprinting database , we search for the first time the parentally imprinted genes that are reported to be involved in the reproductive process including endometriosis .
this database search identified all the existing publications on the imprinting events . in the current version ,
the database contains 150 human imprinted genes , including information such as gene name , aliases , gene location and expressed allele .
particular emphasis was given on the imprinting genes associated with female reproduction , including endometriosis .
additional information was manually collected by keyword searches of the biomedical literature database pubmed ( http://www.ncbi.nlm.nih.gov/pubmed ) .
supplementary data show biological functions of a total of 150 imprinted genes , with preferential expression from the paternal or maternal allele
. biological functions of each imprinting gene were manually searched by pubmed . by analyzing the cellular functions of these 150 imprinted genes , they play an essential role in 1 .
multiple metabolism pathways including diabetes ( mackay and temple , 2010 ; el hajj et al . , 2013 ; santin and eirizik , 2013 ; travers et al . , 2013 ; hamed et al . , 2012 )
, hyperphagia ( zhang et al . , 2012 ) , adipogenesis ( hudak and sul , 2013 ) , obesity ( zhang et al . , 2012 ; liu et al . , 2013 ; do et al . , 2013 ) ,
atherosclerosis , cardiovascular disease , myocardial ischemia and reperfusion , hypertension ( karagiannis et al . , 2013 ; small et al .
, 2011 ; zhu et al . , 2009 ) , prader - willi syndrome ( zhang et al . , 2012 ;
2013 ) , angelman syndrome ( rodriguez - jato et al . , 2013 ; mabb et al . , 2011),wilms tumor ( jacobi et al .
, 2013 ; hubertus et al . , 2013 ; xin et al . , 2000 ) and beckwith - wiedemann syndrome ( zhang et al . , 2012 ;
, 2000 diabetes ( mackay and temple , 2010 ; el hajj et al . , 2013
; santin and eirizik , 2013 ; travers et al . , 2013 ; hamed et al . , 2012 ) ,
hyperphagia ( zhang et al . , 2012 ) , adipogenesis ( hudak and sul , 2013 ) , obesity ( zhang et al . , 2012 ; liu et al . , 2013 ; do et al . , 2013 ) , atherosclerosis , cardiovascular disease , myocardial ischemia and reperfusion , hypertension ( karagiannis et al . , 2013 ; small et al . , 2011 ; zhu et al . , 2009 ) , prader - willi syndrome ( zhang et al . , 2012
, 2013 ; gallagher et al . , 2002 ; rieusset et al . , 2013 ) , angelman syndrome ( rodriguez - jato et al . , 2013 ; mabb et al . ,
2011 ) , wilms tumor ( jacobi et al . , 2013 ; hubertus et al .
, 2013 ; xin et al . , 2000 ) and beckwith - wiedemann syndrome ( zhang et al . , 2012 ;
, 2013 ; mabb et al . , 2011 ; hubertus et al . , 2013
the emotional , social and neurological behavior and the appearance of certain neurodegenerative diseases such as schizophrenia , parkinsonism , huntington disease , auitism , and mature synaptic function ( becanovic et al . ,
2010 ; gos , 2013 ) , 3 . malignancies such as wilms tumor , rhabdomyosarcoma , adrenocortical carcinoma , and lung , ovarian , and breast cancer ( jacobi et al . , 2013 ; hubertus et al . , 2013 ; xin et al .
; zhong et al . , 2012 ; kim et al . , 2012 ; britschgi et al . , 2013 ; ho et al
female reproductive system including placental development , fetal growth , infertile , decidualization and endometrial function , menarche , puberty , pregnancy loss such as spontaneous miscarriages or fetal deaths , preeclampsia , endometriosis , sexual behaviors , and spermatogenesis ( albertsen et al . , 2013 ;
; wu et al . , 2007 ; peltomki and btzow , 2011 ; he et al . , 2004 ; li et al . , 2011
; brandelli and passos , 1998 ; wetendorf and demayo , 2012 ; sonderegger et al . , 2010 ; monteiro et al . , 2014 ;
tiberi et al . , 2010 ; li and wang , 2009 ; kang et al .
, 2010 ; choi et al . , 2013 ; vinatier et al . , 2000 ; nyholt et al . ,
other important spheres including developmental ( jacobi et al . , 2013 ; wetendorf and demayo , 2012 ; kajimura et al . , 2010 ;
2012 ; godfrey et al . , 2011 ) , transport ( balsa et al . , 2012 ; burger et al . ,
2010 ) , regulatory ( hubertus et al . , 2013 ; sturrock et al . , 2013 ) , transcriptional , g - protein signaling processes , inflammatory responses ( santin and eirizik , 2013 ; arnett et al . , 2007 ; liu et al . ,
2013 ) , oxidative stress ( srinivasan and avadhani , 2012 ) , dna replication and transcription ( nasu et al . , 2011 ; haggarty et al . ,
; du et al . , 2013 ; schwertman et al . , 2013 ; tobi et al . , 2011 ;
2013 ) , chromatin remodeling ( du et al . , 2013 ) , bone and skeletal diseases ( nakabayashi et al . , 2004 ) ,
, 2013 ; nakabayashi et al . , 2004 ; devaney et al . , 2007 ) , energy expenditure ( zhou et al . , 2012 ) , eye development and hypothyroidism ( figure 1(fig . 1 ) and figure 2(fig .
; bergman et al . , 2013 ; lambertini et al . , 2012 ; godfrey et al . , 2011 ) , transport ( balsa et al . , 2012 ; burger et al . , 2010 ) , regulatory ( hubertus et al . , 2013 ; sturrock et al . , 2013 ) , transcriptional , g - protein signaling processes , inflammatory responses ( santin and eirizik , 2013 ; arnett et al . , 2007
; liu et al . , 2013 ) , oxidative stress ( srinivasan and avadhani , 2012 ) , dna replication and transcription ( nasu et al . , 2011 ; haggarty et al . ,
; du et al . , 2013 ; schwertman et al . , 2013 ; tobi et al . , 2011 ;
2013 ) , chromatin remodeling ( du et al . , 2013 ) , bone and skeletal diseases ( nakabayashi et al . , 2004 ) , muscle function ( karagiannis et al . , 2013 ; nakabayashi et al . , 2004 ; devaney et al . , 2007 ) , energy expenditure ( zhou et al . , 2012 ) , eye development and hypothyroidism ( figure 1(fig . 1 ) and figure 2(fig .
2 ) ) . aberrant genomic imprinting is an important epigenetic process involved in regulating metabolic disease , the emotional and social behavior , malignancies , placental and fetal growth , reproductive disorders and other important biological processes in later life . among 150
differentially expressed parentally imprinted genes , we extracted ten genes that showed a reproductive biology in the set of imprinted genes in human .
they include paternally expressed genes ( diras3 [ diras family , gtp - binding ras - like 3 ] , bmp8b [ bone morphogenetic protein 8b ] , cyp1b1 [ cytochrome p450 , family 1 , subfamily b , polypeptide 1 ] , zfat [ zinc finger and at hook domain containing ] , igf2 [ insulin - like growth factor-2 ] , mimt1 [ mer1 repeat containing imprinted transcript 1 ] , or mir296 [ microrna 296 ] ) and maternally expressed genes ( dvl1 [ dishevelled segment polarity protein 1 ] , fgfrl1 [ fibroblast growth factor receptor - like 1 ] , or cdkn1c [ cyclin - dependent kinase inhibitor 1c ( p57 , kip2 ) ] ) ( table 1(tab .
1 ) ) . biological functions of seven paternally expressed genes associated with female reproduction ( table 1(tab .
1 ) ) : diras3 , diras family , gtp - binding ras - like 3 diras3 is a member of the ras superfamily , and appears to be a putative tumor suppressor gene ( http://www.ncbi.nlm.nih.gov/gene/9077 ) .
up - regulation of diras3 expression is associated with infertility and endometriosis ( li et al . , 2011 ) .
bmp8b , bone morphogenetic protein 8b bmp is a part of the transforming growth factor - beta ( tgfb ) superfamily that induces ectopic bone growth ( http://www.ncbi.nlm.nih.gov/gene/656 ) .
progesterone - dependent ihh , wnt , and bmp signaling pathways within the endometrium play a role in decidualization , implantation and embryo attachment ( wetendorf and demayo , 2012 ) .
cyp1b1 , cytochrome p450 , family 1 , subfamily b , polypeptide 1 the cytochrome p450 metabolizes procarcinogens and synthesizes cholesterol , steroids and other lipids ( http://www.ncbi.nlm.nih.gov/gene/1545 ) .
the gene polymorphisms of cyp1b1 in exon 2 codon 119 are also an associated risk factor for endometriosis ( li and wang , 2009 ) .
zfat , zinc finger and at hook domain containing zfat - as1 , zfat antisense rna 1 the encoded protein , zfat , is a dna binding protein and functions as a transcriptional regulator involved in cell survival and apoptosis ( http://www.ncbi.nlm.nih.gov/gene/57623 ) .
snps of the zfat gene are associated with an increased risk of autoimmune thyroid disease .
zfat expressed in syncytiotrophoblasts is downregulated in placentas from preeclampsia ( barbaux et al . , 2012 ) .
the zfat - as1 gene encodes a small antisense rna that regulates the sense strand locus , zfat ( http://www.ncbi.nlm.nih.gov/gene/594840 ) igf2 , insulin - like growth factor 2 ( somatomedin a ) the igf2 gene is involved in cell proliferation , growth , migration , differentiation and survival .
epigenetic changes at this locus are associated with wilms tumor , beckwith - wiedemann syndrome , rhabdomyosarcoma , silver - russell syndrome and cardiovascular disease ( bergman et al . ,
mimt1 , mer1 repeat containing imprinted transcript 1 mimt1 is a non - protein coding gene that forms part of the imprinted peg3 ( paternally expressed gene 3 ) domain .
loss of paternal mimt1 expression results in the phenotype of late term abortion and stillbirth in cattle ( flisikowski et al . , 2010 ) .
several mirnas including mir296 are found to be dysregulated in placenta of preeclampsia patients ( choi et al . , 2013 ) .
dna methylation of gnasas gene might be associated with small for gestational age and myocardial infarction among women ( tobi et al . ,
dvl1 , disheveled segment polarity protein 1 wnt stimulation induces recruitment of dvl to the g - protein coupled frizzled ( fzd ) receptors ( kawano et al . , 2011 ) .
dvl plays a key role in relaying cellular information for several developmental pathways such as cell proliferation , migration , polarity , terminal differentiation , and the self - renewal of stem cells ( dillman et al . , 2013 ) .
dvl1 encodes a cytoplasmic phosphoprotein and is a substrate of nr1i2 ( nuclear receptor subfamily 1 , group i , member 2 ) , which is a family of serine / threonine kinases that have been associated with differentiation of epithelial and neuronal cells ( http://www.ncbi.nlm.nih.gov/gene/1855 ) ( elbert et al . , 2006 ) .
charcot - marie - tooth disease has been mapped to the same region as dvl1 .
this disease is the hereditary neuropathy characterized by muscular atrophy and weakness in the distal parts of the legs ( ostern et al . , 2013 ) .
failures in wnt signalling are a cause of infertility and endometriosis ( sonderegger et al . , 2010 ) .
fgfrl1 , fibroblast growth factor receptor - like 1 fgfrl1 influences mitogenesis and differentiation ( http://www.ncbi.nlm.nih.gov/gene/53834 ) .
the fgf2 754c / g polymorphism may be closely associated with a risk of developing endometriosis ( kang et al . , 2010 ) .
cdkn1c , cyclin - dependent kinase inhibitor 1c ( p57 , kip2 ) the encoded protein is a strong g1 cyclin / cdk - dependent inhibitor and a negative regulator of cell proliferation , suggesting a tumor suppressor candidate ( http://www.ncbi.nlm.nih.gov/gene/1028 ) .
cdkn1c plays a role in endometrial stromal cell differentiation in the process of decidualization ( qian et al . , 2005 ) .
these ten genes are mainly associated with the control of resource usage and reproductive biology such as not only endometriosis , but also abortion , stillbirth , infertility , decidualization , preeclampsia , metabolic syndrome , diabetes , coronary heart disease , eye development , autoimmune thyroid disease , tumor suppression , wilms tumor , beckwith - wiedemann syndrome , rhabdomyosarcoma , silver - russell syndrome and charcot - marie - tooth disease ( supplementary data ) .
dvl1 , disheveled segment polarity protein 1 wnt stimulation induces recruitment of dvl to the g - protein coupled frizzled ( fzd ) receptors ( kawano et al . , 2011 ) .
dvl plays a key role in relaying cellular information for several developmental pathways such as cell proliferation , migration , polarity , terminal differentiation , and the self - renewal of stem cells ( dillman et al . , 2013 ) .
dvl1 encodes a cytoplasmic phosphoprotein and is a substrate of nr1i2 ( nuclear receptor subfamily 1 , group i , member 2 ) , which is a family of serine / threonine kinases that have been associated with differentiation of epithelial and neuronal cells ( http://www.ncbi.nlm.nih.gov/gene/1855 ) ( elbert et al . , 2006 ) .
charcot - marie - tooth disease has been mapped to the same region as dvl1 .
this disease is the hereditary neuropathy characterized by muscular atrophy and weakness in the distal parts of the legs ( ostern et al . ,
failures in wnt signalling are a cause of infertility and endometriosis ( sonderegger et al . , 2010 ) .
fgfrl1 , fibroblast growth factor receptor - like 1 fgfrl1 influences mitogenesis and differentiation ( http://www.ncbi.nlm.nih.gov/gene/53834 ) .
the fgf2 754c / g polymorphism may be closely associated with a risk of developing endometriosis ( kang et al . , 2010 ) .
cdkn1c , cyclin - dependent kinase inhibitor 1c ( p57 , kip2 ) the encoded protein is a strong g1 cyclin / cdk - dependent inhibitor and a negative regulator of cell proliferation , suggesting a tumor suppressor candidate ( http://www.ncbi.nlm.nih.gov/gene/1028 ) .
cdkn1c plays a role in endometrial stromal cell differentiation in the process of decidualization ( qian et al . , 2005 ) .
these ten genes are mainly associated with the control of resource usage and reproductive biology such as not only endometriosis , but also abortion , stillbirth , infertility , decidualization , preeclampsia , metabolic syndrome , diabetes , coronary heart disease , eye development , autoimmune thyroid disease , tumor suppression , wilms tumor , beckwith - wiedemann syndrome , rhabdomyosarcoma , silver - russell syndrome and charcot - marie - tooth disease ( supplementary data ) .
using the genomic imprinting database , we identified 10 imprinted genes , of which 7 were paternally expressed , and found that these genes are associated with female reproductive functions , including decidualization ( bmp8b and cdkn1c ) , implantation ( bmp8b ) , embryo attachment ( bmp8b ) , abortion ( mimt1 ) , stillbirth ( mimt1 and mir296 ) , preeclampsia ( zfat ) , infertility ( diras3 and dvl1 ) and endometriosis ( diras3 , cyp1b1 , igf2 , dvl1 and fgfrl1 ) .
these parentally imprinted genes are necessary for female reproductive system such as normal endometrial development , decidualization , placentation and endometriosis . as described previously , the endometriosis susceptibility genes include growth factors ( diras3 , igf2 and fgfrl1 ) , wnt signal transduction ( dvl1 ) , metabolism ( cyp1b1 ) , and stress response and detoxification ( cyp1b1 ) ( khan et al . , 2012 ; kobayashi et al . , 2013 )
. some biological aspects of endometriosis may be explained from a dysregulation of parentally imprinted gene .
recent advances in sequencing , profiling and pathway technologies allow genome - scale approaches to endometriosis - susceptibility gene discovery , which enables us to look for evidence in support of the genetic hypothesis ( albertsen et al . , 2013 ;
, 2013 ; nasu et al . , 2011 ; guo , 2009 ; coln - daz et al . , 2012 ; kobayashi et al . , 2013 ; kawano et al . ,
the previous study has shed new light on the overlapping genetic and epigenetic signatures between endometriosis development and insufficient decidualization process , indicating that a number of genes are essential for the decidualization and implantation processes , but up - regulation of a small number of them , including igf , igfbp , prl , hoxa10 , foxo1 , c / ebpbeta , il11 and lif , are important for this process ( kobayashi et al . , 2013 ) .
downregulation of the specific genes related to embryogenesis ( the downstream targets of hoxa10 ) and immuno - endocrine behavior ( il11 , lif , tgf - beta , fkbp4 , cox2 , pgs , foxo1 and c / ebpbeta ) might appear critical to the development of endometriosis ( izawa et al . , 2013 ; nasu et al . , 2011 ; guo , 2009 ; coln - daz et al . , 2012
wu et al . , 2007 ; peltomki and btzow , 2011 ; tiberi et al . , 2010 ; vinatier et al . ,
2000 ; nyholt et al . , 2012 ; calicchio et al . , 2013 ; ) .
kobayashi et al . ( 2013 ) reported that the upregulated genes in endometriosis may evolve for the benefit of the endometrial growth , whereas the downregulated genes evolve as a protective mechanism for the endometrial decidualization .
the irreversible programming or epigenetics may cause insufficient decidualization , which in turn results in infertility and endometriosis .
previous studies identified several susceptibility genes that have highlighted the important role of endometriosis development , including igf , igfbp , prl , hoxa10 , foxo1 , c / ebpbeta , il11 , lif , tgf - beta , fkbp4 , cox2 , and prostaglandins ( izawa et al . , 2013 ; nasu et al . , 2011 ; guo , 2009 ; coln - daz et al . , 2012
; khan et al . 2012 ; kobayashi et al . , 2013 ; kawano et al . , 2011 ; wu et al . , 2007 ; peltomki and btzow , 2011 ; tiberi et al . , 2010 ; vinatier et al
we tried to summarize the recent literature that supports a direct or indirect relationship between the novel candidate imprinting genes and the previously reported endometriosis susceptibility genes .
epigenetic changes induced by various environmental stress factors including nutrition or ecosystem components play a role in interactions between exposed species and chemicals .
chemicals such as 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) and benzo(a)pyrene modulate the mrna levels of cyp1b1 , c / ebpbeta , il11 and prl ( cao et al . , 2011 ; vogel and matsumura , 2013 ; ueng et al . , 2005 ) .
cyp1b1 is responsible for tumor progression in estrogen receptor - positive breast and endometrial cancers via estrogen metabolism .
allelic polymorphisms at codons 119 and 432 of cyp1b1 gene increases the risk of estrogen - dependent cancer ( sasaki et al . , 2003 ) .
cyp1b1 , igf2 and hoxa10 genes are reported to be hypermethylated in breast and gastric cancer ( kang et al . , 2008 ; park et al . , 2012
cyp1b1 is transcriptionally regulated by steroidogenic factor-1 ( sf-1 ) ( tsuchiya et al . , 2006 ) or pge2 , the main product of cox-2 ( yuan et al . , 2012 )
esr2 and smads , downstream signaling cascades of tgf - beta , participates in the establishment of parent - of - origin - specific expression of igf2 ( pathak et al . , 2009 ; szab et al . , 2004 ; bergstrm et al . ,
igf2 induced through the transactivation of c / ebpbeta is involved in prl signaling ( tao et al . , 2013 ;
lif inhibits the glial cell - derived neurotrophic factor ( gdnf)-dependent alteration of the genomic imprinting of igf2 in mice ( jung et al . , 2010 ) .
dvl1 is a downstream molecule of wnt signaling ( li et al . , 2013 ) .
diras3 modulates estrogen and progesterone receptor expression and inhibits prl - induced mammary gland development and lactation , which results in decreased fertility ( xu et al . , 2000 ) .
diras3 also induces cdh1 expression and acts as a tumor suppressor gene ( lyu et al . , 2013 ) .
taken together , esr2 and tgf - beta downstream targets , smads , co - localize to the igf2 imprinting control region ( bergstrm et al .
no convincing evidence has been provided to suggest that these imprinting genes would control the endometriosis susceptibility genes , although some indirect indications are available . at this time
, no attempt was made to ascertain whether any of these 10 genes had a
role or had a role of merely a by - stander in the development of endometriosis .
since there are no known driver genes in endometriosis , complex genomic alterations may be responsible for the endometriosis phenotype .
the quest for driver imprinting genes or complex genomic alterations can now open new avenues to better understand the mechanisms of endometriosis development . in conclusion , this study supports the possibility that aberrant epigenetic dysregulation of specific imprinting genes may contribute to endometriosis predisposition .
further investigations are needed to provide biological evidence for the direct association between the novel candidate imprinting genes and the previously reported endometriosis susceptibility genes .
the present review was supported by grant - in - aid for scientific research from the ministry of education , science , and culture of japan to the department of obstetrics and gynecology , nara medical university ( to h.k . ) .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | purpose : much work has been carried out to investigate the genetic and epigenetic basis of endometriosis and proposed that endometriosis has been described as an epigenetic disease . the purpose of this study was to extract the imprinting genes that are associated with endometriosis development .
methods : the information on the imprinting genes can be accessed publicly from a web - based interface at http://www.geneimprint.com/site/genes-by-species .
results : in the current version , the database contains 150 human imprinted genes derived from the literature .
we searched gene functions and their roles in particular biological processes or events , such as development and pathogenesis of endometriosis . from the genomic imprinting database
, we picked 10 genes that were highly associated with female reproduction ; prominent among them were paternally expressed genes ( diras3 , bmp8b , cyp1b1 , zfat , igf2 , mimt1 , or mir296 ) and maternally expressed genes ( dvl1 , fgfrl1 , or cdkn1c ) .
these imprinted genes may be associated with reproductive biology such as endometriosis , pregnancy loss , decidualization process and preeclampsia .
discussion : this study supports the possibility that aberrant epigenetic dysregulation of specific imprinting genes may contribute to endometriosis predisposition . | Introduction
Materials and Methods
Results
Biological functions of three maternally expressed genes associated with female reproduction (Table 1)
Discussion
Acknowledgements
Conflict of interest
Supplementary Material | although endometriosis occurs in ~10 % of women of reproductive age and in ~50 % of women with infertility , the etiology is poorly understood . there is accumulating evidence supporting a concept that endometriosis is a disease associated with a genetic ( albertsen et al . , 2010 ) and also an epigenetic disorder ( izawa et al . genetic mechanisms have been ascribed important roles in endometriosis ( albertsen et al . , 2013 ; xiao et al . ,
genetic and network - based pathway analysis of endometrial and endometriotic tissues revealed that the unique endometriosis susceptibility genes include genes encoding cell cycle , growth factors , signal transduction , transcription factors , hormones , cytokines , chemokines and ( pro)inflammation , proteases , cell adhesion and motility , stress response and detoxification , immune response and metabolism ( khan et al . kobayashi et al . ( 2013 ) recently showed the overlapping genetic signatures between endometriosis development and decidualization process , suggesting that insufficient decidualization may underline this disorder . epigenetic alterations reported to date in endometriosis include the genomic dna methylation of progesterone receptor ( pgr)-b , e - cadherin ( cdh1 ) , homeobox a10 ( hoxa10 ) ( cakmak and taylor , 2011 ) , estrogen receptor - beta ( esr2 ) , steroidogenic factor-1 ( nr5a1 ) , aromatase ( cyp19a1 ) ( nasu et al . , 2011 ) , igfbp-1 ( cakmak and taylor , 2011 ) , leukemia inhibitory factor ( lif ) ( cakmak and taylor , 2011 ) and dna - methyltransferase ( dnmts ) ( wu et al . imprinted genes are expressed mainly from one parental allele due to an epigenetic mechanism while the other allele is inactivated . the paternally expressed / maternally imprinted genes such as insulin - like growth factor ( igf)-2 were related to promoting cell proliferation , differentiation and metabolism , and are involved in regulating placental size and birth weight ( haggarty et al . maternally expressed / paternally imprinted genes reduce the flow of resources to the fetus and are associated with fetal growth restriction , supporting the " parental conflict hypothesis".the previous studies have not yet provided convincing evidence for any susceptibility genomic imprinting genes of endometriosis . in the present study , from the genomic imprinting database , we search for the first time the parentally imprinted genes that are reported to be involved in the reproductive process including endometriosis . this database search identified all the existing publications on the imprinting events . in the current version ,
the database contains 150 human imprinted genes , including information such as gene name , aliases , gene location and expressed allele . particular emphasis was given on the imprinting genes associated with female reproduction , including endometriosis . supplementary data show biological functions of a total of 150 imprinted genes , with preferential expression from the paternal or maternal allele
. by analyzing the cellular functions of these 150 imprinted genes , they play an essential role in 1 . , 2013 ; hamed et al . , 2012 ;
2013 ) , angelman syndrome ( rodriguez - jato et al . , 2000 ) and beckwith - wiedemann syndrome ( zhang et al . , 2012 ) ,
hyperphagia ( zhang et al . , 2013 ) , angelman syndrome ( rodriguez - jato et al . ,
2011 ) , wilms tumor ( jacobi et al . , 2013 ; xin et al . , 2000 ) and beckwith - wiedemann syndrome ( zhang et al . , 2013
the emotional , social and neurological behavior and the appearance of certain neurodegenerative diseases such as schizophrenia , parkinsonism , huntington disease , auitism , and mature synaptic function ( becanovic et al . ,
2010 ; gos , 2013 ) , 3 . malignancies such as wilms tumor , rhabdomyosarcoma , adrenocortical carcinoma , and lung , ovarian , and breast cancer ( jacobi et al . , 2013 ; ho et al
female reproductive system including placental development , fetal growth , infertile , decidualization and endometrial function , menarche , puberty , pregnancy loss such as spontaneous miscarriages or fetal deaths , preeclampsia , endometriosis , sexual behaviors , and spermatogenesis ( albertsen et al . , 2010 ; li and wang , 2009 ; kang et al . , 2010 ; choi et al . , 2011 ; haggarty et al . , 2013 ) , bone and skeletal diseases ( nakabayashi et al . , 2012 ) , eye development and hypothyroidism ( figure 1(fig . 1 ) and figure 2(fig . , 2011 ; haggarty et al . , 2013 ; schwertman et al . , 2012 ) , eye development and hypothyroidism ( figure 1(fig . 1 ) and figure 2(fig . 2 ) ) . aberrant genomic imprinting is an important epigenetic process involved in regulating metabolic disease , the emotional and social behavior , malignancies , placental and fetal growth , reproductive disorders and other important biological processes in later life . among 150
differentially expressed parentally imprinted genes , we extracted ten genes that showed a reproductive biology in the set of imprinted genes in human . they include paternally expressed genes ( diras3 [ diras family , gtp - binding ras - like 3 ] , bmp8b [ bone morphogenetic protein 8b ] , cyp1b1 [ cytochrome p450 , family 1 , subfamily b , polypeptide 1 ] , zfat [ zinc finger and at hook domain containing ] , igf2 [ insulin - like growth factor-2 ] , mimt1 [ mer1 repeat containing imprinted transcript 1 ] , or mir296 [ microrna 296 ] ) and maternally expressed genes ( dvl1 [ dishevelled segment polarity protein 1 ] , fgfrl1 [ fibroblast growth factor receptor - like 1 ] , or cdkn1c [ cyclin - dependent kinase inhibitor 1c ( p57 , kip2 ) ] ) ( table 1(tab . biological functions of seven paternally expressed genes associated with female reproduction ( table 1(tab . 1 ) ) : diras3 , diras family , gtp - binding ras - like 3 diras3 is a member of the ras superfamily , and appears to be a putative tumor suppressor gene ( http://www.ncbi.nlm.nih.gov/gene/9077 ) . up - regulation of diras3 expression is associated with infertility and endometriosis ( li et al . bmp8b , bone morphogenetic protein 8b bmp is a part of the transforming growth factor - beta ( tgfb ) superfamily that induces ectopic bone growth ( http://www.ncbi.nlm.nih.gov/gene/656 ) . cyp1b1 , cytochrome p450 , family 1 , subfamily b , polypeptide 1 the cytochrome p450 metabolizes procarcinogens and synthesizes cholesterol , steroids and other lipids ( http://www.ncbi.nlm.nih.gov/gene/1545 ) . zfat , zinc finger and at hook domain containing zfat - as1 , zfat antisense rna 1 the encoded protein , zfat , is a dna binding protein and functions as a transcriptional regulator involved in cell survival and apoptosis ( http://www.ncbi.nlm.nih.gov/gene/57623 ) . snps of the zfat gene are associated with an increased risk of autoimmune thyroid disease . zfat expressed in syncytiotrophoblasts is downregulated in placentas from preeclampsia ( barbaux et al . , 2012 ) . the zfat - as1 gene encodes a small antisense rna that regulates the sense strand locus , zfat ( http://www.ncbi.nlm.nih.gov/gene/594840 ) igf2 , insulin - like growth factor 2 ( somatomedin a ) the igf2 gene is involved in cell proliferation , growth , migration , differentiation and survival . epigenetic changes at this locus are associated with wilms tumor , beckwith - wiedemann syndrome , rhabdomyosarcoma , silver - russell syndrome and cardiovascular disease ( bergman et al . ,
mimt1 , mer1 repeat containing imprinted transcript 1 mimt1 is a non - protein coding gene that forms part of the imprinted peg3 ( paternally expressed gene 3 ) domain . loss of paternal mimt1 expression results in the phenotype of late term abortion and stillbirth in cattle ( flisikowski et al . dna methylation of gnasas gene might be associated with small for gestational age and myocardial infarction among women ( tobi et al . ,
dvl1 , disheveled segment polarity protein 1 wnt stimulation induces recruitment of dvl to the g - protein coupled frizzled ( fzd ) receptors ( kawano et al . , 2011 ) . dvl plays a key role in relaying cellular information for several developmental pathways such as cell proliferation , migration , polarity , terminal differentiation , and the self - renewal of stem cells ( dillman et al . , 2013 ) . dvl1 encodes a cytoplasmic phosphoprotein and is a substrate of nr1i2 ( nuclear receptor subfamily 1 , group i , member 2 ) , which is a family of serine / threonine kinases that have been associated with differentiation of epithelial and neuronal cells ( http://www.ncbi.nlm.nih.gov/gene/1855 ) ( elbert et al . charcot - marie - tooth disease has been mapped to the same region as dvl1 . this disease is the hereditary neuropathy characterized by muscular atrophy and weakness in the distal parts of the legs ( ostern et al . fgfrl1 , fibroblast growth factor receptor - like 1 fgfrl1 influences mitogenesis and differentiation ( http://www.ncbi.nlm.nih.gov/gene/53834 ) . the fgf2 754c / g polymorphism may be closely associated with a risk of developing endometriosis ( kang et al . cdkn1c plays a role in endometrial stromal cell differentiation in the process of decidualization ( qian et al . these ten genes are mainly associated with the control of resource usage and reproductive biology such as not only endometriosis , but also abortion , stillbirth , infertility , decidualization , preeclampsia , metabolic syndrome , diabetes , coronary heart disease , eye development , autoimmune thyroid disease , tumor suppression , wilms tumor , beckwith - wiedemann syndrome , rhabdomyosarcoma , silver - russell syndrome and charcot - marie - tooth disease ( supplementary data ) . dvl1 , disheveled segment polarity protein 1 wnt stimulation induces recruitment of dvl to the g - protein coupled frizzled ( fzd ) receptors ( kawano et al . dvl plays a key role in relaying cellular information for several developmental pathways such as cell proliferation , migration , polarity , terminal differentiation , and the self - renewal of stem cells ( dillman et al . dvl1 encodes a cytoplasmic phosphoprotein and is a substrate of nr1i2 ( nuclear receptor subfamily 1 , group i , member 2 ) , which is a family of serine / threonine kinases that have been associated with differentiation of epithelial and neuronal cells ( http://www.ncbi.nlm.nih.gov/gene/1855 ) ( elbert et al . charcot - marie - tooth disease has been mapped to the same region as dvl1 . this disease is the hereditary neuropathy characterized by muscular atrophy and weakness in the distal parts of the legs ( ostern et al . fgfrl1 , fibroblast growth factor receptor - like 1 fgfrl1 influences mitogenesis and differentiation ( http://www.ncbi.nlm.nih.gov/gene/53834 ) . the fgf2 754c / g polymorphism may be closely associated with a risk of developing endometriosis ( kang et al . , 2010 ) . cdkn1c plays a role in endometrial stromal cell differentiation in the process of decidualization ( qian et al . these ten genes are mainly associated with the control of resource usage and reproductive biology such as not only endometriosis , but also abortion , stillbirth , infertility , decidualization , preeclampsia , metabolic syndrome , diabetes , coronary heart disease , eye development , autoimmune thyroid disease , tumor suppression , wilms tumor , beckwith - wiedemann syndrome , rhabdomyosarcoma , silver - russell syndrome and charcot - marie - tooth disease ( supplementary data ) . using the genomic imprinting database , we identified 10 imprinted genes , of which 7 were paternally expressed , and found that these genes are associated with female reproductive functions , including decidualization ( bmp8b and cdkn1c ) , implantation ( bmp8b ) , embryo attachment ( bmp8b ) , abortion ( mimt1 ) , stillbirth ( mimt1 and mir296 ) , preeclampsia ( zfat ) , infertility ( diras3 and dvl1 ) and endometriosis ( diras3 , cyp1b1 , igf2 , dvl1 and fgfrl1 ) . these parentally imprinted genes are necessary for female reproductive system such as normal endometrial development , decidualization , placentation and endometriosis . as described previously , the endometriosis susceptibility genes include growth factors ( diras3 , igf2 and fgfrl1 ) , wnt signal transduction ( dvl1 ) , metabolism ( cyp1b1 ) , and stress response and detoxification ( cyp1b1 ) ( khan et al . , 2012 ; kobayashi et al . some biological aspects of endometriosis may be explained from a dysregulation of parentally imprinted gene . recent advances in sequencing , profiling and pathway technologies allow genome - scale approaches to endometriosis - susceptibility gene discovery , which enables us to look for evidence in support of the genetic hypothesis ( albertsen et al . , 2011 ; guo , 2009 ; coln - daz et al . ,
the previous study has shed new light on the overlapping genetic and epigenetic signatures between endometriosis development and insufficient decidualization process , indicating that a number of genes are essential for the decidualization and implantation processes , but up - regulation of a small number of them , including igf , igfbp , prl , hoxa10 , foxo1 , c / ebpbeta , il11 and lif , are important for this process ( kobayashi et al . downregulation of the specific genes related to embryogenesis ( the downstream targets of hoxa10 ) and immuno - endocrine behavior ( il11 , lif , tgf - beta , fkbp4 , cox2 , pgs , foxo1 and c / ebpbeta ) might appear critical to the development of endometriosis ( izawa et al . the irreversible programming or epigenetics may cause insufficient decidualization , which in turn results in infertility and endometriosis . previous studies identified several susceptibility genes that have highlighted the important role of endometriosis development , including igf , igfbp , prl , hoxa10 , foxo1 , c / ebpbeta , il11 , lif , tgf - beta , fkbp4 , cox2 , and prostaglandins ( izawa et al . 2012 ; kobayashi et al . , 2013 ; kawano et al . , 2011 ; wu et al . , 2010 ; vinatier et al
we tried to summarize the recent literature that supports a direct or indirect relationship between the novel candidate imprinting genes and the previously reported endometriosis susceptibility genes . chemicals such as 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) and benzo(a)pyrene modulate the mrna levels of cyp1b1 , c / ebpbeta , il11 and prl ( cao et al . , 2011 ; vogel and matsumura , 2013 ; ueng et al . cyp1b1 , igf2 and hoxa10 genes are reported to be hypermethylated in breast and gastric cancer ( kang et al . , 2006 ) or pge2 , the main product of cox-2 ( yuan et al . , 2012 )
esr2 and smads , downstream signaling cascades of tgf - beta , participates in the establishment of parent - of - origin - specific expression of igf2 ( pathak et al . ,
igf2 induced through the transactivation of c / ebpbeta is involved in prl signaling ( tao et al . , 2013 ;
lif inhibits the glial cell - derived neurotrophic factor ( gdnf)-dependent alteration of the genomic imprinting of igf2 in mice ( jung et al . , 2010 ) . diras3 modulates estrogen and progesterone receptor expression and inhibits prl - induced mammary gland development and lactation , which results in decreased fertility ( xu et al . no convincing evidence has been provided to suggest that these imprinting genes would control the endometriosis susceptibility genes , although some indirect indications are available . at this time
, no attempt was made to ascertain whether any of these 10 genes had a
role or had a role of merely a by - stander in the development of endometriosis . since there are no known driver genes in endometriosis , complex genomic alterations may be responsible for the endometriosis phenotype . the quest for driver imprinting genes or complex genomic alterations can now open new avenues to better understand the mechanisms of endometriosis development . in conclusion , this study supports the possibility that aberrant epigenetic dysregulation of specific imprinting genes may contribute to endometriosis predisposition . further investigations are needed to provide biological evidence for the direct association between the novel candidate imprinting genes and the previously reported endometriosis susceptibility genes . the present review was supported by grant - in - aid for scientific research from the ministry of education , science , and culture of japan to the department of obstetrics and gynecology , nara medical university ( to h.k . ) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | [
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] |
pelvic fractures are uncommon injuries that account for 78% of fractures in adults , associated with high energy traumas such as motor vehicle accidents and falls .
approximately , 68.3% of pelvic fractures are complex and serious unstable fractures , and the mortality rate is up to 19% .
selection of a fixation method for unstable pelvic fracture remains a challenging problem for orthopedic surgeons .
therefore , treatment of an unstable fracture must restore the stability and continuity of the posterior pelvic ring .
minimally invasive internal fixation is a definitive method . due to the development of surgical fixation techniques , it has a great improvement to treat this kind of injuries , including percutaneous iliosacral ( is ) screws , posterior transiliac plating , triangular osteosynthesis , and minimally invasive adjustable plate ( miap ) .
these fixations are used in a clinical practice commonly and have a lot of advantages . however , none is proved to be the best method in clinical and experiments .
is screw fixation into the first sacral body is one of the most frequently used methods with satisfactory results .
however , is screw fixation is a highly technical procedure that requires extensive surgical experience and always needs a long learning curve .
some investigators reported that the average fluoroscopy time for is screw fixation ranges from 26 s to 5.7 min using a c - arm .
in addition , this construct has been shown to be clinically unreliable for percutaneous is screw fixation of unstable sacral fractures in which there was a residual gap at the fracture site . in recent years ,
chen et al . introduced a novel miap , which was based on the structural characteristics of the posterior pelvic ring ( patent no .
chen et al . constructed vertical sacral fracture models , which were fastened to an electroforce 3520-at bose biomechanical testing machine in sitting position and fixed with three kinds of internal fixation .
they demonstrated the translation and rotation stiffness of these fractures fixed with miap were inferior to that fixed with two is screws , but the difference was not significant .
clinically , to avoid long - term complications associated with bed rest , patients treated with internal fixation were asked to walk earlier because of strong fixation .
therefore , we hypothesize that the fractures fixed with miap combined with one si screw are stabler than that with two si screws .
the aim of this biomechanical experiment was to compare the stability of posterior pelvic ring disruptions fixed with these two kinds of internal fixation and provide a basis for the clinical application .
the study was approved by the hospital ethics committee of the third hospital of hebei medical university ( shijiazhuang , hebei , china ) .
six embalmed adult male cadaver pelvises ( provided by the department of anatomy of hebei medical university ) were used for biomechanical investigations .
the average age of the specimens was 41.3 years ( range , 2852 years ) .
the inclusion criteria for pelvis specimens were as follows : ( 1 ) specimens that were proven to have osteoporosis using an osteocore 3 dual energy x - ray osteodensitometer ( medilink company , parc de la mediterranee , france ) were excluded .
( 2 ) specimens from patients with tuberculosis , rheumatism , cancer and other diseases , and anatomic variations were excluded .
( 3 ) the fourth and fifth lumbar vertebrae must be intact , and both femurs must be amputated at the transition of the upper to the middle third .
ligaments ( anterior , posterior , interosseal sacroiliac ligament , sacrospinous ligament , and sacrotuberous ligament ) were left intact .
tianjin , china ) is composed of an adjustable connection bar and two z - shaped brackets .
each z - shaped bracket consists of an upper wing , lower wing , and web plate .
the connection is made up of a hexagonal tube and two custom - made eye bolts , which can be stretched or shortened by rotating the hexagonal tube .
the z - shaped bracket was positioned with the upper wing on the dorsal surface of the posterior superior iliac spines and the lower wing close to the dorsal surface of sacrum .
some long cancellous screws were inserted through the holes of the upper and lower wings to secure the z - shaped bracket on the ilium and sacrum , respectively . in tile c fractures ,
the anterior and posterior pelvic rings are disrupted , leading to translation and rotation instability .
anteriorly , a symphysis pubis rupture was made by cutting through the symphysis pubis , which was stabilized with a four- or five - hole plate placed on the cranial aspect of the symphysis .
posteriorly , a denis type ii fracture was manipulated by an oscillating saw passing through the left sacral foramen .
after the reduction of sacral fractures , two kinds of internal fixation were implanted . in this study , two kinds of internal fixation were used in turn as follows :
miap combined with one si screw group : the posterior side of the ilium and sacrum was exposed , and an appropriate miap ( tianjin zhengtian medical instrument company ltd ) was chosen .
and then , one 2.0-mm kirschner wires was inserted through the ipsilateral external surface of the ilium and into the first sacral vertebral body .
fluoroscopy ( anteroposterior , inlet , and outlet views ) was used to confirm appropriate screw placement .
one 7.3-mm cannulated , partially threaded , and cancellous is screw ( tianjin zhengtian medical instrument company ltd ) was inserted into the first sacral vertebral body along kirschner wire .
computed tomography ( ct ) scan was used to confirm appropriate screw placement [ figure 2]two is screws group : to remove the miap .
then , the other 7.3-mm cannulated , partially threaded , and cancellous is screw was also inserted into the first sacral vertebral body .
miap combined with one si screw group : the posterior side of the ilium and sacrum was exposed , and an appropriate miap ( tianjin zhengtian medical instrument company ltd ) was chosen .
and then , one 2.0-mm kirschner wires was inserted through the ipsilateral external surface of the ilium and into the first sacral vertebral body .
fluoroscopy ( anteroposterior , inlet , and outlet views ) was used to confirm appropriate screw placement .
one 7.3-mm cannulated , partially threaded , and cancellous is screw ( tianjin zhengtian medical instrument company ltd ) was inserted into the first sacral vertebral body along kirschner wire .
computed tomography ( ct ) scan was used to confirm appropriate screw placement [ figure 2 ] two is screws group : to remove the miap .
then , the other 7.3-mm cannulated , partially threaded , and cancellous is screw was also inserted into the first sacral vertebral body .
( a ) posterior view of the specimen and grating displacement sensor ; ( b ) anteroposterior radiograph of the pelvis .
( a ) posterior view of the specimen and grating displacement sensor ; ( b ) anteroposterior radiograph of the pelvis .
bilateral distal femurs of the specimens were fixed in the homemade clamp using type ii denture - based self - curing denture acrylic .
the pelvic specimens were fastened in the electroforce 3520-at bose biomechanical testing machine ( bose corporation , eden prairie , usa ) at a standing neutral position . and both anterior superior iliac spines and ventral surface of the pubic symphysis were covered by the same plane .
the positioning pins of a grating displacement sensor ( guangzhou lokshun cnc equipment ltd . ,
guangzhou , china ) were set to enclose an angle parallel to the posterior inferior iliac spine ( piis ) and perpendicular to the fracture gap , which was fastened to the testing machine .
after each kind of internal fixation was implanted , a vertical load of 200 n was applied to eliminate creep .
the vertical cyclic load was between 0 and 500 n and increased at a rate of 10 n / s .
the displacement of the specimens was recorded at vertical loads of 100 , 200 , 300 , 400 , and 500 n by the testing machine .
shifts in the fracture gap were recorded at a vertical load of 500 n by the grating displacement sensor . the pelvic fracture model was constructed and fixed by the same researcher . designing the direction and location of the internal fixation prior to implantation and using x - ray fluoroscopy after the procedure can reduce the influence of prefixation on subsequent operations .
displacement and shifts in the fracture gap were compared by paired - samples t - test .
six embalmed adult male cadaver pelvises ( provided by the department of anatomy of hebei medical university ) were used for biomechanical investigations .
the average age of the specimens was 41.3 years ( range , 2852 years ) .
the inclusion criteria for pelvis specimens were as follows : ( 1 ) specimens that were proven to have osteoporosis using an osteocore 3 dual energy x - ray osteodensitometer ( medilink company , parc de la mediterranee , france ) were excluded .
( 2 ) specimens from patients with tuberculosis , rheumatism , cancer and other diseases , and anatomic variations were excluded .
( 3 ) the fourth and fifth lumbar vertebrae must be intact , and both femurs must be amputated at the transition of the upper to the middle third .
ligaments ( anterior , posterior , interosseal sacroiliac ligament , sacrospinous ligament , and sacrotuberous ligament ) were left intact . ( 4 ) hip joints and pubic symphysis must be intact .
tianjin , china ) is composed of an adjustable connection bar and two z - shaped brackets .
each z - shaped bracket consists of an upper wing , lower wing , and web plate .
the connection is made up of a hexagonal tube and two custom - made eye bolts , which can be stretched or shortened by rotating the hexagonal tube .
the z - shaped bracket was positioned with the upper wing on the dorsal surface of the posterior superior iliac spines and the lower wing close to the dorsal surface of sacrum .
some long cancellous screws were inserted through the holes of the upper and lower wings to secure the z - shaped bracket on the ilium and sacrum , respectively .
in tile c fractures , the anterior and posterior pelvic rings are disrupted , leading to translation and rotation instability .
anteriorly , a symphysis pubis rupture was made by cutting through the symphysis pubis , which was stabilized with a four- or five - hole plate placed on the cranial aspect of the symphysis .
posteriorly , a denis type ii fracture was manipulated by an oscillating saw passing through the left sacral foramen .
after the reduction of sacral fractures , two kinds of internal fixation were implanted . in this study , two kinds of internal fixation were used in turn as follows :
miap combined with one si screw group : the posterior side of the ilium and sacrum was exposed , and an appropriate miap ( tianjin zhengtian medical instrument company ltd ) was chosen .
and then , one 2.0-mm kirschner wires was inserted through the ipsilateral external surface of the ilium and into the first sacral vertebral body .
fluoroscopy ( anteroposterior , inlet , and outlet views ) was used to confirm appropriate screw placement .
one 7.3-mm cannulated , partially threaded , and cancellous is screw ( tianjin zhengtian medical instrument company ltd ) was inserted into the first sacral vertebral body along kirschner wire .
computed tomography ( ct ) scan was used to confirm appropriate screw placement [ figure 2]two is screws group : to remove the miap .
then , the other 7.3-mm cannulated , partially threaded , and cancellous is screw was also inserted into the first sacral vertebral body .
miap combined with one si screw group : the posterior side of the ilium and sacrum was exposed , and an appropriate miap ( tianjin zhengtian medical instrument company ltd ) was chosen .
and then , one 2.0-mm kirschner wires was inserted through the ipsilateral external surface of the ilium and into the first sacral vertebral body .
fluoroscopy ( anteroposterior , inlet , and outlet views ) was used to confirm appropriate screw placement .
one 7.3-mm cannulated , partially threaded , and cancellous is screw ( tianjin zhengtian medical instrument company ltd ) was inserted into the first sacral vertebral body along kirschner wire .
computed tomography ( ct ) scan was used to confirm appropriate screw placement [ figure 2 ] two is screws group : to remove the miap .
then , the other 7.3-mm cannulated , partially threaded , and cancellous is screw was also inserted into the first sacral vertebral body .
( a ) posterior view of the specimen and grating displacement sensor ; ( b ) anteroposterior radiograph of the pelvis .
( a ) posterior view of the specimen and grating displacement sensor ; ( b ) anteroposterior radiograph of the pelvis .
bilateral distal femurs of the specimens were fixed in the homemade clamp using type ii denture - based self - curing denture acrylic .
the pelvic specimens were fastened in the electroforce 3520-at bose biomechanical testing machine ( bose corporation , eden prairie , usa ) at a standing neutral position . and both anterior superior iliac spines and ventral surface of the pubic symphysis were covered by the same plane .
the positioning pins of a grating displacement sensor ( guangzhou lokshun cnc equipment ltd . ,
guangzhou , china ) were set to enclose an angle parallel to the posterior inferior iliac spine ( piis ) and perpendicular to the fracture gap , which was fastened to the testing machine .
after each kind of internal fixation was implanted , a vertical load of 200 n was applied to eliminate creep .
the vertical cyclic load was between 0 and 500 n and increased at a rate of 10 n / s .
the displacement of the specimens was recorded at vertical loads of 100 , 200 , 300 , 400 , and 500 n by the testing machine .
shifts in the fracture gap were recorded at a vertical load of 500 n by the grating displacement sensor . the pelvic fracture model was constructed and fixed by the same researcher . designing the direction and location of the internal fixation prior to implantation and using x - ray fluoroscopy after the procedure can reduce the influence of prefixation on subsequent operations .
displacement and shifts in the fracture gap were compared by paired - samples t - test .
during the experimental period , the specimens were fixed at a neutral position without any obvious obliquity or fracture . loosening , breakage , and avulsion of the internal fixation were not observed .
displacements were recorded by the bose biomechanical workstation under vertical load and shifts in the fracture gap were simultaneously recorded by a researcher .
the load - displacement scatter graph of the specimens was a smooth straight line , which indicates that the deformation observed in the specimens was elastic [ figure 4 ] .
load - displacement scatter graph of the specimens indicates that the deformation of specimens was elastic . under the vertical load of 100 , 200 , 300 , 400 , and 500 n ,
the average displacement of the specimens fixed with two is screws was 0.692 , 1.249 , 1.669 , 2.061 , and 2.421 mm , respectively . and
the average displacement of the specimens fixed with miap combined with one is screw was 0.46 , 0.735 , 1.377 , 1.823 , and 2.215 mm , respectively , which was significantly lower than that of specimens fixed with two is screws under corresponding load ( p < 0.05 ) [ table 1 ] .
displacement of the pelvis fixed with two kinds of internal fixation under vertical load ( meansd , n = 6 ) is : iliosacral ; miap : minimally invasive adjustable plate . under the vertical load of 500 n
, the shift in the fracture gap of specimens fixed with miap combined with one is screw was 0.261 0.095 mm , and that of specimens fixed with two is screws was 0.809 0.170 mm .
unstable pelvic ring injuries are always intricate and challenging problems for traumatologists and orthopedic surgeons because of the associated significant morbidity and mortality .
early reduction and rigid fixation are very important for the successful management of this lesion . with the rapid development in medical technology ,
percutaneous placement of is screws is an attractive procedure with the well - known advantage of providing a sufficient biomechanical stability .
miap was also applied using a minimally invasive procedure , and the incidence of iatrogenic injury involving blood vessels and nerves was low .
chen et al . indicated that posterior pelvic ring disruption fixed with miap was as stable as that fixed with two is screws . in order to achieve greater stability ,
we propose a new method , miap combined with one si screw , for treating the posterior pelvic ring fractures . in this study , we designed a biomechanical experiment to compare the stability of posterior pelvic ring disruptions fixed with the two methods under physiological conditions .
posterior injuries are composed of a dislocation or fracture of the sacroiliac joint or vertical fracture of the sacrum .
anteriorly , the injury may consist of disruption of the symphysis pubis or unilateral or bilateral fractures of the pubic rami . in this study , we manufactured a tile c pelvic fracture , the most frequent sacral fracture , by cutting the sacrum through the sacral foramen using an oscillating saw and cutting the symphysis pubis using a scalpel .
some scholars considered that conservative treatment is most effective when the separation of the symphysis pubis was < 2.5 cm .
however , clinical research showed that fixing posterior pelvic ring simply was prone to the inclination of pelvis , pain , and nonunion .
given that the sacroiliac complex has an important load - bearing function , it could be fixed by rigid internal fixation after reduction to achieve better effects . in our study , compared with two is screws fixation , we tested a new method , miap combined with one si screw , for attaining the most rigid fixation . in order to avoid inaccuracies caused by implanting sequence of internal fixation ,
and then , we removed the miap and inserted the second is screw into the first sacral vertebral body .
we fixed the samples at a standing neutral position and used a peak load of 500 n , which corresponded to the average weight of the upper body of an adult .
twenty cycles were carried out to determine the stability of posterior pelvic ring fixed with two kinds of internal fixation .
only examined ten cycles in biomechanical tests . under vertical load , the shifts in the fracture gap in tile c injuries were not limited to vertical ( z - axis ) shifts but also included rotational ( x - axis ) and translational ( y - axis ) shifts .
shifts arising along three axes were calculated , and the shifts in the fracture gap on the level of the piis were measured using the grating displacement sensor , which was used to reflect the stability of pelvic fractures fixed with two kinds of internal fixation . the vertical displacement recorded by the biomechanical testing machine reflected the total displacement of the entire structure , however the two kinds of internal fixation were fixed in the same specimen in turn , therefore , our data were comparable .
the results of this study indicate that using two is screws and miap combined with one si screw in the treatment of tile c pelvic ring injuries restore most of the stability of the pelvic ring .
the miap conforms to the anatomical structure of the posterior pelvic ring , which is used to fixate the posterior pelvic ring fractures without prebending .
it is functioned as a suspension bridge structure similar to the sacroiliac complex that plays a very important role in the human skeletal system by linking up the trunk and lower limbs .
biomechanical study suggested that the longitudinal sacral fractures fixed with miap were as stable as that with two is screws under vertical and torsional load .
under 600 n vertical load , the maximal displacement of the plevis fixed with miap was 1.30 mm on average , similar to the average displacement of 1.27 mm fixed with two is screws ; the torque required for 4 rotation of the pelvis fixed with miap was 7.81 nm on average , similar to the average torque of 7.82 nm .
it was attached to the dorsal part of sacroiliac joints and fixed the ilium and sacrum through long cancellous screws .
miap can be used to reduce separated or compressed fractures by rotating the connection bar .
chen et al . treated patients with denis type ii sacral fractures using miap , and yielded satisfactory clinical outcomes .
two is screws placed in the first sacral vertebral body to stabilize the posterior pelvic ring was considered to be the strongest fixation .
central fixation technique that provides strong fixation because is screw placement into the s1 body penetrates through three layers of cortical bone .
although some biomechanical studies suggested that two is screws provided better fixation than only one screw for stabilizing the posterior pelvic ring , placing of the single screw was considered sufficient .
therefore , miap combined with one is screw , equal to posterior fixation combined with central fixation , can provide an excellent biomechanical stability . in our study
, we found that the stability of the posterior pelvic ring fixed by miap combined with one screw was better than that fixed by two is screws .
percutaneous two is screws fixation has such advantages as short incisions , low soft tissue complications , and less blood loss .
however , two is screws fixation remains a technically difficult manipulation because of limitations of medical facilities , clinical experience , and variations of the anatomical structure .
although computer - navigated is screw insertion can reduce the operation time , malposition rate , and radiation exposure , its widespread application has been limited because the huge facilities and expensive equipment are required .
c - arm fluoroscopy is still commonly used in practice , under which it is difficult to identify safe zone .
sacral dysmorphism is not rare , occurring in 3040% of adult patients , and the upper sacral safe zone is 36% smaller than that in normals .
routine one is screw insertion into the first sacral vertebral body was safe , but the placement of two is screws was considered to be difficult . in addition , is screw can not effectively reduce the compressed sacral fractures . in our approach ,
miap was implanted firstly , which can be used to reduce separated or compressed fractures .
and then , one is screw was inserted easily for increasing the stability of pelvis .
this procedure is helpful in promoting its widespread application in most hospitals in the world .
we implanted two kinds of internal fixation in the same specimen in turn to reduce the influence caused by skeletal age and bone , but the implantation and removal of internal fixation were bound to affect the strength of posterior pelvic ring . in addition , only six specimens were used in this study because the acquisition of a large number of specimens was difficult . in future study , we should increase the sample size and compare the stability of the posterior pelvic ring fixed with these kinds of internal fixation . in conclusion , the stability of tile c pelvic fractures fixed with miap combined with one is screw was better than that fixed with two is screws under vertical load .
this new method could be used in the clinical treatment of longitudinal fractures of the posterior pelvic ring .
this study was funded by the grants from the national natural science foundation of china ( no .
this study was funded by the grants from the national natural science foundation of china ( no . | background : unstable pelvic fractures are complex and serious injuries .
selection of a fixation method for these fractures remains a challenging problem for orthopedic surgeons .
this study aimed to compare the stability of tile c pelvic fractures fixed with two iliosacral ( is ) screws and minimally invasive adjustable plate ( miap ) combined with one is screw.methods:this study was a biomechanical experiment .
six embalmed specimens of the adult pelvis were used .
the soft tissue was removed from the specimens , and the spines from the fourth lumbar vertebra to the proximal one - third of both femurs were retained .
the pubic symphysis , bilateral sacroiliac joints and ligaments , bilateral hip joints , bilateral sacrotuberous ligaments , and bilateral sacrospinous ligaments were intact .
tile c pelvic fractures were made on the specimens .
the symphysis pubis was fixed with a plate , and the fracture on the posterior pelvic ring was fixed with two kinds of internal fixation in turn .
the specimens were placed in a biomechanical machine at a standing neutral posture
. a cyclic vertical load of up to 500 n was applied , and displacement was recorded .
shifts in the fracture gap were measured by a grating displacement sensor . statistical analysis used :
paired - samples t-test.results:under the vertical load of 100 , 200 , 300 , 400 , and 500 n , the average displacement of the specimens fixed with miap combined with one is screw was 0.46 , 0.735 , 1.377 , 1.823 , and 2.215 mm , respectively , which was significantly lower than that of specimens fixed with two is screws under corresponding load ( p < 0.05 ) . under the vertical load of 500 n
, the shift in the fracture gap of specimens fixed with miap combined with one is screw was 0.261 0.095 mm , and that of specimens fixed with two is screws was 0.809 0.170 mm .
the difference was significant ( p < 0.05).conclusion : the stability of tile c pelvic fractures fixed with miap combined with one is screw was better than that fixed with two is screws . | I
M
Preparation and preservation of specimens
Structure of minimally invasive adjustable plate
Modeling Type C pelvic ring injury and fixation of specimens
Measurements
Statistical analysis
R
D
Financial support and sponsorship
Conflicts of interest | pelvic fractures are uncommon injuries that account for 78% of fractures in adults , associated with high energy traumas such as motor vehicle accidents and falls . approximately , 68.3% of pelvic fractures are complex and serious unstable fractures , and the mortality rate is up to 19% . selection of a fixation method for unstable pelvic fracture remains a challenging problem for orthopedic surgeons . therefore , treatment of an unstable fracture must restore the stability and continuity of the posterior pelvic ring . minimally invasive internal fixation is a definitive method . due to the development of surgical fixation techniques , it has a great improvement to treat this kind of injuries , including percutaneous iliosacral ( is ) screws , posterior transiliac plating , triangular osteosynthesis , and minimally invasive adjustable plate ( miap ) . is screw fixation into the first sacral body is one of the most frequently used methods with satisfactory results . some investigators reported that the average fluoroscopy time for is screw fixation ranges from 26 s to 5.7 min using a c - arm . in addition , this construct has been shown to be clinically unreliable for percutaneous is screw fixation of unstable sacral fractures in which there was a residual gap at the fracture site . introduced a novel miap , which was based on the structural characteristics of the posterior pelvic ring ( patent no . constructed vertical sacral fracture models , which were fastened to an electroforce 3520-at bose biomechanical testing machine in sitting position and fixed with three kinds of internal fixation . they demonstrated the translation and rotation stiffness of these fractures fixed with miap were inferior to that fixed with two is screws , but the difference was not significant . therefore , we hypothesize that the fractures fixed with miap combined with one si screw are stabler than that with two si screws . the aim of this biomechanical experiment was to compare the stability of posterior pelvic ring disruptions fixed with these two kinds of internal fixation and provide a basis for the clinical application . the study was approved by the hospital ethics committee of the third hospital of hebei medical university ( shijiazhuang , hebei , china ) . six embalmed adult male cadaver pelvises ( provided by the department of anatomy of hebei medical university ) were used for biomechanical investigations . the average age of the specimens was 41.3 years ( range , 2852 years ) . ( 3 ) the fourth and fifth lumbar vertebrae must be intact , and both femurs must be amputated at the transition of the upper to the middle third . the connection is made up of a hexagonal tube and two custom - made eye bolts , which can be stretched or shortened by rotating the hexagonal tube . the z - shaped bracket was positioned with the upper wing on the dorsal surface of the posterior superior iliac spines and the lower wing close to the dorsal surface of sacrum . some long cancellous screws were inserted through the holes of the upper and lower wings to secure the z - shaped bracket on the ilium and sacrum , respectively . in tile c fractures ,
the anterior and posterior pelvic rings are disrupted , leading to translation and rotation instability . anteriorly , a symphysis pubis rupture was made by cutting through the symphysis pubis , which was stabilized with a four- or five - hole plate placed on the cranial aspect of the symphysis . after the reduction of sacral fractures , two kinds of internal fixation were implanted . in this study , two kinds of internal fixation were used in turn as follows :
miap combined with one si screw group : the posterior side of the ilium and sacrum was exposed , and an appropriate miap ( tianjin zhengtian medical instrument company ltd ) was chosen . and then , one 2.0-mm kirschner wires was inserted through the ipsilateral external surface of the ilium and into the first sacral vertebral body . fluoroscopy ( anteroposterior , inlet , and outlet views ) was used to confirm appropriate screw placement . one 7.3-mm cannulated , partially threaded , and cancellous is screw ( tianjin zhengtian medical instrument company ltd ) was inserted into the first sacral vertebral body along kirschner wire . then , the other 7.3-mm cannulated , partially threaded , and cancellous is screw was also inserted into the first sacral vertebral body . miap combined with one si screw group : the posterior side of the ilium and sacrum was exposed , and an appropriate miap ( tianjin zhengtian medical instrument company ltd ) was chosen . one 7.3-mm cannulated , partially threaded , and cancellous is screw ( tianjin zhengtian medical instrument company ltd ) was inserted into the first sacral vertebral body along kirschner wire . computed tomography ( ct ) scan was used to confirm appropriate screw placement [ figure 2 ] two is screws group : to remove the miap . then , the other 7.3-mm cannulated , partially threaded , and cancellous is screw was also inserted into the first sacral vertebral body . ( a ) posterior view of the specimen and grating displacement sensor ; ( b ) anteroposterior radiograph of the pelvis . ( a ) posterior view of the specimen and grating displacement sensor ; ( b ) anteroposterior radiograph of the pelvis . bilateral distal femurs of the specimens were fixed in the homemade clamp using type ii denture - based self - curing denture acrylic . the pelvic specimens were fastened in the electroforce 3520-at bose biomechanical testing machine ( bose corporation , eden prairie , usa ) at a standing neutral position . and both anterior superior iliac spines and ventral surface of the pubic symphysis were covered by the same plane . the positioning pins of a grating displacement sensor ( guangzhou lokshun cnc equipment ltd . ,
guangzhou , china ) were set to enclose an angle parallel to the posterior inferior iliac spine ( piis ) and perpendicular to the fracture gap , which was fastened to the testing machine . after each kind of internal fixation was implanted , a vertical load of 200 n was applied to eliminate creep . the vertical cyclic load was between 0 and 500 n and increased at a rate of 10 n / s . the displacement of the specimens was recorded at vertical loads of 100 , 200 , 300 , 400 , and 500 n by the testing machine . shifts in the fracture gap were recorded at a vertical load of 500 n by the grating displacement sensor . designing the direction and location of the internal fixation prior to implantation and using x - ray fluoroscopy after the procedure can reduce the influence of prefixation on subsequent operations . displacement and shifts in the fracture gap were compared by paired - samples t - test . six embalmed adult male cadaver pelvises ( provided by the department of anatomy of hebei medical university ) were used for biomechanical investigations . the average age of the specimens was 41.3 years ( range , 2852 years ) . ( 2 ) specimens from patients with tuberculosis , rheumatism , cancer and other diseases , and anatomic variations were excluded . ( 3 ) the fourth and fifth lumbar vertebrae must be intact , and both femurs must be amputated at the transition of the upper to the middle third . ( 4 ) hip joints and pubic symphysis must be intact . the connection is made up of a hexagonal tube and two custom - made eye bolts , which can be stretched or shortened by rotating the hexagonal tube . the z - shaped bracket was positioned with the upper wing on the dorsal surface of the posterior superior iliac spines and the lower wing close to the dorsal surface of sacrum . some long cancellous screws were inserted through the holes of the upper and lower wings to secure the z - shaped bracket on the ilium and sacrum , respectively . in tile c fractures , the anterior and posterior pelvic rings are disrupted , leading to translation and rotation instability . anteriorly , a symphysis pubis rupture was made by cutting through the symphysis pubis , which was stabilized with a four- or five - hole plate placed on the cranial aspect of the symphysis . after the reduction of sacral fractures , two kinds of internal fixation were implanted . in this study , two kinds of internal fixation were used in turn as follows :
miap combined with one si screw group : the posterior side of the ilium and sacrum was exposed , and an appropriate miap ( tianjin zhengtian medical instrument company ltd ) was chosen . fluoroscopy ( anteroposterior , inlet , and outlet views ) was used to confirm appropriate screw placement . one 7.3-mm cannulated , partially threaded , and cancellous is screw ( tianjin zhengtian medical instrument company ltd ) was inserted into the first sacral vertebral body along kirschner wire . then , the other 7.3-mm cannulated , partially threaded , and cancellous is screw was also inserted into the first sacral vertebral body . miap combined with one si screw group : the posterior side of the ilium and sacrum was exposed , and an appropriate miap ( tianjin zhengtian medical instrument company ltd ) was chosen . fluoroscopy ( anteroposterior , inlet , and outlet views ) was used to confirm appropriate screw placement . one 7.3-mm cannulated , partially threaded , and cancellous is screw ( tianjin zhengtian medical instrument company ltd ) was inserted into the first sacral vertebral body along kirschner wire . computed tomography ( ct ) scan was used to confirm appropriate screw placement [ figure 2 ] two is screws group : to remove the miap . then , the other 7.3-mm cannulated , partially threaded , and cancellous is screw was also inserted into the first sacral vertebral body . ( a ) posterior view of the specimen and grating displacement sensor ; ( b ) anteroposterior radiograph of the pelvis . ( a ) posterior view of the specimen and grating displacement sensor ; ( b ) anteroposterior radiograph of the pelvis . bilateral distal femurs of the specimens were fixed in the homemade clamp using type ii denture - based self - curing denture acrylic . the pelvic specimens were fastened in the electroforce 3520-at bose biomechanical testing machine ( bose corporation , eden prairie , usa ) at a standing neutral position . and both anterior superior iliac spines and ventral surface of the pubic symphysis were covered by the same plane . the positioning pins of a grating displacement sensor ( guangzhou lokshun cnc equipment ltd . ,
guangzhou , china ) were set to enclose an angle parallel to the posterior inferior iliac spine ( piis ) and perpendicular to the fracture gap , which was fastened to the testing machine . after each kind of internal fixation was implanted , a vertical load of 200 n was applied to eliminate creep . the vertical cyclic load was between 0 and 500 n and increased at a rate of 10 n / s . the displacement of the specimens was recorded at vertical loads of 100 , 200 , 300 , 400 , and 500 n by the testing machine . shifts in the fracture gap were recorded at a vertical load of 500 n by the grating displacement sensor . designing the direction and location of the internal fixation prior to implantation and using x - ray fluoroscopy after the procedure can reduce the influence of prefixation on subsequent operations . displacement and shifts in the fracture gap were compared by paired - samples t - test . during the experimental period , the specimens were fixed at a neutral position without any obvious obliquity or fracture . loosening , breakage , and avulsion of the internal fixation were not observed . displacements were recorded by the bose biomechanical workstation under vertical load and shifts in the fracture gap were simultaneously recorded by a researcher . the load - displacement scatter graph of the specimens was a smooth straight line , which indicates that the deformation observed in the specimens was elastic [ figure 4 ] . load - displacement scatter graph of the specimens indicates that the deformation of specimens was elastic . under the vertical load of 100 , 200 , 300 , 400 , and 500 n ,
the average displacement of the specimens fixed with two is screws was 0.692 , 1.249 , 1.669 , 2.061 , and 2.421 mm , respectively . and
the average displacement of the specimens fixed with miap combined with one is screw was 0.46 , 0.735 , 1.377 , 1.823 , and 2.215 mm , respectively , which was significantly lower than that of specimens fixed with two is screws under corresponding load ( p < 0.05 ) [ table 1 ] . displacement of the pelvis fixed with two kinds of internal fixation under vertical load ( meansd , n = 6 ) is : iliosacral ; miap : minimally invasive adjustable plate . under the vertical load of 500 n
, the shift in the fracture gap of specimens fixed with miap combined with one is screw was 0.261 0.095 mm , and that of specimens fixed with two is screws was 0.809 0.170 mm . unstable pelvic ring injuries are always intricate and challenging problems for traumatologists and orthopedic surgeons because of the associated significant morbidity and mortality . miap was also applied using a minimally invasive procedure , and the incidence of iatrogenic injury involving blood vessels and nerves was low . indicated that posterior pelvic ring disruption fixed with miap was as stable as that fixed with two is screws . in order to achieve greater stability ,
we propose a new method , miap combined with one si screw , for treating the posterior pelvic ring fractures . in this study , we designed a biomechanical experiment to compare the stability of posterior pelvic ring disruptions fixed with the two methods under physiological conditions . posterior injuries are composed of a dislocation or fracture of the sacroiliac joint or vertical fracture of the sacrum . anteriorly , the injury may consist of disruption of the symphysis pubis or unilateral or bilateral fractures of the pubic rami . in this study , we manufactured a tile c pelvic fracture , the most frequent sacral fracture , by cutting the sacrum through the sacral foramen using an oscillating saw and cutting the symphysis pubis using a scalpel . some scholars considered that conservative treatment is most effective when the separation of the symphysis pubis was < 2.5 cm . however , clinical research showed that fixing posterior pelvic ring simply was prone to the inclination of pelvis , pain , and nonunion . in our study , compared with two is screws fixation , we tested a new method , miap combined with one si screw , for attaining the most rigid fixation . in order to avoid inaccuracies caused by implanting sequence of internal fixation ,
and then , we removed the miap and inserted the second is screw into the first sacral vertebral body . we fixed the samples at a standing neutral position and used a peak load of 500 n , which corresponded to the average weight of the upper body of an adult . twenty cycles were carried out to determine the stability of posterior pelvic ring fixed with two kinds of internal fixation . under vertical load , the shifts in the fracture gap in tile c injuries were not limited to vertical ( z - axis ) shifts but also included rotational ( x - axis ) and translational ( y - axis ) shifts . shifts arising along three axes were calculated , and the shifts in the fracture gap on the level of the piis were measured using the grating displacement sensor , which was used to reflect the stability of pelvic fractures fixed with two kinds of internal fixation . the vertical displacement recorded by the biomechanical testing machine reflected the total displacement of the entire structure , however the two kinds of internal fixation were fixed in the same specimen in turn , therefore , our data were comparable . the results of this study indicate that using two is screws and miap combined with one si screw in the treatment of tile c pelvic ring injuries restore most of the stability of the pelvic ring . the miap conforms to the anatomical structure of the posterior pelvic ring , which is used to fixate the posterior pelvic ring fractures without prebending . it is functioned as a suspension bridge structure similar to the sacroiliac complex that plays a very important role in the human skeletal system by linking up the trunk and lower limbs . biomechanical study suggested that the longitudinal sacral fractures fixed with miap were as stable as that with two is screws under vertical and torsional load . under 600 n vertical load , the maximal displacement of the plevis fixed with miap was 1.30 mm on average , similar to the average displacement of 1.27 mm fixed with two is screws ; the torque required for 4 rotation of the pelvis fixed with miap was 7.81 nm on average , similar to the average torque of 7.82 nm . it was attached to the dorsal part of sacroiliac joints and fixed the ilium and sacrum through long cancellous screws . two is screws placed in the first sacral vertebral body to stabilize the posterior pelvic ring was considered to be the strongest fixation . although some biomechanical studies suggested that two is screws provided better fixation than only one screw for stabilizing the posterior pelvic ring , placing of the single screw was considered sufficient . therefore , miap combined with one is screw , equal to posterior fixation combined with central fixation , can provide an excellent biomechanical stability . in our study
, we found that the stability of the posterior pelvic ring fixed by miap combined with one screw was better than that fixed by two is screws . percutaneous two is screws fixation has such advantages as short incisions , low soft tissue complications , and less blood loss . however , two is screws fixation remains a technically difficult manipulation because of limitations of medical facilities , clinical experience , and variations of the anatomical structure . although computer - navigated is screw insertion can reduce the operation time , malposition rate , and radiation exposure , its widespread application has been limited because the huge facilities and expensive equipment are required . sacral dysmorphism is not rare , occurring in 3040% of adult patients , and the upper sacral safe zone is 36% smaller than that in normals . routine one is screw insertion into the first sacral vertebral body was safe , but the placement of two is screws was considered to be difficult . and then , one is screw was inserted easily for increasing the stability of pelvis . we implanted two kinds of internal fixation in the same specimen in turn to reduce the influence caused by skeletal age and bone , but the implantation and removal of internal fixation were bound to affect the strength of posterior pelvic ring . in addition , only six specimens were used in this study because the acquisition of a large number of specimens was difficult . in future study , we should increase the sample size and compare the stability of the posterior pelvic ring fixed with these kinds of internal fixation . in conclusion , the stability of tile c pelvic fractures fixed with miap combined with one is screw was better than that fixed with two is screws under vertical load . this new method could be used in the clinical treatment of longitudinal fractures of the posterior pelvic ring . this study was funded by the grants from the national natural science foundation of china ( no . this study was funded by the grants from the national natural science foundation of china ( no . | [
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this rapidly evolving technology is now routinely applied in many laboratories for protein expression profiling , biomarker discovery / validation , posttranslational modification mapping , and complex disease research .
the depth of sampling and sensitivity and the scan speed of mass spectrometers have improved tremendously . but critical sample preparation steps which could impact the success of proteomics experiments , for example , the digestion of proteins into peptides prior to the mass spectrometry step , have not improved or received the kind of attention that instrumentation receives .
the variant of proteomics termed shotgun proteomics , also known as bottom - up proteomics , is one of the most widely applied proteomic strategies which rely on the digestion of proteins into peptides prior to mass spectrometry . in shotgun proteomics , an enzyme such as trypsin is added to the protein sample and the mixture is incubated at 37c for a specified time or overnight at ambient room temperature to digest proteins .
trypsin cleaves proteins at the c - terminus of lysine ( k ) or arginine ( r ) residues , except when either residue is followed by a proline ( p ) .
the resulting peptides are analyzed by mass spectrometry and the mass - to - charge ( m / z ) ratio generated is used to establish protein identities by reference to an appropriate database .
the protein digestion step is recognized as one of the single most important sample preparation steps that can directly affect the outcome of all proteomics experiments . since the central objective of all shotgun proteomics is to identify all proteins present in the sample in a single run
, it is desirable and very important to optimize all pre - analytical and sample preparation steps especially the protein digestion step , to ensure that there is run - to - run reproducibility and the maximum possible number of proteins in each sample will be identified in each run .
zhang and li ( 2004 ) reports that the type of protein , complexity of sample mixture , presence of surfactants , and impurities in the reaction mixture can affect the efficiency of tryptic digestion and hence the outcome of the experiment .
considering that trypsin cleaves proteins at the c - terminus of lysine or arginine residues except when either residue is followed by a proline , it is expected that the time required to sufficiently digest proteins with trypsin will vary amongst different protein types , sample complexity , and reaction condition .
that means the addition of trypsin ( or any selected enzyme ) to a protein mixture and incubation of the reaction mixture for a specified time period is not a guarantee that the enzyme will sufficiently digest all classes of protein(s ) present within the time allowed for optimal proteomics outcome .
in fact the study of klammer and maccoss published in 2006 clearly demonstrated that the number of unique proteins identified in plasma is primarily determined by the quality and completeness of the tryptic digestion step , and karuso et al .
recommend that proteins should be optimally digested prior to mass spectrometry , to avoid the wastage of valuable instrument time and generation of results that will be difficult to interpret .
but most laboratories add trypsin to samples and incubate for a time period with the hope that the digestion obtained will be sufficient for optimal mass spectrometry .
no attempt is typically made to determine if the digestion is sufficient before the mass spectrometry step .
experience in our laboratory indicate that the same batch of trypsin often digests different protein samples to different degrees within a specified time due to sample complexity , sequence differences , and specific activity of the enzyme . considering that under the same conditions , two different protein samples exposed to trypsin
may not be digested to the same extent , using a fixed digestion time will not result in optimal digestion of all types of proteins .
therefore , to objectively compare mass spectrometry results , the tryptic digestion step must be standardized and a better method for standardization should be based on the extent rather than duration of digestion .
it is reported that routine monitoring of tryptic digestion for understanding extent or percentage of protein digestion is not performed in most laboratories partly because the methods available to monitor protein digestion including hplc , circular dichroism , sds - page , mass spectrometry , and the use of fluorescent dyes are both time and sample consuming , and expensive to perform on a regular basis , or require special and expensive equipment [ 5 , 6 ] .
methods for protein digestion monitoring that rely on the use of a mass spectrometer is unattractive and challenging because of the cost and time for the mass spectrometry analysis , database search to identify the protein sequence and deducing sample independent metrics that can be used to monitor the completion of trypsin digestion .
additionally , because of scanty scientific literature on the subject , many proteomics scientists are unaware of the significance of insufficient and excessive digestion on the outcome of proteomics experiments , and many erroneously believe that the only requirement for a good proteomics result is access to a state - of - the - art high - end mass spectrometer .
we were motivated to conduct this study because our laboratory routinely performs shotgun proteomics of plasma and serum samples using label - free lc / ms / ms and itraq technologies , and we observe differences in protein identification that were attributed to inconsistent trypsin digestion .
since reproducibility and maximum protein identification are essential in quantitative proteomics , we presumed that using a method that is independent of mass spectrometry to standardize the tryptic digestion step will improve reproducibility and outcome .
this paper describes the evaluation of a novel protein digestion monitoring ( prodm ) kit , as a method for monitoring trypsin digestion prior to mass spectrometry .
the results obtained from our experiments demonstrate that the number of proteins identified and the protein sequence coverage are affected by the extent of protein digestion , and prodm can be used to precisely determine the extent of protein digestion before the mass spectrometry step .
the ammonium bicarbonate , acetonitrile , and trifluoroethanol were purchased from fisher scientific , pittsburgh , pa , iodoacetamide ( iaa ) was purchased from ge healthcare biosciences , piscataway , nj , formic acid from sigma , st .
louis , mo , and -mercaptoethanol was purchased from amresco , solon , oh . the agilent protein 80 chip kit was purchased from agilent technologies , santa clara , ca . the proteomics grade plasma ( pgpt ) and serum ( pgst ) tubes for collection of blood , protein digestion monitoring kit ( prodm ) , and total protein assay reagent ( topa ) were supplied by itsi - biosciences , johnstown , pa , and picofrit c18 nanospray column was purchased from new objective , woburn , ma .
blood was collected from a 45-year - old male donor using the pgpt and pgst tubes to eliminate ex vivo changes that could skew the data .
plasma and serum were isolated according to standard plasma and serum isolation protocols , aliquoted in 1 ml amounts to avoid repeated freeze / thaw of the same aliquot , and stored at 80c until used .
prior to use , samples were thawed on ice and the total protein content was determined for all samples using the topa kit .
all experiments were performed in duplicate and all results presented are averages of two separate readings .
plasma and serum proteins were digested with trypsin in a buffer system containing trifluoroethanol ( tfe ) .
briefly , 50 l of plasma or serum was added to a clean microfuge tube containing 50 l of tfe , and the mixture vortexed briefly .
the sample was reduced with dtt ( 10 mm ) and alkylated with iaa ( 20 mm ) .
400 l of hplc grade water was added to dilute the tfe and prevent the interference from tfe .
the ph was checked and appropriate amount of 1 m ammonium bicarbonate was added so that the final concentration of ammonium bicarbonate was 50 mm and the ph was above 8.0 .
trypsin ( 5% w / w ) was added and an aliquot representing time zero was removed and processed immediately .
subsequently , the reaction mixture was incubated for 8 h and 24 h at 37c . at the end of the incubation ,
the time zero , 8 h , and 24 h samples were analyzed as described below . in a parallel experiment serum and plasma
were digested as described above for 2 h , 8 h , and 24 h and the digested samples were analyzed by mass spectrometry .
the sequence coverage of three model proteins , namely , albumin , haptoglobin , and serotransferrin was determined and compared to elucidate the effect of digestion extent on protein sequence coverage .
plasma and serum samples were analyzed with the prodm kit according to the manufacturers ' protocol .
briefly , prodm kit contains ready - to - use reagents including ( i ) a standard buffer ( urea - tris , buffer ph 8.5 ) , ( ii ) reaction buffer ( tris - buffer , ph 8.5 ) , ( iii ) reaction quencher ( buffered phosphoric acid ) , and ( iv ) modified topa colorimetric reagent ( itsi - biosciences , johnstown , pa ) .
the samples collected at time zero and the samples collected after 8 h and 24 h digestion with trypsin were independently processed to determine the extent of protein digestion
. 10 l of the reaction mixture was transferred , at zero time and after 8 h and 24 h incubation , to a fresh eppendorf tube containing 2 l of reaction quencher .
the mixture was vortexed briefly to mix , colorimetric reagent was added , and the absorbance was read at 595 nm within 13 min of adding the color reagent .
the % protein digested ( % pd ) was calculated with an application running in ms excel .
the application is based on the formula % pd = [ ( a1(t0,l ) a2(tx , l))/a1(t0,l)]100 , where % pd is the percentage of protein digested by the enzyme into peptides within a specific incubation time interval ; a1(t0,l ) is the absorbance of the first aliquot at time zero ; and a2(tx , l ) is the absorbance of the second aliquot after 8 h or 24 h digestion . to screen the samples with the agilent bioanalyzer protein 80 chip , 50 l each of ( a ) time zero , ( b ) 8 h digested , and ( c )
24 h digested serum and plasma samples were transferred to fresh tubes , dried down in a speedvac , and resuspended with 30 l of distilled and deionized ( milli - q ) water .
then , 4 l of the resuspended sample containing 3.4 g of total protein was transferred to a clean tube and 2 l of agilent sample buffer supplemented with 3.5% v / v of -mercaptoethanol was added .
the samples and 6 l of protein ladder included in the protein 80 kit were heated for 5 min at 95c and diluted with 84 l of milli - q water before electrophoresis .
for electrophoresis , the agilent protein 80 chip was primed according to the manufacturer 's instructions and 6 l of all samples was loaded into separate sample wells and the chip was run on the agilent bioanalyzer 2100 using the protein 80 assay program .
following trypsin digestion for 8 h and 24 h , the digestion mixtures were acidified with 5% ( v / v ) formic acid and dried down in a speedvac .
the dried sample was reconstituted in 2% acetonitrile/0.1% formic acid and loaded onto a picofrit c18 nanospray column using a thermo scientific surveyor autosampler operated in no waste injection mode .
peptides were eluted from the column using a linear acetonitrile gradient from 2% to 40% over 60 minutes into a ltq xl mass spectrometer ( thermo scientific ) via a nanospray source with the spray voltage set to 1.8 kv and the ion transfer capillary set at 180c .
a data - dependent top 5 method was used where a full ms scan from m / z 4001500 was followed by ms / ms scans on the five most abundant ions . protein identification and the number of missed cleavages
briefly , the raw data files were searched utilizing sequest algorithm in proteome discoverer 1.3 ( thermo scientific ) against the most recent species - specific fasta database for human downloaded from ncbi .
trypsin was the selected enzyme and we allowed for up to three missed cleavages per peptide .
precursor and fragment ion peaks were searched with a mass tolerance of 5000 ppm and 2 da , respectively .
proteins were identified when unique peptides had x - correlation scores greater than 1.5 , 2.0 , and 2.5 for respective charge states of + 1 , + 2 , and + 3 . to test for optimal - specificity and nonspecific cleaving of proteins
the database was searched with full and semispecificity setting for trypsin . since , contaminating chymotrypsin activity may contribute to generation of nontryptic peptides a similar search was also conducted using chymotrypsin with full specificity . to identify other parameters that may be different as a result of different tryptic digestion times , we compared the m / z distribution and ion intensities in 8 h and 24 h samples by plotting 2d density maps consisting of time ( x - axis ) versus m / z ( y - axis ) versus relative abundance ( z - axis ) for both samples using xcalibur 2.1 ( thermo scientific ) .
proteome discoverer 1.3 was utilized to plot the graph of m / z versus peptide length to demonstrate the distribution of charges in the digested plasma and serum samples .
the primary goal of this study was to determine if prodm , a novel protein digestion monitoring kit , can be used to monitor the pre - analytical trypsin digestion step to identify the precise extent of protein digestion that gives the best protein identification and sequence coverage outcome . since prodm is a colorimetric method we also analyzed the samples with the agilent bioanalyzer protein 80 chip , to benchmark and verify the prodm data . to determine how the extent of digestion affects the outcome of the mass spectrometry data , we analyzed the digested samples by tandem mass spectrometry and compared ( a ) the number of proteins identified and ( b ) protein sequence coverage .
the prodm analysis revealed that about 46% of plasma and serum proteins were digested after 8 h incubation , whereas after 24 h digestion , 56% of proteins were digested in plasma and 50% in serum ( table 1 ) .
this simple colorimetric method required 10 l of sample and less than 15 min to complete . although the mechanism of action of prodm has not been fully elucidated , preliminary studies indicate that the modified topa reagent used in the reaction binds to full length proteins and not peptides .
the color at time zero ( no digestion ) when it is expected that there will be more full length proteins is consistently more intense ( higher absorbance ) than the color after tryptic digestion ( lower absorbance ) when fewer full length proteins are expected .
this reduction in color intensity ( and absorbance ) correlates with the disappearance ( or reduction ) in peak height of higher molecular weight proteins as revealed by sds - page ( results not presented ) and agilent bioanalyzer data ( figure 1 ) .
therefore , the data for % protein digested is the result of undigested and partially digested proteins in the samples at the time of sample collection .
thus , prodm will provide information on the difference between time intervals and can not differentiate between enzymatic and nonenzymatic digestions .
analysis of aliquots of the digested samples with the agilent bioanalyzer protein 80 chip revealed extensive digestion of abundant proteins , especially albumin ( figure 1 ) .
the bioanalyzer also showed that there are still partially digested proteins particularly in the 20 kda to 50 kda region after 24 h incubation ( figure 1 ) .
the visible peaks around 6.5 kda ( figure 1 ) in the 8 h samples are products of partial tryptic digestion of larger proteins .
this assumption is plausible because these peaks are significantly lower in the 24 h digested samples ( figure 1 ) .
the agilent bioanalyzer data correlated with the prodm results and provided a gel - based verification of the presence of undigested proteins after 8 h and 24 h digestion with trypsin .
this means that prodm , which gives the precise percentage of proteins digested , can be used to determine the extent of trypsin digestion that will give the best proteomics outcome .
the goal of all shotgun proteomics experiments is to identify as many proteins as possible with high confidence , and the more the percentage of sequence coverage the higher the confidence . to determine how the tryptic digestion step can affect the outcome of shotgun proteomics
, we analyzed the samples digested for different extents and compared the number of proteins identified and sequence coverage of three proteins commonly found in plasma and serum .
as shown in table 1 , a total of 991 and 733 peptides were sequenced in 8 h and 24 h digested plasma samples , whereas a total of 897 and 693 peptides were sequenced in 8 h and 24 h digested serum samples , respectively .
the corresponding total number of unique proteins identified in plasma in the 8 h and 24 h samples was 125 and 118 , and in serum this was 127 and 107 , respectively ( table 1 ) .
this represents about 26% reduction in the number of peptides sequenced and 6% drop in the number of proteins identified in 24 h digested samples compared to 8 h digested samples , respectively . in serum 127 proteins were identified in 8 h digested samples and 107 were identified in 24 h digested samples ( table 1 ) .
this represents about 16% reduction in the number of proteins identified in 24 h digested samples compared to 8 h digested samples .
the finding that longer digestion times can result in reduced number of identified proteins has been previously reported , and the 125 unique proteins we identified in plasma after 8 h tryptic digestion are comparable to the 150 proteins identified by zimmerman et al . using a comparable lc
/ ms / ms equipment and approach , suggesting that our finding could be generalized to some extent . since
24 h digestion apparently resulted in fewer number of identified proteins compared to 8 h , the prodm data obtained in this experiment shows that ~46% digestion of plasma and serum yields greater numbers of identified proteins compared to 24 h digestion which resulted in 50% protein digestion . in a parallel experiment
, we used albumin , haptoglobin , and serotransferrin as models to gain insights into the potential effect of the % of digestion on protein sequence coverage .
these three proteins have different molecular weights , and together they account for over 70% of the total proteins in serum . as shown in figure 2 , the average sequence coverage for the three proteins in the 2 h , 8 h , and 24 h samples for serum were 52% , 45% , and 45% , respectively , and for plasma were 54% , 47% , and 42% , respectively .
specifically , in plasma , the sequence coverage in the 2 h samples ranged from about 47% ( haptoglobin ) to 61% ( albumin ) and the coverage in 24 h samples ranged from about 30% ( haptoglobin ) to 52% ( albumin ) . in serum ,
the coverage in the 2 h samples ranged from 32% ( haptoglobin ) to 70% ( albumin ) , whereas the coverage in the 24 h samples ranged from 31% ( haptoglobin ) to 61% ( albumin ) .
it was interesting to observe that the average coverage in 2 h was consistently higher than the coverage in 24 h for the three proteins in serum and plasma .
this finding is noteworthy because it suggests that excessive digestion may not only result in reduced number of the total proteins identified , but could also lead to lower confidence due to a reduced protein sequence coverage .
if optimal sequence coverage is required , then prodm could be used to determine the least percentage of digestion that gives the best coverage for the target protein .
additional benefits to finding and using the shortest digestion time that will give the best results include the ability to perform more experiments per day and savings on labor cost . to determine why longer digestion times resulted in fewer number of proteins identified and less sequence coverage we postulated that the number of missed cleavages in the 8 h and 24 h digested samples will be different .
we were interested in missed cleavages because it could provide insights into the specific activity of the enzyme as the digestion progressed .
interestingly , there was no dramatic difference in the number of missed cleavages between the 8 h and 24 h samples , except for 2 missed cleavages , where for plasma 2.3% was detected in 8 h and 0.8% was detected in 24 h and for serum 0.8% was detected in 8 h and 1.5% was detected in 24 h ( figure 3 ) .
the absence of a major difference in the number of zero missed cleavages between 8 h and 24 h digestion is remarkable and requires more study . to determine if this phenomenon is related to reproducibility between mass spectrometry runs , a plot consisting of time ( x - axis ) versus m / z ( y - axis ) versus relative abundance ( z - axis ) was used to compare the 8 h and 24 h digested samples .
figure 4 shows only a subtle difference between 8 h and 24 h samples for plasma and serum indicating that there was good run - run reproducibility .
thus , the difference in the number of proteins identified is apparently not likely due to reproducibility .
the finding that 24 h digestion compared to 8 h digestion leads to a decrease in the number of proteins identified and % protein sequence coverage underscores the importance of optimizing and standardizing the enzymatic digestion step prior to mass spectrometry .
one postulate is that extended digestion leads to poor identifications because of loss of enzyme specificity over time . if this is the case , then a search of the database using chymotrypsin or semitrypsin as the selected enzyme should produce a new set of identified proteins .
indeed , a database search with chymotrypsin using the raw ms / ms files identified a new set of proteins and resulted in a 12% increase in the coverage for albumin in the 24 h sample .
this demonstrates the presence of more nontryptic peptides in the 24 h samples compared to 8 h samples .
it is therefore likely that fewer proteins were identified in the 24 h samples probably because of the loss of enzyme specificity , which resulted in the production of non-/semitryptic peptides .
the charge state of peptides in the 8 h and 24 h samples were compared because the presence of peptides with higher charges is an indication of the presence of longer peptides .
as shown in table 2 , 72% of all peptides identified in plasma and 86% of all peptides identified in serum in the 8 h samples had + 2 charge states .
the 24 h samples contained a higher number of + 3 charges indicating that there were more longer peptides compared to the 8 h samples .
the graphical illustration of the distribution of charges obtained by plotting the xcorr versus peptide length clearly shows the presence of more + 2 charge states in the 8 h samples compared to 24 h ( figure 5 ) .
the finding that 24 h samples contained longer peptides is interesting since they should ideally have fewer undigested peptides due to enzymatic activity .
it is therefore likely that overall , while there is likely a reduction in the total enzyme activity and a loss of specific activity in 24 h samples , there are other random or nonspecific activities at play that result in the presence of relatively more peptides that are longer and nontryptic peptides in 24 h samples . since protein identification by mass spectrometry is optimal for + 2 charge state peptides compared to + 3 , the presence of longer peptides in 24 h samples could partly explain why fewer number of proteins were identified in the 24 h digested samples under our experimental conditions .
the tryptic digestion step is a critical and the most time consuming sample preparation step .
although many studies including that of klammer and maccoss demonstrate that the trypsin digestion step is a limiting factor in the efficiency of protein identification by mass spectrometry , many researchers still do not optimize or standardize this process , and this raises the question of how the digestion efficiency is evaluated and compared within and between laboratories .
the amino acid sequence coverage ( sq% ) has been reported as a measure that can be used to determine both the completeness of the protein digestion and the detection efficiency of the various tryptic peptides and digestion rate .
but the use of sq% might be misleading because different mass spectrometers and different search parameters may reveal different sq% .
furthermore , since a high sq% obtained from tryptic peptides without missed cleavages indicates a more complete digest than the same high sq% obtained from tryptic peptides with many missed cleavages , it is important to relate sq% to the degree of missed cleavages of the peptides used to calculate this value . it is reported that digestion efficiency can be determined by searching for the possible presence of intact protein in the total ion chromatogram .
we believe that a less complicated and demanding method for monitoring the efficiency and sufficiency of trypsin digestion that is independent of the mass spectrometry step will be more practical and easily implemented by many laboratories .
we realize that shotgun proteomics does not necessarily require complete digestion of proteins into peptides to be successful .
however , for reproducibility and to be able to objectively compare the results of experiments within and between laboratories it will be necessary to standardize , in addition to the other parameters , the trypsin digestion step using an objective method .
obviously , the extent of digestion that will produce optimal protein identification and sequence coverage will have to be empirically determined for each sample type , when a new batch of enzyme is acquired or when a new or modified protocol is to be used . in this experiment ,
digestion of plasma and serum under the same conditions resulted in different extents of digestion and proteomics outcomes .
this means that for improved efficiency , consistency and reproducibility the exact duration of digestion , the sweet spot , that produces the best amount of identified proteins and sequence coverage should be determined for each shotgun proteomics experiment . to monitor protein digestion routinely , a fast and inexpensive method that requires a small amount of digested sample
the bioanalyzer has the advantage of being a sensitive , reproducible , and well accepted method for protein analysis .
it provides an electrophoresis image that will show the presence or absence of protein peaks .
however , the prodm kit proved to be a faster and less expensive method for monitoring tryptic digestion compared to the bioanalyzer .
specifically , in addition to the cost of the bioanalyzer reader which could be as high as $ 17,000 , a protein-80 chip for 110 samples was purchased for about $ 36 , and a minimum of 60 min was required for one analysis .
conversely , prodm only required an in - house bench top spectrophotometer , the cost per sample was less than $ 3.00 , and less than 15 min was required for one analysis
. a direct comparison of the prodm and bioanalyzer approach therefore shows that the bioanalyzer approach requires about 20 l more digested sample , the cost of each assay was about 10 times more than that of prodm , and the time required to complete the assay was at least 4 times more than the prodm process .
hence prodm , a colorimetric method could be a better , and an alternative method for screening tryptic digest to identify the extent of digestion that yields optimal proteomics results .
prodm could help take away the guess work and avoid time / sample wastage due to insufficient or excessive digestion .
taken together , this study demonstrates the need to monitor and standardize the protein digestion step to improve ( a ) reproducibility , ( b ) protein identification efficiency , and ( c ) protein sequence coverage .
this is particularly critical in proteomics applications like protein expression profiling , quantitative proteomics , and biomarker identification , where run - run reproducibility is vitally important and the more the number of proteins identified the better .
we observed that the extent of protein digestion influenced the mass spectrometry results , indicating that the protein digestion step needs to be optimized to improve the success of proteomics experiments and prevent the wastage of valuable samples and time .
furthermore , the extent of protein(s ) digested , rather than the duration of trypsin digestion , may be a more objective method for standardizing and comparing results , since the type of protein , complexity of the protein mixture , and specific activity of the enzyme will affect the total time required to achieve the extent of digestion that will yield the best proteomics outcome .
prodm , a simple colorimetric assay which allows the precise calculation of the percentage of proteins digested in samples , could help laboratories monitor protein digestion prior to mass spectrometry , identify digestion conditions that yield the best outcome , standardize the digestion step , and optimize protein identification and sequence coverage .
it is expected that this paper will stimulate additional studies that will increase our understanding of the significance of monitoring and standardizing the protein digestion step . | tryptic digestion is an important preanalytical step in shotgun proteomics because inadequate or excessive digestion can result in a failed or incomplete experiment .
unfortunately , this step is not routinely monitored before mass spectrometry because methods available for protein digestion monitoring either are time / sample consuming or require expensive equipment . to determine if a colorimetric method ( prodm kit ) can be used to identify the extent of tryptic digestion that yields the best proteomics outcome , plasma and serum digested for 8 h and 24 h were screened with prodm , bioanalyzer , and lc / ms / ms , and the effect of digestion on the number of proteins identified and sequence coverage was compared .
about 6% and 16% less proteins were identified when > 50% of proteins were digested in plasma and serum , respectively , compared to when ~46% of proteins were digested .
average sequence coverage for albumin , haptoglobin , and serotransferrin after 2 h , 8 h , and 24 h digestion was 52% , 45% , and 45% for serum and 54% , 47% , and 42% for plasma , respectively .
this paper reiterates the importance of optimizing the tryptic digestion step and demonstrates the extent to which prodm can be used to monitor and standardize protein digestion to achieve better proteomics outcomes . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion | this rapidly evolving technology is now routinely applied in many laboratories for protein expression profiling , biomarker discovery / validation , posttranslational modification mapping , and complex disease research . the variant of proteomics termed shotgun proteomics , also known as bottom - up proteomics , is one of the most widely applied proteomic strategies which rely on the digestion of proteins into peptides prior to mass spectrometry . in shotgun proteomics , an enzyme such as trypsin is added to the protein sample and the mixture is incubated at 37c for a specified time or overnight at ambient room temperature to digest proteins . the resulting peptides are analyzed by mass spectrometry and the mass - to - charge ( m / z ) ratio generated is used to establish protein identities by reference to an appropriate database . the protein digestion step is recognized as one of the single most important sample preparation steps that can directly affect the outcome of all proteomics experiments . since the central objective of all shotgun proteomics is to identify all proteins present in the sample in a single run
, it is desirable and very important to optimize all pre - analytical and sample preparation steps especially the protein digestion step , to ensure that there is run - to - run reproducibility and the maximum possible number of proteins in each sample will be identified in each run . zhang and li ( 2004 ) reports that the type of protein , complexity of sample mixture , presence of surfactants , and impurities in the reaction mixture can affect the efficiency of tryptic digestion and hence the outcome of the experiment . in fact the study of klammer and maccoss published in 2006 clearly demonstrated that the number of unique proteins identified in plasma is primarily determined by the quality and completeness of the tryptic digestion step , and karuso et al . no attempt is typically made to determine if the digestion is sufficient before the mass spectrometry step . considering that under the same conditions , two different protein samples exposed to trypsin
may not be digested to the same extent , using a fixed digestion time will not result in optimal digestion of all types of proteins . therefore , to objectively compare mass spectrometry results , the tryptic digestion step must be standardized and a better method for standardization should be based on the extent rather than duration of digestion . it is reported that routine monitoring of tryptic digestion for understanding extent or percentage of protein digestion is not performed in most laboratories partly because the methods available to monitor protein digestion including hplc , circular dichroism , sds - page , mass spectrometry , and the use of fluorescent dyes are both time and sample consuming , and expensive to perform on a regular basis , or require special and expensive equipment [ 5 , 6 ] . methods for protein digestion monitoring that rely on the use of a mass spectrometer is unattractive and challenging because of the cost and time for the mass spectrometry analysis , database search to identify the protein sequence and deducing sample independent metrics that can be used to monitor the completion of trypsin digestion . additionally , because of scanty scientific literature on the subject , many proteomics scientists are unaware of the significance of insufficient and excessive digestion on the outcome of proteomics experiments , and many erroneously believe that the only requirement for a good proteomics result is access to a state - of - the - art high - end mass spectrometer . we were motivated to conduct this study because our laboratory routinely performs shotgun proteomics of plasma and serum samples using label - free lc / ms / ms and itraq technologies , and we observe differences in protein identification that were attributed to inconsistent trypsin digestion . since reproducibility and maximum protein identification are essential in quantitative proteomics , we presumed that using a method that is independent of mass spectrometry to standardize the tryptic digestion step will improve reproducibility and outcome . this paper describes the evaluation of a novel protein digestion monitoring ( prodm ) kit , as a method for monitoring trypsin digestion prior to mass spectrometry . the results obtained from our experiments demonstrate that the number of proteins identified and the protein sequence coverage are affected by the extent of protein digestion , and prodm can be used to precisely determine the extent of protein digestion before the mass spectrometry step . the proteomics grade plasma ( pgpt ) and serum ( pgst ) tubes for collection of blood , protein digestion monitoring kit ( prodm ) , and total protein assay reagent ( topa ) were supplied by itsi - biosciences , johnstown , pa , and picofrit c18 nanospray column was purchased from new objective , woburn , ma . plasma and serum were isolated according to standard plasma and serum isolation protocols , aliquoted in 1 ml amounts to avoid repeated freeze / thaw of the same aliquot , and stored at 80c until used . plasma and serum proteins were digested with trypsin in a buffer system containing trifluoroethanol ( tfe ) . subsequently , the reaction mixture was incubated for 8 h and 24 h at 37c . at the end of the incubation ,
the time zero , 8 h , and 24 h samples were analyzed as described below . in a parallel experiment serum and plasma
were digested as described above for 2 h , 8 h , and 24 h and the digested samples were analyzed by mass spectrometry . the sequence coverage of three model proteins , namely , albumin , haptoglobin , and serotransferrin was determined and compared to elucidate the effect of digestion extent on protein sequence coverage . plasma and serum samples were analyzed with the prodm kit according to the manufacturers ' protocol . the samples collected at time zero and the samples collected after 8 h and 24 h digestion with trypsin were independently processed to determine the extent of protein digestion
. 10 l of the reaction mixture was transferred , at zero time and after 8 h and 24 h incubation , to a fresh eppendorf tube containing 2 l of reaction quencher . the application is based on the formula % pd = [ ( a1(t0,l ) a2(tx , l))/a1(t0,l)]100 , where % pd is the percentage of protein digested by the enzyme into peptides within a specific incubation time interval ; a1(t0,l ) is the absorbance of the first aliquot at time zero ; and a2(tx , l ) is the absorbance of the second aliquot after 8 h or 24 h digestion . to screen the samples with the agilent bioanalyzer protein 80 chip , 50 l each of ( a ) time zero , ( b ) 8 h digested , and ( c )
24 h digested serum and plasma samples were transferred to fresh tubes , dried down in a speedvac , and resuspended with 30 l of distilled and deionized ( milli - q ) water . following trypsin digestion for 8 h and 24 h , the digestion mixtures were acidified with 5% ( v / v ) formic acid and dried down in a speedvac . a data - dependent top 5 method was used where a full ms scan from m / z 4001500 was followed by ms / ms scans on the five most abundant ions . protein identification and the number of missed cleavages
briefly , the raw data files were searched utilizing sequest algorithm in proteome discoverer 1.3 ( thermo scientific ) against the most recent species - specific fasta database for human downloaded from ncbi . proteins were identified when unique peptides had x - correlation scores greater than 1.5 , 2.0 , and 2.5 for respective charge states of + 1 , + 2 , and + 3 . to identify other parameters that may be different as a result of different tryptic digestion times , we compared the m / z distribution and ion intensities in 8 h and 24 h samples by plotting 2d density maps consisting of time ( x - axis ) versus m / z ( y - axis ) versus relative abundance ( z - axis ) for both samples using xcalibur 2.1 ( thermo scientific ) . proteome discoverer 1.3 was utilized to plot the graph of m / z versus peptide length to demonstrate the distribution of charges in the digested plasma and serum samples . the primary goal of this study was to determine if prodm , a novel protein digestion monitoring kit , can be used to monitor the pre - analytical trypsin digestion step to identify the precise extent of protein digestion that gives the best protein identification and sequence coverage outcome . to determine how the extent of digestion affects the outcome of the mass spectrometry data , we analyzed the digested samples by tandem mass spectrometry and compared ( a ) the number of proteins identified and ( b ) protein sequence coverage . the prodm analysis revealed that about 46% of plasma and serum proteins were digested after 8 h incubation , whereas after 24 h digestion , 56% of proteins were digested in plasma and 50% in serum ( table 1 ) . the agilent bioanalyzer data correlated with the prodm results and provided a gel - based verification of the presence of undigested proteins after 8 h and 24 h digestion with trypsin . this means that prodm , which gives the precise percentage of proteins digested , can be used to determine the extent of trypsin digestion that will give the best proteomics outcome . the goal of all shotgun proteomics experiments is to identify as many proteins as possible with high confidence , and the more the percentage of sequence coverage the higher the confidence . to determine how the tryptic digestion step can affect the outcome of shotgun proteomics
, we analyzed the samples digested for different extents and compared the number of proteins identified and sequence coverage of three proteins commonly found in plasma and serum . as shown in table 1 , a total of 991 and 733 peptides were sequenced in 8 h and 24 h digested plasma samples , whereas a total of 897 and 693 peptides were sequenced in 8 h and 24 h digested serum samples , respectively . the corresponding total number of unique proteins identified in plasma in the 8 h and 24 h samples was 125 and 118 , and in serum this was 127 and 107 , respectively ( table 1 ) . this represents about 26% reduction in the number of peptides sequenced and 6% drop in the number of proteins identified in 24 h digested samples compared to 8 h digested samples , respectively . in serum 127 proteins were identified in 8 h digested samples and 107 were identified in 24 h digested samples ( table 1 ) . this represents about 16% reduction in the number of proteins identified in 24 h digested samples compared to 8 h digested samples . the finding that longer digestion times can result in reduced number of identified proteins has been previously reported , and the 125 unique proteins we identified in plasma after 8 h tryptic digestion are comparable to the 150 proteins identified by zimmerman et al . using a comparable lc
/ ms / ms equipment and approach , suggesting that our finding could be generalized to some extent . since
24 h digestion apparently resulted in fewer number of identified proteins compared to 8 h , the prodm data obtained in this experiment shows that ~46% digestion of plasma and serum yields greater numbers of identified proteins compared to 24 h digestion which resulted in 50% protein digestion . in a parallel experiment
, we used albumin , haptoglobin , and serotransferrin as models to gain insights into the potential effect of the % of digestion on protein sequence coverage . as shown in figure 2 , the average sequence coverage for the three proteins in the 2 h , 8 h , and 24 h samples for serum were 52% , 45% , and 45% , respectively , and for plasma were 54% , 47% , and 42% , respectively . specifically , in plasma , the sequence coverage in the 2 h samples ranged from about 47% ( haptoglobin ) to 61% ( albumin ) and the coverage in 24 h samples ranged from about 30% ( haptoglobin ) to 52% ( albumin ) . in serum ,
the coverage in the 2 h samples ranged from 32% ( haptoglobin ) to 70% ( albumin ) , whereas the coverage in the 24 h samples ranged from 31% ( haptoglobin ) to 61% ( albumin ) . it was interesting to observe that the average coverage in 2 h was consistently higher than the coverage in 24 h for the three proteins in serum and plasma . this finding is noteworthy because it suggests that excessive digestion may not only result in reduced number of the total proteins identified , but could also lead to lower confidence due to a reduced protein sequence coverage . if optimal sequence coverage is required , then prodm could be used to determine the least percentage of digestion that gives the best coverage for the target protein . to determine why longer digestion times resulted in fewer number of proteins identified and less sequence coverage we postulated that the number of missed cleavages in the 8 h and 24 h digested samples will be different . interestingly , there was no dramatic difference in the number of missed cleavages between the 8 h and 24 h samples , except for 2 missed cleavages , where for plasma 2.3% was detected in 8 h and 0.8% was detected in 24 h and for serum 0.8% was detected in 8 h and 1.5% was detected in 24 h ( figure 3 ) . the absence of a major difference in the number of zero missed cleavages between 8 h and 24 h digestion is remarkable and requires more study . to determine if this phenomenon is related to reproducibility between mass spectrometry runs , a plot consisting of time ( x - axis ) versus m / z ( y - axis ) versus relative abundance ( z - axis ) was used to compare the 8 h and 24 h digested samples . figure 4 shows only a subtle difference between 8 h and 24 h samples for plasma and serum indicating that there was good run - run reproducibility . thus , the difference in the number of proteins identified is apparently not likely due to reproducibility . the finding that 24 h digestion compared to 8 h digestion leads to a decrease in the number of proteins identified and % protein sequence coverage underscores the importance of optimizing and standardizing the enzymatic digestion step prior to mass spectrometry . indeed , a database search with chymotrypsin using the raw ms / ms files identified a new set of proteins and resulted in a 12% increase in the coverage for albumin in the 24 h sample . this demonstrates the presence of more nontryptic peptides in the 24 h samples compared to 8 h samples . it is therefore likely that fewer proteins were identified in the 24 h samples probably because of the loss of enzyme specificity , which resulted in the production of non-/semitryptic peptides . the charge state of peptides in the 8 h and 24 h samples were compared because the presence of peptides with higher charges is an indication of the presence of longer peptides . as shown in table 2 , 72% of all peptides identified in plasma and 86% of all peptides identified in serum in the 8 h samples had + 2 charge states . the 24 h samples contained a higher number of + 3 charges indicating that there were more longer peptides compared to the 8 h samples . the graphical illustration of the distribution of charges obtained by plotting the xcorr versus peptide length clearly shows the presence of more + 2 charge states in the 8 h samples compared to 24 h ( figure 5 ) . since protein identification by mass spectrometry is optimal for + 2 charge state peptides compared to + 3 , the presence of longer peptides in 24 h samples could partly explain why fewer number of proteins were identified in the 24 h digested samples under our experimental conditions . the tryptic digestion step is a critical and the most time consuming sample preparation step . although many studies including that of klammer and maccoss demonstrate that the trypsin digestion step is a limiting factor in the efficiency of protein identification by mass spectrometry , many researchers still do not optimize or standardize this process , and this raises the question of how the digestion efficiency is evaluated and compared within and between laboratories . the amino acid sequence coverage ( sq% ) has been reported as a measure that can be used to determine both the completeness of the protein digestion and the detection efficiency of the various tryptic peptides and digestion rate . obviously , the extent of digestion that will produce optimal protein identification and sequence coverage will have to be empirically determined for each sample type , when a new batch of enzyme is acquired or when a new or modified protocol is to be used . in this experiment ,
digestion of plasma and serum under the same conditions resulted in different extents of digestion and proteomics outcomes . this means that for improved efficiency , consistency and reproducibility the exact duration of digestion , the sweet spot , that produces the best amount of identified proteins and sequence coverage should be determined for each shotgun proteomics experiment . to monitor protein digestion routinely , a fast and inexpensive method that requires a small amount of digested sample
the bioanalyzer has the advantage of being a sensitive , reproducible , and well accepted method for protein analysis . however , the prodm kit proved to be a faster and less expensive method for monitoring tryptic digestion compared to the bioanalyzer . a direct comparison of the prodm and bioanalyzer approach therefore shows that the bioanalyzer approach requires about 20 l more digested sample , the cost of each assay was about 10 times more than that of prodm , and the time required to complete the assay was at least 4 times more than the prodm process . hence prodm , a colorimetric method could be a better , and an alternative method for screening tryptic digest to identify the extent of digestion that yields optimal proteomics results . prodm could help take away the guess work and avoid time / sample wastage due to insufficient or excessive digestion . taken together , this study demonstrates the need to monitor and standardize the protein digestion step to improve ( a ) reproducibility , ( b ) protein identification efficiency , and ( c ) protein sequence coverage . this is particularly critical in proteomics applications like protein expression profiling , quantitative proteomics , and biomarker identification , where run - run reproducibility is vitally important and the more the number of proteins identified the better . we observed that the extent of protein digestion influenced the mass spectrometry results , indicating that the protein digestion step needs to be optimized to improve the success of proteomics experiments and prevent the wastage of valuable samples and time . furthermore , the extent of protein(s ) digested , rather than the duration of trypsin digestion , may be a more objective method for standardizing and comparing results , since the type of protein , complexity of the protein mixture , and specific activity of the enzyme will affect the total time required to achieve the extent of digestion that will yield the best proteomics outcome . prodm , a simple colorimetric assay which allows the precise calculation of the percentage of proteins digested in samples , could help laboratories monitor protein digestion prior to mass spectrometry , identify digestion conditions that yield the best outcome , standardize the digestion step , and optimize protein identification and sequence coverage . it is expected that this paper will stimulate additional studies that will increase our understanding of the significance of monitoring and standardizing the protein digestion step . | [
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gingival thickness ( gt ) is determined by the shape and size of the dental root , and contour of the alveolar bone .
it can be classified into two types : thick and thin.12 a gt of 1 mm is classified as belonging to the thin biotype , while a gt of > 1 mm is classified as belonging to the thick biotype.1 gt is considered as an important factor in the success of periodontal and orthodontic treatment.34 careful evaluation of gt is important during the treatment planning stage in order to prevent pathological periodontal problems , such as gingival recession due to orthodontic treatment.25 it has been reported that movements of teeth made within the anatomical limits of the alveolar bone by applying controlled orthodontic force do not cause any pathological problems .
dehiscence and fenestrations have been observed as a result of tooth movements exceeding the anatomical limits of the alveolar bone , and it has been noted that this causes gingival recession , especially in individuals who display the thin gingival biotype ; nevertheless , this is largely dependent on the extent to which the alveolar bone supports the loss of the gingiva.67 long - term studies evaluating the association between gingival recession and orthodontic treatment have led to the conclusion that gingival recession is more prevalent in individuals who have been treated orthodontically , as opposed to those who have not received such treatment.68 in addition , the body of scholarly research demonstrates that mandibular incisors are more prone to gingival recession than other teeth.6 in studies evaluating the association between lower incisor protrusion and orthodontic treatment , it has been stated that there is no statistically significant relationship between these two factors.910 however , yared et al.11 investigated the relationship between gingival recession and the health status of periodontal tissue , the type and amount of tooth movement , the width of keratinized gingiva ( wkg ) , and gt , and concluded that gingival recession is characterized by a greater prevalence in certain contexts . specifically , the condition is more frequent when gt is < 0.5 mm , wkg is < 2 mm , and lower incisors protrude at an angle that is greater than 95. the researchers also noted that gt is a factor with greater importance than protrusion movement . although a number of studies have been published that relate to an evaluation of the correlation between gt and malocclusions , no studies in the extant literature have evaluated the association between keratinized gingival width and malocclusions .
therefore , the aim of the present study was to investigate the relationship of gt and wkg with different malocclusion groups and levels of crowding .
the null hypothesis is that the gt and wkg of lower anterior teeth will change on the basis of the different malocclusion groups and levels of crowding .
a total of 187 subjects ( 121 females and 66 males ) were enrolled in the present study , all of whom presented at the department of orthodontics , faculty of dentistry , yznc yl university between june 2014 and june 2015 for orthodontic treatment . following the provision of a description of the study , written and informed consent
the study was commenced after obtaining the approval of the research ethics committee of faculty of medicine in yznc yl university ( b.30.2.yyu.0.01.00.00/141 ) .
the exclusion criteria were as follows : history of previous orthodontic treatment ; the presence of attachment loss or a pocket deeper than 4 mm ; congenital anomaly ; dental structural disorder ; crowns or extensive restoration ; pregnancy or lactation ; any systemic problems and related medications that could have an impact on the thickness of gingival tissues ; the administration of antibiotic premedication due to any disturbance within the recent six months ; and smoking .
correspondingly , periodontally healthy subjects with complete permanent dentition ( with the exception of third molars ) were included in the study .
the participants were divided into three groups ; angle class i malocclusion , angle class ii malocclusion , and angle class iii malocclusion .
for the angle class i relationship , the mesio - buccal cusp of the maxillary first permanent molar occluded with the mesio - buccal groove of the mandibular first permanent molar .
additionally , further attention was directed towards the distal surface of the disto - buccal cusp of the upper first permanent molar and the way in which it contacted with the mesial surface of the mesio - buccal cusp of the lower second molar ; furthermore , the mesio - palatal cusp of the upper first permanent molar occluded with the central fossa of the lower first permanent molar . the mesio - buccal grove of the mandibular first permanent molar was positioned distally to the mesio - buccal cusp of the maxillary first permanent molar in the angle class ii relationship and mesially in the angle class iii relationship.12 each angle classification group was divided into subgroups according to the level of dental crowding in the mandibular anterior region .
the mesio - distal width of each tooth , including the canine teeth , was measured from plaster models with a boley gauge . where the contact points were broken , the required space for each tooth was calculated by subtracting the mesio - distal width of the tooth from the available space .
this study determined the level of crowding by the sum of the lack of all space , and it was classified in three ways ; mild ( 03 mm ) , moderate ( 46 mm ) , and severe ( > 6 mm).13 measurements of the plaque index ( pi),14 gingival index ( gi),15 and probing depth ( pd ) of the periodontal pocket were conducted from the mesial and distal surfaces ; furthermore , this took place from the vestibular midpoint and palatinal midpoint of the subjects ' mandibular anterior teeth using a periodontal probe ( pqw7 , williams ; hu - friedy mfg .
the wkg was measured from the mucogingival junction to the free gingival margin at the buccal area of the mandibular anterior teeth .
the gt of each patient was assessed by a single researcher ( yk ) prior to the orthodontic treatment .
measurements were carried out from two points on the buccal aspect of the mandibular anterior teeth ( canine canine ) : apical to the free gingival margin and coronal to the mucogingival junction ( figure 1 ) . after marking the measurement points with a marking pen , xylocaine spray ( vemcaine 10% lidocaine ; vem , istanbul , turkey ) was administered to the patient or alternatively , a local anesthetic ( maxicaine , lidocaine hydrochloride ; vem ) was used to reduce pain when necessary . for the patients for whom a local anesthetic was necessary ,
the anesthetic solution was injected slowly at a dose of 0.1 ml to prevent an anesthesia - related increase of the mucosal volume .
the required measurements were performed 1020 minutes after the injection was administered , and took place from the marked points by perpendicularly inserting a 10-mm endodontic spreader ( golden star medical co. , ltd . ,
this involved the placement of a silicone stopper to the gingiva until the alveolar bone was reached .
since the application of excessive force would cause the spreader to cross the soft tissue and go through the alveolar bone , careful attention was paid to apply a light force that was limited only to the soft tissue .
all measurements were repeated two times at 10-minute intervals by the same researcher and the average result was recorded as the final measurement for thickness at each location .
it is notable that intra - examiner agreement was high ( pearson correlation coefficient = 0.901 , p < 0.001 ) .
in addition , the random measurement error was calculated with dahlberg 's formula , and it was observed that these error values ranged from 0.034 to 0.022 .
the gt of each tooth was determined by the arithmetic mean of the gt values obtained from the apical part of the gingival sulcus and the coronal aspect of the mucogingival junction .
the gingival biotype of the mandibular anterior teeth was determined by the ratio of the sum of gt of the mandibular anterior teeth to the number of teeth .
if the obtained measurement values were less than 1 mm , the gingiva was classified as a thin biotype ; where the obtained values were greater than 1 mm , it was classified as a thick biotype .
statistical analysis was carried out using the program ibm spss statistics for windows , version 22.0 ( ibm co. , armonk , ny , usa ) . the sample size was determined by considering the minimum 80% power value and the 5% type i error .
descriptive statistics for the considered parameters were presented as mean , standard deviation , and maximum and minimum values .
additionally , the kolmogorov - smirnov test was performed to determine the normality of the variables , and levene 's test was also used to determine the homogeneity of variances .
after these tests , factorial variance analysis was performed to determine whether any differences existed with regard to the angle classification and the level of crowding . following the factorial variance analysis ,
duncan 's multiple - range test was performed to assess the nature of the crowding groups and the different classes of angle classification . in turn , the chi - square test was implemented in order to identify the nature of the relationship between the gingival biotype , angle classification , the level of crowding , and gender .
statistical analysis was carried out using the program ibm spss statistics for windows , version 22.0 ( ibm co. , armonk , ny , usa ) . the sample size was determined by considering the minimum 80% power value and the 5% type i error .
descriptive statistics for the considered parameters were presented as mean , standard deviation , and maximum and minimum values .
additionally , the kolmogorov - smirnov test was performed to determine the normality of the variables , and levene 's test was also used to determine the homogeneity of variances .
after these tests , factorial variance analysis was performed to determine whether any differences existed with regard to the angle classification and the level of crowding . following the factorial variance analysis ,
duncan 's multiple - range test was performed to assess the nature of the crowding groups and the different classes of angle classification . in turn , the chi - square test was implemented in order to identify the nature of the relationship between the gingival biotype , angle classification , the level of crowding , and gender .
there was no statistically significant difference between the genders in terms of number and the mean age of patients .
the study group consisted of a total of 187 patients aged between 10 and 28.2 years , of whom 121 were female ( mean age , 17.5 4.25 years ) and 66 were male ( mean age , 15.8 3.17 years ) .
no statistically significant difference was found between the genders in terms of number and mean age of patients .
the number of patients in the angle class i , ii , and iii malocclusion groups was 76 ( 40.6% ) , 80 ( 42.8% ) , and 31 ( 16.6% ) , respectively .
in addition , the number of patients in the mild , moderate , and severe crowding groups was 101 ( 54.0% ) , 50 ( 26.7% ) , and 36 ( 19.3% ) , respectively ( table 1 ) .
there was no statistically significant difference between the groups in terms of the number of patients . in tables 2 , 3 , and 4 , the vertical columns describe the crowding levels and the horizontal columns describe the angle classification groups .
the distribution of pi , gi , and pd measurements , used in determining the periodontal status of patients , are displayed in table 2 , according to angle classification and the level of crowding .
the pi values were significantly higher in the severe crowding group than in the mild crowding group ; this was also the case in the angle class iii malocclusion group , compared to the angle class ii malocclusion group ( p = 0.042 ) .
in addition , pd values were higher in terms of statistical significance in the angle class iii malocclusion group than they were in the angle class i malocclusion group ( p = 0.101 ) . the wkg of the lower anterior teeth according to angle classification and the level of crowding is displayed in table 3 .
no statistically significant difference was observed between the wkg of the lower anterior teeth according to angle classification .
the wkg values for the mandibular left central and lateral incisors and the mandibular right lateral incisor were higher in the severe crowding group ( p < 0.05 ) .
in addition , it should also be noted that , while the wkg of the mandibular right central incisor was higher in the severe crowding group , the difference was not statistically significant .
the wkg values of mandibular canines were higher in the mild crowding group ( p < 0.05 ) .
table 4 summarizes and displays the gt of the lower anterior teeth on the basis of angle classification and the level of crowding .
it was observed that teeth in the lower anterior jaw displayed thin gingival biotype , and yet there was no statistically significant difference observed between angle classification and the gts of the mandibular left central and lateral incisors , and the mandibular canines .
however , the gts of the mandibular right central and lateral incisors was observed to be significantly lower in the angle class iii malocclusion group ( p < 0.05 ) .
the gts of the mandibular left central and lateral incisors , and the mandibular right lateral incisor were significantly higher in the severe crowding group ( p < 0.05 ) .
notably , although the gt of the mandibular right central incisor was higher in the severe crowding group , the difference was not statistically significant .
the gts of the mandibular canines were higher in terms of statistical significance in the mild crowding group ( p < 0.05 ) .
table 5 presents an overview of the mean gt of the mandibular anterior region on the basis of angle classification and the level of crowding .
it was determined that the mean gt of the mandibular anterior region is 0.71 0.17 mm and , moreover , displays the thin gingival biotype .
it did not exhibit any significant association with angle classification or the level of crowding ( p = 0.140 and 0.321 , respectively ) .
there is a range of risk factors associated with gingival recession , especially in the area of the mandibular incisors during orthodontic treatment .
these include the following : the age of the patient , the health status of the periodontal tissues , the duration of the treatment , the amount and type of tooth movement , the wkg , and gt.916 nevertheless , owing to the lack of research that has been conducted in order to evaluate each of these parameters , a range of conflicting opinions exist regarding the effect of mandibular incisor protrusion on periodontal tissues.916 one group of researchers has published the claim that such orthodontic tooth movements are risk factors for gingival recession , due to the fact that the buccal alveolar bone of mandibular incisors is thin.1117 contrastingly , another group has reported there is no such relation between the two.91018 two notable studies , by wennstrm et al.18 and yared et al.,11 stated that among the parameters evaluated at the planning stage of orthodontic treatment , gt is a more important factor than wkg and protrusion movement .
therefore , the aim of the present study was to evaluate the relationship that exists between wkg and gt , both of which are considered to be significant risk factors for gingival recession that may be seen in the mandibular anterior region with different malocclusion groups and levels of crowding .
literature review demonstrated that visual assessment,1 ultrasonic devices,19 the parallel profile radiography technique,20 cone - beam computed tomography,13 periodontal probes,13 and transgingival probing121 are the techniques that have been used for the purpose of determining gt .
the most frequently used techniques in modern orthodontic practice for gt measurements are periodontal probing and transgingival probing . however , in studies which compare the reliability of periodontal probing in relation to transgingival probing,22 cone beam computed tomography,320 and parallel profile radiography techniques,19 it has been determined that this technique is not reliable .
contrastingly , in studies that compared transgingival probing with surgical flap operations21 and cone - beam computed tomography,20 transgingival probing has been found to be a reliable way in which to derive measurements of gt .
it is for this reason that transgingival probing , which facilitates the evaluation of the gingiva at two points in millimeters , is preferred in our study .
it has been found that gt is affected by changes in the position of teeth during the eruption period.23 furthermore , it has been determined that this effect is decreased as age increases ; this is because as connective tissue becomes denser , the cell count decreases , the epithelium becomes thinner , and keratinization increases.24 it has been observed that age groups were constituted differently in studies which investigated the relation between gt and age .
the present study group consisted of subjects aged less than 29 years who had all of their permanent teeth erupted , meaning that gt would not be influenced to a considerable degree by age - related factors.25 a range of opinions exists in the extant literature relating to the role that wkg plays in the maintenance of periodontal health during orthodontic treatment .
closs et al.26 investigated the relationship between the initial wkg and gingival recession by examining 209 subjects treated with fixed orthodontic appliances , and concluded that no significant difference existed in the initial wkg for individuals who did or did not have gingival recession .
however , it is important to note that , in this study , the mean wkg of all teeth was greater than 2 mm .
in addition , while yared et al.11 suggested that a wkg of less than 2 mm is inadequate to maintain periodontal health , coatoam et al.17 suggested that such a width is adequate for individuals with good oral hygiene . in light of these results , it is important to note the findings of the present study : namely , that a wkg for mandibular anterior teeth of between 2.01 1.33 3.90 1.70 mm was found to be adequate for the maintenance of periodontal health .
given this , it has been determined that , when the thickness of the gingiva is decreased in the bucco - lingual direction , the apicocoronal height also decreases.18 accordingly , the wkg of the thin gingival biotype is less than that of the thick gingival biotype.13 it has also been determined that the tooth germs of the mandibular permanent canines are positioned in the same direction as the mandibular primary canine roots , thereby meaning that the wkg and gt of these teeth is less than that of mandibular incisor teeth .
this is due to the fact that frequent eruptions on the vestibular position take place if there is insufficient space for them.182327 consistent with these findings , the present study observed that the wkg and gt of mandibular canines are less than those of mandibular incisors .
however , the difference between the gt of mandibular canines and that of tooth number 31 was not significant .
the tooth germs of mandibular permanent incisors are positioned lingually with respect to the mandibular primary incisors .
thus , there is a tendency for the mandibular permanent incisors to erupt somewhat lingually and in an irregular position , even in children who have normal dental arches .
furthermore , this position can not be corrected in the event of crowding.27 in addition , the extant literature reports that the tooth germs of mandibular lateral incisors are positioned more lingually compared to the tooth germs of mandibular central incisors and , in cases where crowding is an issue , this facilitates eruption in a more lingual position than mandibular central incisors.27 moreover , the wkg and gt of lingually erupting teeth are reported as being higher.1823 consistent with these results , the present study demonstrated that the wkg and gt of mandibular lateral incisors are higher than those of mandibular central incisors . when the level of crowding increases , it is expected that the wkg and gt of the mandibular canines will decrease .
this is primarily owing to the fact that they erupt in a more vestibular position and , in addition , the wkg and gt of the mandibular central and lateral incisors increases ; this also takes place because they erupt in a more lingual position . in the extant literature relating to the wkg
, no findings have been published regarding the relation between the wkg of each mandibular anterior tooth and the level of crowding .
our study indicates that the wkg of the mandibular left central and lateral incisors and mandibular right lateral incisor are significantly higher in the severe crowding group ( p < 0.05 ) , compared to the other groups .
although the wkg of the mandibular right central incisor is higher in the severe crowding group , the difference is not statistically significant .
the wkg of the mandibular canines are significantly higher in the mild crowding group ( p < 0.05 ) , compared to the other groups .
relatively few studies have been published in the extant literature that focus on evaluating the relationship between the gingival biotype and the level of crowding . of the available studies , zawawi and
al - zahrani28 stated that there is no significant association between the level of crowding and gt in the mandibular anterior region . in this study , it was observed that periodontal probing was used to measure gt ; the space analysis was performed by including only mandibular incisors ; and only the mandibular central incisor was taken as a reference to detect the gingival biotype of subjects . in our study , the relationship of the gt of each mandibular anterior tooth with the level of crowding was evaluated , primarily because the gt may be subject to variation depending on the position of the teeth in the dental arch.1823 in addition , space analysis was performed by including the canine teeth , and gts of the mandibular left central and lateral incisors and mandibular right lateral incisor were observed to be significantly higher in the severe crowding group ( p < 0.05 ) . although the gt of the mandibular right central incisor is higher in the severe crowding group , this difference is not statistically significant . in addition , it is noteworthy that the gt of the mandibular canines are significantly higher in the mild crowding group ( p < 0.05 ) .
the present study displayed no statistically significant relationship between the wkg and the angle classification in the mandibular anterior region .
furthermore , it is worth noting that no study currently exists in the extant literature that has evaluated the relationship between these two variables .
a limited number of studies exist in the literature that have evaluated the association of gingival biotype with different dental malocclusion groups . among these ,
zawawi et al.2 studied 200 individuals and reported that no statistically significant relationship was observed between the gingival biotype and angle classification .
their study used periodontal probing to determine the gingival biotype and only the maxillary central incisor was used as a reference to determine the gingival biotype of subjects .
however , matarese et al.,29 in a study focusing on 76 individuals , assessed biotype by employing periodontal probing at the mid - facial aspect of the maxillary central , lateral incisors , and canines , and found that no statistically significant relationship existed between the gingival biotype and angle classification .
the researchers also noted that the gt is subject to change on the basis of tooth position , facial characteristics , and profile , thereby warranting further study to evaluate the impact of these parameters .
consistent with these results , our study showed that there is no statistically significant relationship between the mean gt of the mandibular anterior region and angle classification .
it was also found that there is no statistically significant relationship of the gt of the mandibular left central and lateral incisors , and that of the mandibular canines with angle classification .
the gt values of the mandibular right central and lateral incisors are statistically lower in the angle class iii malocclusion group ( p < 0.05 ) .
it is important to acknowledge the current study 's limitations , and the primary limitation of this research has been the sole examination and evaluation of the relationship of the gt of the mandibular anterior jaw with angle classification and the level of crowding .
given the fact that other parameters have an impact on gt , including vertical and sagittal skeletal relationship , tooth position , and overjet / overbite , further study to evaluate their impact is recommended .
the first critical finding of the current study is the observation that teeth in the lower anterior jaw display thin gingival biotype .
second , it has been found that , when the level of crowding increases , there is a corresponding increase in the wkg and gt of the central and lateral incisors ; simultaneously , there is a corresponding decrease in the wkg and gt of canines .
third , there is no association between the angle classification and the mean gt of the mandibular anterior region .
the final conclusion is that the gt of the mandibular right central and lateral incisors is lower in the angle class iii group . | objectiveto evaluate the relationship of gingival thickness ( gt ) and the width of keratinized gingiva ( wkg ) with different malocclusion groups and the level of crowding.methodsa total of 187 periodontally healthy subjects ( 121 females and 66 males ) who presented at the faculty of dentistry in yznc yl university for orthodontic treatment were enrolled in the study .
the individuals involved in the study were divided into three groups ; angle class i malocclusion , angle class ii malocclusion , and angle class iii malocclusion .
each group was classified as mild , moderate , or severe according to the level of crowding .
wkg was determined as the distance between the mucogingival junction and the free gingival margin .
gt was determined by the transgingival probing technique .
factorial variance analysis and the duncan multiple comparison test were employed to identify the extent to which a difference was apparent between the groups according to these parameters.resultsit was determined that teeth in the mandibular anterior region display the thin gingival biotype .
wkg and gt were observed as being higher at the mandibular incisor teeth in the severe crowding group and at the mandibular canine teeth in the mild crowding group .
the gt of the mandibular right central and lateral incisors was found to be thinner in the angle class iii group.conclusionswithin the limits of this study , the results demonstrate that , there is no significant relationship of wkg and the mean gt in the mandibular anterior region according to the angle classification . | INTRODUCTION
MATERIALS AND METHODS
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION | gingival thickness ( gt ) is determined by the shape and size of the dental root , and contour of the alveolar bone . it can be classified into two types : thick and thin.12 a gt of 1 mm is classified as belonging to the thin biotype , while a gt of > 1 mm is classified as belonging to the thick biotype.1 gt is considered as an important factor in the success of periodontal and orthodontic treatment.34 careful evaluation of gt is important during the treatment planning stage in order to prevent pathological periodontal problems , such as gingival recession due to orthodontic treatment.25 it has been reported that movements of teeth made within the anatomical limits of the alveolar bone by applying controlled orthodontic force do not cause any pathological problems . dehiscence and fenestrations have been observed as a result of tooth movements exceeding the anatomical limits of the alveolar bone , and it has been noted that this causes gingival recession , especially in individuals who display the thin gingival biotype ; nevertheless , this is largely dependent on the extent to which the alveolar bone supports the loss of the gingiva.67 long - term studies evaluating the association between gingival recession and orthodontic treatment have led to the conclusion that gingival recession is more prevalent in individuals who have been treated orthodontically , as opposed to those who have not received such treatment.68 in addition , the body of scholarly research demonstrates that mandibular incisors are more prone to gingival recession than other teeth.6 in studies evaluating the association between lower incisor protrusion and orthodontic treatment , it has been stated that there is no statistically significant relationship between these two factors.910 however , yared et al.11 investigated the relationship between gingival recession and the health status of periodontal tissue , the type and amount of tooth movement , the width of keratinized gingiva ( wkg ) , and gt , and concluded that gingival recession is characterized by a greater prevalence in certain contexts . therefore , the aim of the present study was to investigate the relationship of gt and wkg with different malocclusion groups and levels of crowding . the null hypothesis is that the gt and wkg of lower anterior teeth will change on the basis of the different malocclusion groups and levels of crowding . a total of 187 subjects ( 121 females and 66 males ) were enrolled in the present study , all of whom presented at the department of orthodontics , faculty of dentistry , yznc yl university between june 2014 and june 2015 for orthodontic treatment . following the provision of a description of the study , written and informed consent
the study was commenced after obtaining the approval of the research ethics committee of faculty of medicine in yznc yl university ( b.30.2.yyu.0.01.00.00/141 ) . correspondingly , periodontally healthy subjects with complete permanent dentition ( with the exception of third molars ) were included in the study . the participants were divided into three groups ; angle class i malocclusion , angle class ii malocclusion , and angle class iii malocclusion . for the angle class i relationship , the mesio - buccal cusp of the maxillary first permanent molar occluded with the mesio - buccal groove of the mandibular first permanent molar . the mesio - buccal grove of the mandibular first permanent molar was positioned distally to the mesio - buccal cusp of the maxillary first permanent molar in the angle class ii relationship and mesially in the angle class iii relationship.12 each angle classification group was divided into subgroups according to the level of dental crowding in the mandibular anterior region . this study determined the level of crowding by the sum of the lack of all space , and it was classified in three ways ; mild ( 03 mm ) , moderate ( 46 mm ) , and severe ( > 6 mm).13 measurements of the plaque index ( pi),14 gingival index ( gi),15 and probing depth ( pd ) of the periodontal pocket were conducted from the mesial and distal surfaces ; furthermore , this took place from the vestibular midpoint and palatinal midpoint of the subjects ' mandibular anterior teeth using a periodontal probe ( pqw7 , williams ; hu - friedy mfg . the wkg was measured from the mucogingival junction to the free gingival margin at the buccal area of the mandibular anterior teeth . the gt of each patient was assessed by a single researcher ( yk ) prior to the orthodontic treatment . measurements were carried out from two points on the buccal aspect of the mandibular anterior teeth ( canine canine ) : apical to the free gingival margin and coronal to the mucogingival junction ( figure 1 ) . the gt of each tooth was determined by the arithmetic mean of the gt values obtained from the apical part of the gingival sulcus and the coronal aspect of the mucogingival junction . the gingival biotype of the mandibular anterior teeth was determined by the ratio of the sum of gt of the mandibular anterior teeth to the number of teeth . after these tests , factorial variance analysis was performed to determine whether any differences existed with regard to the angle classification and the level of crowding . following the factorial variance analysis ,
duncan 's multiple - range test was performed to assess the nature of the crowding groups and the different classes of angle classification . in turn , the chi - square test was implemented in order to identify the nature of the relationship between the gingival biotype , angle classification , the level of crowding , and gender . after these tests , factorial variance analysis was performed to determine whether any differences existed with regard to the angle classification and the level of crowding . following the factorial variance analysis ,
duncan 's multiple - range test was performed to assess the nature of the crowding groups and the different classes of angle classification . in turn , the chi - square test was implemented in order to identify the nature of the relationship between the gingival biotype , angle classification , the level of crowding , and gender . the study group consisted of a total of 187 patients aged between 10 and 28.2 years , of whom 121 were female ( mean age , 17.5 4.25 years ) and 66 were male ( mean age , 15.8 3.17 years ) . the number of patients in the angle class i , ii , and iii malocclusion groups was 76 ( 40.6% ) , 80 ( 42.8% ) , and 31 ( 16.6% ) , respectively . in addition , the number of patients in the mild , moderate , and severe crowding groups was 101 ( 54.0% ) , 50 ( 26.7% ) , and 36 ( 19.3% ) , respectively ( table 1 ) . in tables 2 , 3 , and 4 , the vertical columns describe the crowding levels and the horizontal columns describe the angle classification groups . the distribution of pi , gi , and pd measurements , used in determining the periodontal status of patients , are displayed in table 2 , according to angle classification and the level of crowding . the pi values were significantly higher in the severe crowding group than in the mild crowding group ; this was also the case in the angle class iii malocclusion group , compared to the angle class ii malocclusion group ( p = 0.042 ) . in addition , pd values were higher in terms of statistical significance in the angle class iii malocclusion group than they were in the angle class i malocclusion group ( p = 0.101 ) . the wkg of the lower anterior teeth according to angle classification and the level of crowding is displayed in table 3 . no statistically significant difference was observed between the wkg of the lower anterior teeth according to angle classification . the wkg values for the mandibular left central and lateral incisors and the mandibular right lateral incisor were higher in the severe crowding group ( p < 0.05 ) . in addition , it should also be noted that , while the wkg of the mandibular right central incisor was higher in the severe crowding group , the difference was not statistically significant . the wkg values of mandibular canines were higher in the mild crowding group ( p < 0.05 ) . table 4 summarizes and displays the gt of the lower anterior teeth on the basis of angle classification and the level of crowding . it was observed that teeth in the lower anterior jaw displayed thin gingival biotype , and yet there was no statistically significant difference observed between angle classification and the gts of the mandibular left central and lateral incisors , and the mandibular canines . however , the gts of the mandibular right central and lateral incisors was observed to be significantly lower in the angle class iii malocclusion group ( p < 0.05 ) . the gts of the mandibular left central and lateral incisors , and the mandibular right lateral incisor were significantly higher in the severe crowding group ( p < 0.05 ) . notably , although the gt of the mandibular right central incisor was higher in the severe crowding group , the difference was not statistically significant . the gts of the mandibular canines were higher in terms of statistical significance in the mild crowding group ( p < 0.05 ) . table 5 presents an overview of the mean gt of the mandibular anterior region on the basis of angle classification and the level of crowding . it was determined that the mean gt of the mandibular anterior region is 0.71 0.17 mm and , moreover , displays the thin gingival biotype . it did not exhibit any significant association with angle classification or the level of crowding ( p = 0.140 and 0.321 , respectively ) . there is a range of risk factors associated with gingival recession , especially in the area of the mandibular incisors during orthodontic treatment . these include the following : the age of the patient , the health status of the periodontal tissues , the duration of the treatment , the amount and type of tooth movement , the wkg , and gt.916 nevertheless , owing to the lack of research that has been conducted in order to evaluate each of these parameters , a range of conflicting opinions exist regarding the effect of mandibular incisor protrusion on periodontal tissues.916 one group of researchers has published the claim that such orthodontic tooth movements are risk factors for gingival recession , due to the fact that the buccal alveolar bone of mandibular incisors is thin.1117 contrastingly , another group has reported there is no such relation between the two.91018 two notable studies , by wennstrm et al.18 and yared et al.,11 stated that among the parameters evaluated at the planning stage of orthodontic treatment , gt is a more important factor than wkg and protrusion movement . therefore , the aim of the present study was to evaluate the relationship that exists between wkg and gt , both of which are considered to be significant risk factors for gingival recession that may be seen in the mandibular anterior region with different malocclusion groups and levels of crowding . it has been found that gt is affected by changes in the position of teeth during the eruption period.23 furthermore , it has been determined that this effect is decreased as age increases ; this is because as connective tissue becomes denser , the cell count decreases , the epithelium becomes thinner , and keratinization increases.24 it has been observed that age groups were constituted differently in studies which investigated the relation between gt and age . closs et al.26 investigated the relationship between the initial wkg and gingival recession by examining 209 subjects treated with fixed orthodontic appliances , and concluded that no significant difference existed in the initial wkg for individuals who did or did not have gingival recession . however , it is important to note that , in this study , the mean wkg of all teeth was greater than 2 mm . in light of these results , it is important to note the findings of the present study : namely , that a wkg for mandibular anterior teeth of between 2.01 1.33 3.90 1.70 mm was found to be adequate for the maintenance of periodontal health . given this , it has been determined that , when the thickness of the gingiva is decreased in the bucco - lingual direction , the apicocoronal height also decreases.18 accordingly , the wkg of the thin gingival biotype is less than that of the thick gingival biotype.13 it has also been determined that the tooth germs of the mandibular permanent canines are positioned in the same direction as the mandibular primary canine roots , thereby meaning that the wkg and gt of these teeth is less than that of mandibular incisor teeth . this is due to the fact that frequent eruptions on the vestibular position take place if there is insufficient space for them.182327 consistent with these findings , the present study observed that the wkg and gt of mandibular canines are less than those of mandibular incisors . however , the difference between the gt of mandibular canines and that of tooth number 31 was not significant . thus , there is a tendency for the mandibular permanent incisors to erupt somewhat lingually and in an irregular position , even in children who have normal dental arches . furthermore , this position can not be corrected in the event of crowding.27 in addition , the extant literature reports that the tooth germs of mandibular lateral incisors are positioned more lingually compared to the tooth germs of mandibular central incisors and , in cases where crowding is an issue , this facilitates eruption in a more lingual position than mandibular central incisors.27 moreover , the wkg and gt of lingually erupting teeth are reported as being higher.1823 consistent with these results , the present study demonstrated that the wkg and gt of mandibular lateral incisors are higher than those of mandibular central incisors . when the level of crowding increases , it is expected that the wkg and gt of the mandibular canines will decrease . this is primarily owing to the fact that they erupt in a more vestibular position and , in addition , the wkg and gt of the mandibular central and lateral incisors increases ; this also takes place because they erupt in a more lingual position . in the extant literature relating to the wkg
, no findings have been published regarding the relation between the wkg of each mandibular anterior tooth and the level of crowding . our study indicates that the wkg of the mandibular left central and lateral incisors and mandibular right lateral incisor are significantly higher in the severe crowding group ( p < 0.05 ) , compared to the other groups . although the wkg of the mandibular right central incisor is higher in the severe crowding group , the difference is not statistically significant . the wkg of the mandibular canines are significantly higher in the mild crowding group ( p < 0.05 ) , compared to the other groups . relatively few studies have been published in the extant literature that focus on evaluating the relationship between the gingival biotype and the level of crowding . of the available studies , zawawi and
al - zahrani28 stated that there is no significant association between the level of crowding and gt in the mandibular anterior region . in this study , it was observed that periodontal probing was used to measure gt ; the space analysis was performed by including only mandibular incisors ; and only the mandibular central incisor was taken as a reference to detect the gingival biotype of subjects . in our study , the relationship of the gt of each mandibular anterior tooth with the level of crowding was evaluated , primarily because the gt may be subject to variation depending on the position of the teeth in the dental arch.1823 in addition , space analysis was performed by including the canine teeth , and gts of the mandibular left central and lateral incisors and mandibular right lateral incisor were observed to be significantly higher in the severe crowding group ( p < 0.05 ) . although the gt of the mandibular right central incisor is higher in the severe crowding group , this difference is not statistically significant . in addition , it is noteworthy that the gt of the mandibular canines are significantly higher in the mild crowding group ( p < 0.05 ) . the present study displayed no statistically significant relationship between the wkg and the angle classification in the mandibular anterior region . a limited number of studies exist in the literature that have evaluated the association of gingival biotype with different dental malocclusion groups . among these ,
zawawi et al.2 studied 200 individuals and reported that no statistically significant relationship was observed between the gingival biotype and angle classification . however , matarese et al.,29 in a study focusing on 76 individuals , assessed biotype by employing periodontal probing at the mid - facial aspect of the maxillary central , lateral incisors , and canines , and found that no statistically significant relationship existed between the gingival biotype and angle classification . the researchers also noted that the gt is subject to change on the basis of tooth position , facial characteristics , and profile , thereby warranting further study to evaluate the impact of these parameters . consistent with these results , our study showed that there is no statistically significant relationship between the mean gt of the mandibular anterior region and angle classification . it was also found that there is no statistically significant relationship of the gt of the mandibular left central and lateral incisors , and that of the mandibular canines with angle classification . the gt values of the mandibular right central and lateral incisors are statistically lower in the angle class iii malocclusion group ( p < 0.05 ) . it is important to acknowledge the current study 's limitations , and the primary limitation of this research has been the sole examination and evaluation of the relationship of the gt of the mandibular anterior jaw with angle classification and the level of crowding . the first critical finding of the current study is the observation that teeth in the lower anterior jaw display thin gingival biotype . second , it has been found that , when the level of crowding increases , there is a corresponding increase in the wkg and gt of the central and lateral incisors ; simultaneously , there is a corresponding decrease in the wkg and gt of canines . third , there is no association between the angle classification and the mean gt of the mandibular anterior region . the final conclusion is that the gt of the mandibular right central and lateral incisors is lower in the angle class iii group . | [
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historically , the taming of singularities in classical field models has driven a great deal of research .
a particularly elegant example is the nonlinear extension of maxwell electrodynamics introduced by born and infeld to remove the divergence of both the coulomb field and the self - energy of point particles . in this determinantal form of the classical action , the modified field ( the bion )
this specific form of nonlinear electrodynamics arises in the low - energy limit of certain string theories [ 35 ] .
recovering the idea of the determinantal form of the gravitational action suggested by eddington [ 6 , 7 ] , an eddington - inspired born
infeld action ( eibi ) for the gravitational field has been introduced recently [ 8 , 9 ] . in order to avoid troubles with higher - order derivatives and ghosts ,
eibi gravity is formulated in the palatini approach , which means that the metric and connection are regarded as physically independent entities .
this implies that the connection is determined by the field equations , not constrained a priori to any particular form .
hilbert action which might allow one to remove the appearance of singularities , thus avoiding an undesirable feature of einstein s theory of general relativity ( gr ) .
the eibi theory is expected to be in agreement with gr at energies well below the planck scale , which represents the regime where quantum gravitational effects are expected to begin to become important and modify the classical description .
the naturalness of eibi gravity has been argued on the basis of canonical procedures to construct lagrangian densities with second - order field equations .
moreover , this theory is able to avoid cosmological singularities , has been employed to study properties of dark matter and dark energy [ 1315 ] , in the coupling to several kinds of fields , as an alternative to inflation , and to explore the structure of compact stars [ 18 , 19 ] .
when coupled to a perfect fluid with a given equation of state , it has been found that the theory can be interpreted as gr coupled again to a perfect fluid , but with a modified equation of state [ 20 , 21 ] .
though in its determinantal form the eibi theory may appear as lacking an intuitive motivation , here we show that , when applied to elementary systems such as electric fields generated by point - like sources ( or elementary particles ) , the theory boils down to a simple quadratic extension of gr .
this simplification occurs when the stress - energy tensor of the matter possesses certain algebraic properties , namely , when it has two double eigenvalues .
we take advantage of this property to explore in detail the internal structure of the electrovacuum solutions of the theory and find that the central singularity is generically replaced by a wormhole supported by the electric field .
the wormhole structure turns out to be crucial to regularize the total energy stored in the electric field , which occurs in a way that resembles the original born infeld electromagnetic theory . among the solutions of the theory ,
there exist a family ( characterized by a certain charge - to - mass ratio ) for which curvature invariants are finite everywhere .
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\begin{document}$$r_{\mu \nu } \equiv { r^\rho } _ { \mu \rho \nu } $ $ \end{document}rr is the ricci tensor , \documentclass[12pt]{minimal }
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\begin{document}$${r^\alpha } _ { \beta \mu \nu } = \partial _ { \mu } \gamma ^{\alpha } _
_ { \mu \beta } + \gamma ^{\alpha } _ { \mu \lambda } \gamma ^{\lambda } _ { \nu \beta } -\gamma ^{\alpha } _ { \nu \lambda } \gamma ^{\lambda } _ { \mu \beta } $ $ \end{document}r=-+- is the riemann tensor of the connection \documentclass[12pt]{minimal }
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\begin{document}$$ \gamma _ { \mu \nu } ^{\lambda } $ $ \end{document} , which is a priori independent of the metric ( palatini formalism ) , and \documentclass[12pt]{minimal }
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\begin{document}$$s_m$$\end{document}sm is the matter action .
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\begin{document}$$\lambda $ $ \end{document} can be obtained from the field equations and from the leading - order terms of an expansion in \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon \ll 1$$\end{document}1 , which reads2\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \lim _ { \epsilon \rightarrow 0 } s&= \frac{1}{2\kappa ^2 } \int \mathrm{d}^4x \sqrt{-g } [ r\!-\!2\lambda _ { eff } ] \nonumber \\&-\frac{1}{2\kappa ^2 } \int \mathrm{d}^4x \sqrt{-g } \frac{\epsilon } { 2 } \left ( \frac{-r^2}{2 } \!+\ !
\end{aligned}$$\end{document}lim0s=122d4x - g[r-2eff]-122d4x - g2-r22+rr+sm.here \documentclass[12pt]{minimal }
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\begin{document}$$r\equiv g^{\mu \nu } r_{\mu \nu } $ $ \end{document}rgr and \documentclass[12pt]{minimal }
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\begin{document}$$\lambda _ { eff}=\frac{\lambda -1}{\epsilon } $ $ \end{document}eff=-1. therefore , when \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon \rightarrow 0$$\end{document}0 the leading - order in ( 2 ) coincides with gr plus a cosmological constant term , with \documentclass[12pt]{minimal }
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\begin{document}$$\lambda = 1+\epsilon \lambda _ { eff}$$\end{document}=1+eff . the action ( 1 )
can thus be seen as a high - energy modification of einstein s theory , including a cosmological constant as long as \documentclass[12pt]{minimal }
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\begin{document}$$\lambda \ne 1$$\end{document}1 .
variation of ( 1 ) with respect to metric and connection leads to3\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}&\frac{\sqrt{\vert q \vert } } { \sqrt{\vert g \vert } } q^{\mu \nu } -\lambda g^{\mu \nu } = -\kappa ^2 \epsilon t^{\mu \nu } , \end{aligned}$$\end{document}|q||g|q-g=-2t,4\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}&\nabla _ { \alpha } \left ( \sqrt{q } q^{\mu \nu } \right ) = 0 , \end{aligned}$$\end{document}qq=0,where \documentclass[12pt]{minimal }
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\begin{document}$$q^{\mu \nu } $ $ \end{document}q is the inverse of \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q. to obtain these equations we have assumed vanishing torsion , \documentclass[12pt]{minimal }
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\begin{document}$$\gamma _ { [ \mu \nu ] } = 0$$\end{document}[]=0 , as well as \documentclass[12pt]{minimal }
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\begin{document}$$r_{[\mu \nu ] } = 0$$\end{document}r[]=0 , which guarantees the existence of volume invariants preserved by the theory .
the connection equation ( 4 ) is formally identical to that found in the palatini version of gr and implies that \documentclass[12pt]{minimal }
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\begin{document}$$\gamma _ { \mu \nu } ^{\lambda } $ $ \end{document} can be written as the levi - civita connection of \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q , which can be seen as an auxiliary metric tensor associated with the independent connection .
similarly as in other palatini theories , the relation between \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q and \documentclass[12pt]{minimal }
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\begin{document}$$g_{\mu \nu } $ $ \end{document}g is algebraic and given by5\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \hat{q}=\sqrt{|\hat{\sigma } | } \hat{\sigma } ^{-1 } \hat{g } \ , \
\hat{q}^{-1}=\frac{\hat{g}^{-1}\hat{\sigma } } { \sqrt{|\hat{\sigma } | } } , \end{aligned}$$\end{document}q^=|^|^-1g^,q^-1=g^-1^|^|,where we have used a hat to denote matrix representation and have defined \documentclass[12pt]{minimal }
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\begin{document}$$\sqrt{|\hat{\sigma } | } \hat{\sigma } ^{-1}\equiv \hat{i}+\epsilon \hat{p}$$\end{document}|^|^-1i^+p^ , with \documentclass[12pt]{minimal }
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\begin{document}$$\hat{p}$$\end{document}p^ denoting the matrix \documentclass[12pt]{minimal }
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\begin{document}$${p^\alpha } _
\nu \equiv g^{\alpha \mu } r_{\mu \nu } ( \gamma ) $ $ \end{document}pgr( ) .
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\begin{document}$$|\hat{\sigma } |=|\hat{i}+\epsilon \hat{p}|$$\end{document}|^|=|i^+p^| . using this notation and ( 3 )
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\begin{document}$$\begin{aligned } \hat{\sigma } = \lambda \hat{i}-\epsilon \kappa ^2 \hat{t } , \end{aligned}$$\end{document}^=i^-2t^,where \documentclass[12pt]{minimal }
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\begin{document}$${[\hat{t}]_\mu } ^\nu \equiv t^{\nu \alpha } g_{\alpha \mu } $ $ \end{document}[t^]tg. note that ( 6 ) allows one to obtain the relation between \documentclass[12pt]{minimal }
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\begin{document}$$g_{\mu \nu } $ $ \end{document}g and \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q once the matter sources have been specified .
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q can be written in a very compact and convenient form noting that \documentclass[12pt]{minimal }
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\begin{document}$$\hat{q}=\hat{g}+\epsilon \hat{r}$$\end{document}q^=g^+r^ can be written as \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon \hat{r } \hat { q}^{-1}=i-\hat{g } \hat{q}^{-1}$$\end{document}r^q^-1=i - g^q^-1 . using ( 5 ) we find that \documentclass[12pt]{minimal }
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\begin{document}$$\hat{g } \hat{q}^{-1}= \frac{\hat{\sigma } } { \sqrt{|\hat{\sigma } |}}$$\end{document}g^q^-1=^|^| , and taking ( 6 ) we finally get7\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } { r_\mu } ^\nu ( q)=\frac{\kappa ^2}{{|\hat{\sigma } |}^{1/2}}\left [ \mathcal { l}_g { \delta _ \mu } ^\nu + { t_\mu } ^\nu \right ] , \end{aligned}$$\end{document}r(q)=2|^|1/2lg+t,where \documentclass[12pt]{minimal }
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\begin{document}$${r_\mu } ^\nu ( q)= r_{\mu \alpha } ( \gamma ) q^{\alpha \nu } $ $ \end{document}r(q)=r()q , and we have used the fact that the gravity action in ( 1 ) can be written as \documentclass[12pt]{minimal }
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\begin{document}$$s_g=\int \mathrm{d}^4x \sqrt{-g}\mathcal { l}_g$$\end{document}sg=d4x - glg with \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal { l}_g\equiv ( { |\hat{\sigma } |}^{1/2}-\lambda ) / ( \kappa ^2\epsilon ) $ $ \end{document}lg(|^|1/2-)/(2 ) .
it is worth noting that the representation of the field equations given in ( 7 ) seems to be very general , since it is also valid for other families of palatini theories with lagrangians of the form \documentclass[12pt]{minimal }
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\begin{document}$$f(r)/(2\kappa ^2)$$\end{document}f(r)/(22 ) and \documentclass[12pt]{minimal }
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\begin{document}$$f(r , r_{\mu \nu } r^{\mu \nu } ) /(2\kappa ^2)$$\end{document}f(r ,
rr)/(22 ) , which include gr as a particular case ( with \documentclass[12pt]{minimal }
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\begin{document}$$\hat{\sigma } = \hat{i}$$\end{document}^=i^ ) . from ( 7 ) one clearly sees that the metric \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q satisfies a system of second - order differential equations with the matter sources on the right - hand side ( recall from ( 6 ) that \documentclass[12pt]{minimal }
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\begin{document}$$\hat{\sigma } = \hat{\sigma } [ \hat{t}]$$\end{document}^=^[t^ ] ) . since \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q is algebraically related to \documentclass[12pt]{minimal }
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\begin{document}$$g_{\mu \nu } $ $ \end{document}g through ( 5 ) , it follows that \documentclass[12pt]{minimal }
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\begin{document}$$g_{\mu \nu } $ $ \end{document}g also satisfies second - order equations . on the other hand ,
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\begin{document}$$|\hat{\sigma } |=\lambda ^4$$\end{document}|^|=4 , \documentclass[12pt]{minimal }
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\begin{document}$$g_{\mu \nu } $ $ \end{document}g and \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q are identical up to a constant conformal factor and that \documentclass[12pt]{minimal }
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\begin{document}$${r_\mu } ^\nu ( q)=\frac{(\lambda -1)}{\lambda \epsilon } { \delta _ \mu } ^\nu $ $ \end{document}r(q)=(-1) , which is equivalent to \documentclass[12pt]{minimal }
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\begin{document}$$r_{\mu \nu } ( g)=\frac{(\lambda -1)}{\epsilon } g_{\mu \nu } $ $ \end{document}r(g)=(-1)g , thus confirming that \documentclass[12pt]{minimal }
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\begin{document}$$\frac{(\lambda -1)}{\epsilon } $ $ \end{document}(-1) plays the role of an effective cosmological constant in the full theory . since the vacuum theory is equivalent to gr with a cosmological constant , no ghost - like instabilities are present in the theory , which is a rather general property of palatini theories .
we now couple our gravity theory to an electromagnetic field with action8\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } s_m=-\frac{1}{16\pi } \int \mathrm{d}^4x \sqrt{-g } f_{\mu \nu } f^{\mu \nu } , \end{aligned}$$\end{document}sm=-116d4x - gff,where \documentclass[12pt]{minimal }
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\begin{document}$$f_{\mu \nu } = \partial _ { \mu } a_{\nu } -\partial _ { \nu } a_{\mu } $ $ \end{document}f=a-a is the field strength tensor . assuming a spherically symmetric and static electric field , without loss of generality we can choose coordinates such that the line element becomes9\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathrm{d}s^2=-a(x)\mathrm{d}t^2+\frac{1}{a(x)}\mathrm{d}x^2+r^2(x ) \mathrm{d}\omega ^2 .
\end{aligned}$$\end{document}ds2=-a(x)dt2 + 1a(x)dx2+r2(x)d2.it is sometimes useful to use the function \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document}r as a radial coordinate , which turns ( 9 ) into \documentclass[12pt]{minimal }
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\begin{document}$$\mathrm{d}s^2=-a(r)\mathrm{d}t^2+\mathrm{d}r^2/b(r)+r^2\mathrm{d}\omega ^2$$\end{document}ds2=-a(r)dt2+dr2/b(r)+r2d2 .
this replacement is possible as long as the relation between the coordinate \documentclass[12pt]{minimal }
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\begin{document}$$r^2(x)$$\end{document}r2(x ) is monotonic . for reasons that will become clear later
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\begin{document}$$x$$\end{document}x instead of \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document}r in our analysis . with this choice of coordinates
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\begin{document}$$f^{tx}=q / r^2$$\end{document}ftx = q / r2 is the only non - zero component of \documentclass[12pt]{minimal }
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\begin{document}$$f^{\mu \nu } $ $ \end{document}f , where \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}q is an integration constant .
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\begin{document}$${t_{\mu } } ^{\nu } = \frac{q^2}{8\pi r^4 } diag(-1,-1,1,1)$$\end{document}t=q28r4diag(-1,-1,1,1 ) , which , inserted in ( 6 ) , yields10\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } { \sigma _ { \mu } } ^{\nu } = \left ( \begin{array}{l@{\quad } l } \sigma ^{(\epsilon ) } _ { + } \hat{i } & { } \hat{0 } \\ \hat{0 } & { } \sigma ^{(\epsilon ) } _ { - } \hat{i } \end{array } \right ) , \
\sigma ^{(\epsilon ) } _ { \pm } = \lambda \pm \frac{\epsilon \kappa ^2 q^2}{8\pi r^4}. \end{aligned}$$\end{document}=+()i^0 ^ 0^-()i^,()=2q28r4.the field equations ( 7 ) then become11\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } { r_{\mu } } ^{\nu } ( q)=\frac{1}{\epsilon } \left ( \begin{array}{l@{\quad } l } \frac{(\sigma ^{(\epsilon ) } _ { - } -1)}{\sigma ^{(\epsilon ) } _ { - } } \hat{i } & { } \hat{0 } \\ \hat{0 } & { } \frac{(\sigma ^{(\epsilon ) } _ { + } -1)}{\sigma ^{(\epsilon ) } _ { + } } \hat{i } \end{array } \right ) .
\end{aligned}$$\end{document}r(q)=1(-()-1)-()i^0 ^ 0^(+()-1)+()i^.the strategy now consists on solving for \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q and then use ( 5 ) to obtain \documentclass[12pt]{minimal }
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\begin{document}$$g_{\mu \nu } $ $ \end{document}g. to proceed , we write a line element for \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mu \nu } $ $ \end{document}q , which according to ( 5 ) and ( 9 ) takes the form12\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathrm{d}\tilde{s}^2\!=\!-\sigma ^{(\epsilon ) } _ { - } a(x ) \mathrm{d}t^2\!+\!(\sigma ^{(\epsilon ) } _ { -}/a(x))\mathrm{d}x^2\!+\!\sigma ^{(\epsilon ) } _ { + } r^2(x ) \mathrm{d}\omega ^2,\nonumber \\ \end{aligned}$$\end{document}ds~2=--()a(x)dt2+(-()/a(x))dx2++()r2(x)d2,and
rewrite it as13\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathrm{d}\tilde{s}^2=-c(\tilde{r } ) e^{2\psi ( \tilde{r } ) } \mathrm{d}t^2 + 1/c(\tilde{r } ) \mathrm{d}\tilde{r}^2+\tilde{r}^2\mathrm{d}\omega ^2 , \end{aligned}$$\end{document}ds~2=-c(r~)e2(r~)dt2 + 1/c(r~)dr~2+r~2d2,where we have defined \documentclass[12pt]{minimal }
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\begin{document}$$\tilde{r}^2=\sigma ^{(\epsilon ) } _ { + } r^2$$\end{document}r~2=+()r2 , \documentclass[12pt]{minimal }
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\begin{document}$$c(\tilde{r})=\sigma ^{(\epsilon ) } _ { - } a(x)$$\end{document}c(r~)=-()a(x ) , and \documentclass[12pt]{minimal }
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\begin{document}$$(\mathrm{d}\tilde{r}/\mathrm{d}x)^2=(\sigma ^{(\epsilon ) } _ { -})^2$$\end{document}(dr~/dx)2=(-())2 . plugging this ansatz into ( 11 ) , we find that \documentclass[12pt]{minimal }
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\begin{document}$$\psi ( \tilde{r})$$\end{document}(r~ ) is a constant . with the typical ansatz \documentclass[12pt]{minimal }
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\begin{document}$$c=1-\frac{2 g m(\tilde{r})}{c^2\tilde{r}}$$\end{document}c=1 - 2gm(r~)c2r~ we obtain14\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } m_r\equiv \frac{\mathrm{d}m}{\mathrm{d}r}=\frac{r^2(\sigma _ { + } ^{(\epsilon ) } -1)\sigma _ { -}^{(\epsilon ) } } { 2\epsilon \sigma _ { + } ^{(\epsilon ) 1/2 } } , \end{aligned}$$\end{document}mrdmdr = r2(+()-1)-()2+()1/2,where the relation \documentclass[12pt]{minimal }
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\begin{document}$$\mathrm{d}\tilde{r}/\mathrm{d}r=\sigma _ { -}^{(\epsilon ) } /\sqrt{\sigma _ { + } ^{(\epsilon ) } } $ $ \end{document}dr~/dr=-()/+( ) , which follows from \documentclass[12pt]{minimal }
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\begin{document}$$\tilde{r}^2=r^2 \sigma _ { + }
this relation also allows one to find that \documentclass[12pt]{minimal }
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\begin{document}$$(\mathrm{d}r/\mathrm{d}x)^2=\sigma _ { + } ^{(\epsilon ) } $ $ \end{document}(dr / dx)2=+( ) .
we thus conclude that the line element ( 9 ) is completely determined by15\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } a(x)=\frac{1}{\sigma ^{(\epsilon ) } _ { -}}\left ( 1-\frac{2 g m(r)}{c^2r \sqrt{\sigma _ { + }
^{(\epsilon ) } } } \right ) \ , \ \left ( \frac{\mathrm{d}r}{\mathrm{d}x}\right ) ^2=\sigma _ { + } ^{(\epsilon ) } \ , \end{aligned}$$\end{document}a(x)=1-()1 - 2gm(r)c2r+(),drdx2=+(),with \documentclass[12pt]{minimal }
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\begin{document}$$m(r)$$\end{document}m(r ) given by the integration of ( 14 ) .
note that , though this solution is formally equivalent to that obtained by baados and ferreira in , in the following sections we shall discuss the physical properties of those solutions with \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon < 0$$\end{document}<0 .
therefore the claims of regarding the presence of singularities in this theory when \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon > 0$$\end{document}>0 do not apply in our case , as we will explicitly show below . for a more detailed discussion on the influence of signs of the parameters in the theory see .
to discuss the physics behind the above solutions , we note that ( 7 ) , ( 10 ) , ( 14 ) , and ( 15 ) exactly coincide with those corresponding to the quadratic palatini theory16\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } s&= \frac{1}{2\kappa ^2}\int \mathrm{d}^4x \sqrt{-g}\left [ r-2\lambda + a\left ( -\frac{r^2}{2}+r_{\mu \nu } r^{\mu \nu } \right ) \right ] \nonumber \\&+\,s_m \end{aligned}$$\end{document}s=122d4x - gr-2+a - r22+rr+smwith the identifications \documentclass[12pt]{minimal }
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\begin{document}$$\epsilon = -2a$$\end{document}=-2a and \documentclass[12pt]{minimal }
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\begin{document}$$\lambda = 1+\epsilon \lambda $ $ \end{document}=1+ [ compare with ( 2 ) ] .
the reason for this equivalence lies on the algebraic properties of the action ( 1 ) .
given the linear relation between \documentclass[12pt]{minimal }
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\begin{document}$${t_\mu } ^\nu $ $ \end{document}t and \documentclass[12pt]{minimal }
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\begin{document}$${\sigma _ \mu } ^\nu $ $ \end{document} , in a basis in which \documentclass[12pt]{minimal }
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\begin{document}$${t_\mu } ^\nu $ $ \end{document}t is diagonal the matrix \documentclass[12pt]{minimal }
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\begin{document}$$\hat{p}$$\end{document}p^ will also be diagonal .
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\begin{document}$$\hat{p}$$\end{document}p^ has two double eigenvalues ( in our case \documentclass[12pt]{minimal }
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\begin{document}$$\hat{p}=diag[p_1,p_1,p_2,p_2]$$\end{document}p^=diag[p1,p1,p2,p2 ] ) then the fourth - order polynomial defined by \documentclass[12pt]{minimal }
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\begin{document}$$|\hat{\sigma } |=|\hat{i}+\epsilon \hat{p}|$$\end{document}|^|=|i^+p^| turns into the second - order polynomial appearing in ( 16 ) when the squared root is evaluated . note that this quadratic polynomial coincides exactly with the series expansion of ( 2 ) , thus implying that all other higher - order terms vanish identically when \documentclass[12pt]{minimal }
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\begin{document}$$\hat{p}$$\end{document}p^ has this structure .
the intimate relation existing between the quadratic palatini theory and the eibi theory can be used to shed useful new light on the physics of the corresponding solutions . to see this ,
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\begin{document}$$\epsilon = -2l_\epsilon ^2$$\end{document}=-2l2 , with \documentclass[12pt]{minimal }
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\begin{document}$$l_\epsilon $ $ \end{document}l some small length scale , for which exact solutions can be found for arbitrary \documentclass[12pt]{minimal }
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\begin{document}$$\lambda $ $ \end{document} . for simplicity
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\begin{document}$$\lambda = 1$$\end{document}=1 [ 2628 ] .
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\begin{document}$$m(r)$$\end{document}m(r ) appearing in ( 15 ) can be written as \documentclass[12pt]{minimal }
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\begin{document}$$m(r)=m_0(1+\delta _ 1 g(r))$$\end{document}m(r)=m0(1+1g(r ) ) , where \documentclass[12pt]{minimal }
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\begin{document}$$m_0$$\end{document}m0 is an integration constant representing the scharzschild mass of the uncharged case , while17\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \delta _ 1=\frac{1}{2r_s}\sqrt{r_q^3/l_{\epsilon } } \end{aligned}$$\end{document}1=12rsrq3/lis a dimensionless constant that controls the charge - to - mass ratio , and \documentclass[12pt]{minimal }
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\begin{document}$$g(r)$$\end{document}g(r ) encodes the electric field contribution . for convenience ,
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\begin{document}$$q$$\end{document}q by the length scales \documentclass[12pt]{minimal }
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\begin{document}$$r_s=2gm_0/c^2$$\end{document}rs=2gm0/c2 and \documentclass[12pt]{minimal }
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\begin{document}$$r_q^2=\kappa ^2 q^2/4\pi $ $ \end{document}rq2=2q2/4 , and measure the radial function \documentclass[12pt]{minimal }
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\begin{document}$$r(x)$$\end{document}r(x ) in units of \documentclass[12pt]{minimal }
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\begin{document}$$r_c\equiv \sqrt{r_q l_\epsilon } $ $ \end{document}rcrql by defining \documentclass[12pt]{minimal }
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\begin{document}$$z = z(x)=r(x)/r_c$$\end{document}z = z(x)=r(x)/rc . for \documentclass[12pt]{minimal }
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\begin{document}$$z\gg 1$$\end{document}z1 , \documentclass[12pt]{minimal }
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\begin{document}$$g(z)\approx -1/z$$\end{document}g(z)-1/z leads to the expected gr limit \documentclass[12pt]{minimal }
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\begin{document}$$a(x)\approx \left ( 1-\frac{r_s}{r}+\frac{r_q^2}{2r^2}\right ) + o(\frac{r_c^4}{r^4})$$\end{document}a(x)1-rsr+rq22r2+o(rc4r4 ) and , therefore , does not exhibit any pathological behavior1 . for \documentclass[12pt]{minimal }
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\begin{document}$$z\rightarrow 1$$\end{document}z1 , however , the geometry strongly departs from the low - energy limit represented by gr . to understand the relevance of this region
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\begin{document}$$z(x)=r(x)/r_c$$\end{document}z(x)=r(x)/rc:18\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left ( \frac{\mathrm{d}z}{\mathrm{d}x}\right ) ^2=\frac{1}{r_c^2}\left ( 1-\frac{1}{z^4}\right ) \ .
\end{aligned}$$\end{document}dzdx2=1rc21 - 1z4.for \documentclass[12pt]{minimal }
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\begin{document}$$z\gg 1$$\end{document}z1 the relation between \documentclass[12pt]{minimal }
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\begin{document}$$x$$\end{document}x is linear , but \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z reaches a minimum at \documentclass[12pt]{minimal }
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\begin{document}$$z(x_c)=1$$\end{document}z(xc)=1 .
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\begin{document}$$z(x)$$\end{document}z(x ) is a clear signal of the presence of a wormhole .
in fact , ( 18 ) can be integrated to get the curve shown in fig .
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\begin{document}$$\mathrm{d}z/\mathrm{d}x=(1-\frac{1}{z^4})^{1/2}/r_c$$\end{document}dz / dx=(1 - 1z4)1/2/rc if \documentclass[12pt]{minimal }
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\begin{document}$$\mathrm{d}z/\mathrm{d}x=-(1-\frac{1}{z^4})^{1/2}/r_c$$\end{document}dz / dx=-(1 - 1z4)1/2/rc if \documentclass[12pt]{minimal }
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\begin{document}$$x\le x_c$$\end{document}xxc.fig .
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\begin{document}$$z(x)$$\end{document}z(x ) and \documentclass[12pt]{minimal }
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\begin{document}$$\mathrm{d}g/\mathrm{d}x$$\end{document}dg / dx ( with \documentclass[12pt]{minimal }
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\begin{document}$$r_c=1$$\end{document}rc=1 ) .
the wormhole throat is at the minimum of \documentclass[12pt]{minimal }
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\begin{document}$$z(x)$$\end{document}z(x ) ( maximum of \documentclass[12pt]{minimal }
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\begin{document}$$g_x$$\end{document}gx ) plot of \documentclass[12pt]{minimal }
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\begin{document}$$\mathrm{d}g/\mathrm{d}x$$\end{document}dg / dx ( with \documentclass[12pt]{minimal }
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\begin{document}$$r_c=1$$\end{document}rc=1 ) .
the wormhole throat is at the minimum of \documentclass[12pt]{minimal }
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\begin{document}$$z(x)$$\end{document}z(x ) ( maximum of \documentclass[12pt]{minimal }
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\begin{document}$$g_x$$\end{document}gx ) therefore , our space - time can be seen as consisting on two identical pieces connected through a ( spherical ) hole ( the wormhole throat ) of area \documentclass[12pt]{minimal }
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\begin{document}$$a=4\pi r_c^2$$\end{document}a=4rc2 .
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\begin{document}$$z\approx 1$$\end{document}z1 , \documentclass[12pt]{minimal }
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\begin{document}$$g(z)$$\end{document}g(z ) can be expanded as19\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } g(z)\approx \beta + 2\sqrt{z-1}-\frac{11}{6}(z-1)^{3/2}+\cdots \end{aligned}$$\end{document}g(z)+2z-1 - 116(z-1)3/2+where \documentclass[12pt]{minimal }
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\begin{document}$$\beta \approx -1.74804$$\end{document}-1.74804 is an integration constant .
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\begin{document}$$z(x)$$\end{document}z(x ) known , the geometry can be explored in detail .
one then finds that , in general , there exist curvature divergences at \documentclass[12pt]{minimal }
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\begin{document}$$z=1$$\end{document}z=1 , the leading order being dominated by \documentclass[12pt]{minimal }
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\begin{document}$$\sim 1/(z-1)^3$$\end{document}1/(z-1)3 in the case of \documentclass[12pt]{minimal }
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_ { \beta \mu \nu } ( g){r_\alpha } ^{\beta \mu \nu } ( g)$$\end{document}r(g)r(g ) ( which contrasts with the much stronger divergences found in gr , \documentclass[12pt]{minimal }
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\begin{document}$$\sim 1/r^8$$\end{document}1/r8 ) .
however , if the charge - to - mass ratio defined by the constant \documentclass[12pt]{minimal }
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\begin{document}$$\delta _ 1$$\end{document}1 is tuned to the value \documentclass[12pt]{minimal }
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1^*=-1/\beta $ $ \end{document}1=-1/ , the \documentclass[12pt]{minimal }
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\begin{document}$$z=1$$\end{document}z=1 divergences disappear yielding a completely regular geometry .
one can now wonder about the nature of the sources that generate the charge \documentclass[12pt]{minimal }
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\begin{document}$$m_0$$\end{document}m0 that characterize our solutions2 .
as first shown by misner and wheeler , an electric flux through a wormhole can define by itself an electric charge3 without the need for sources of the electric field .
therefore , the charge \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}q appearing in our solutions is entirely given by the electric flux through any two - surface \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal { s}$$\end{document}s enclosing one of the sides of the wormhole , i.e.,20\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } q=\frac{1}{4\pi } \int _ { \mathcal { s } } * f , \end{aligned}$$\end{document}q=14sf , where \documentclass[12pt]{minimal }
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\begin{document}$$*f$$\end{document}f is the two - form dual to faraday s tensor .
the existence of a wormhole where one would naively expect to find the sources poses a more severe challenge to identify the origin of the mass \documentclass[12pt]{minimal }
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\begin{document}$$m_0$$\end{document}m0 .
a tempting guess would be to see \documentclass[12pt]{minimal }
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\begin{document}$$m_0$$\end{document}m0 as related to the energy stored in the electric field .
this idea , however , seems in conflict with the results from minkowski space - time , where the energy of a point - like charged field is defined as \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal { e}_e= 4\pi \int _ 0^{\infty } \mathrm{d}r r^2 q^2/8\pi r^4 $ $ \end{document}ee=40drr2q2/8r4 , and diverges as \documentclass[12pt]{minimal }
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\begin{document}$$r\rightarrow 0$$\end{document}r0 .
historically , this divergence was circumvented by replacing maxwell electrodynamics ( 8) by born infeld theory , whose lagrangian reads , in determinantal form,21\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathcal { l}_{bi}= \beta ^2\left ( \sqrt{-\vert g_{\mu \nu } + \beta ^{-1 } f_{\mu \nu } \vert } - \sqrt{-g } \right ) .
\end{aligned}$$\end{document}lbi=2-|g+-1f|--g.this modification yields a total finite energy for the electromagnetic field,22\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } e_e^{bi}=n_{bi } q^{3/2}(c\beta ^2)^{1/4 } , \end{aligned}$$\end{document}eebi = nbiq3/2(c2)1/4,where \documentclass[12pt]{minimal }
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\begin{document}$$n_{bi}=\frac{\pi ^{3/2 } } { 3\gamma ( 3/4)^2}\approx 1.23605$$\end{document}nbi=3/23(3/4)21.23605 . given
that in our gravitational model the function \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document}r is bounded to \documentclass[12pt]{minimal }
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\begin{document}$$r\ge r_c$$\end{document}rrc , the electric energy could be somehow regularized by the non - trivial topological structure of the space - time . since for an electric field in minkowski space
the action can be written as \documentclass[12pt]{minimal }
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\begin{document}$$s_\mathrm{maxwell}= \int \mathrm{d}t \times \mathcal { e}_e$$\end{document}smaxwell=dtee , to estimate the total electric energy in a gravitational scenario we propose to evaluate the total action as a means to get \documentclass[12pt]{minimal }
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\begin{document}$$s=\int \mathrm{d}t \times ( \mathcal { e}_g+\mathcal { e}_e)$$\end{document}s=dt(eg+ee ) . by doing this
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\begin{document}$$\begin{aligned } s=\frac{4\pi r_c^3}{l_\epsilon ^2 \kappa ^2}\alpha \int \mathrm{d}t , \end{aligned}$$\end{document}s=4rc3l22dt , where \documentclass[12pt]{minimal }
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\begin{document}$$\alpha = \int _
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\begin{document}$$\alpha = \frac{\sqrt{2}\pi ^{3/2 } } { 3\gamma ( 3/4)^2}\approx 1.74804=1/\delta _ 1^*=\sqrt{2 } n_{bi}$$\end{document}=23/23(3/4)21.74804=1/1=2nbi . with simple manipulations
, one finds that the total energy can be written as \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal { e}_t\equiv ( \mathcal { e}_g+\mathcal { e}_e)= 2m_0c^2 \delta _
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\begin{document}$$2$$\end{document}2 stems from the need to integrate on both sides of the wormhole . remarkably , this result is finite , regardless of the value of \documentclass[12pt]{minimal }
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\begin{document}$$\delta _ 1$$\end{document}1 , which implies that the total energy is insensitive to the presence of curvature divergences .
we note that these objects , with their charge having a topological origin and their mass being generated by the electric field , naturally realize the idea of geon ( self - consistent solutions of the sourceless gravito - electromagnetic field equations ) originally introduced by wheeler .
when \documentclass[12pt]{minimal }
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\begin{document}$$\delta _ 1=\delta _ 1^*$$\end{document}1=1 , from ( 17 ) , it is easily seen that the mass spectrum can be written as24\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } m_0=n_{bi}\left ( \frac{n_q}{n_q^c}\right ) ^{3/2}m_p \left ( \frac{l_p}{l_\epsilon } \right ) ^{1/2 } , \end{aligned}$$\end{document}m0=nbinqnqc3/2mplpl1/2,where \documentclass[12pt]{minimal }
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\begin{document}$$n_q \equiv q / e$$\end{document}nqq / e represents the number of charges ( with \documentclass[12pt]{minimal }
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\begin{document}$$e$$\end{document}e being the electron charge ) , \documentclass[12pt]{minimal }
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\begin{document}$$n_q^c=\sqrt{2/\alpha _ { e.m.}}\approx 16.55$$\end{document}nqc=2/e.m.16.55 ( with \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ { e.m.}$$\end{document}e.m .
the fine structure constant ) is the critical number of charges and plays an important role in the existence or not of event horizons [ see paragraph below ] , \documentclass[12pt]{minimal }
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\begin{document}$$m_p$$\end{document}mp is the planck mass , and \documentclass[12pt]{minimal }
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\begin{document}$$l_p$$\end{document}lp is the planck length .
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\begin{document}$$l_\beta ^2\equiv ( 4\pi /\kappa ^2 c\beta ^2)$$\end{document}l2(4/2c2 ) , we can write the expression for \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal { e}_{bi}$$\end{document}ebi given above as25\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \frac{e_{bi}}{c^2}=\sqrt{2}n_{bi}\left ( \frac{n_q}{n_q^c}\right ) ^{3/2}m_p \left ( \frac{l_p}{l_\beta } \right ) ^{1/2 } , \end{aligned}$$\end{document}ebic2=2nbinqnqc3/2mplpl1/2,which , up to a factor \documentclass[12pt]{minimal }
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\begin{document}$$\sqrt{2}$$\end{document}2 , is identical to ( 24 ) with the replacement of the electromagnetic scale \documentclass[12pt]{minimal }
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\begin{document}$$l_\beta $ $ \end{document}l by the gravitational scale \documentclass[12pt]{minimal }
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\begin{document}$$l_\epsilon $ $ \end{document}l. remarkably , ( 24 ) was derived using the maxwell electromagnetic lagrangian .
thus , besides regularizing the geometry , the eibi gravitational theory also regularizes the matter sector in a way almost identical to its electromagnetic counterpart .
a very important aspect of the \documentclass[12pt]{minimal }
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\begin{document}$$\delta _ 1=\delta _ 1^*$$\end{document}1=1 solutions is that , as can be graphically verified ( see for details ) , for \documentclass[12pt]{minimal }
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\begin{document}$$n_q\le n_q^c$$\end{document}nqnqc there is no event horizon , which implies stability of that sector of the theory against hawking radiation . for \documentclass[12pt]{minimal }
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\begin{document}$$n_q > n_q^c$$\end{document}nq > nqc the horizon exists and its location almost coincides with the prediction of gr .
this means that black holes , understood as objects with an event horizon , can be continuously connected with horizonless configurations lying in the lowest part of the charge and mass spectrum .
such states can be naturally identified as black hole remnants and their existence could have deep theoretical implications for the information loss problem and the hawking evaporation process .
note , in addition , that for these remnants the surface \documentclass[12pt]{minimal }
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\begin{document}$$z=1$$\end{document}z=1 is timelike and \documentclass[12pt]{minimal }
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\begin{document}$$s=2m_0c^2\int \mathrm{d}t$$\end{document}s=2m0c2dt coincides with the action of a point - like particle at rest , which suggests that they possess particle - like properties .
in order to test the robustness of the results obtained so far against quantum corrections in the matter sector , one can work within the effective lagrangians approach and consider the coupling of the eibi gravity model to some nonlinear theory of electrodynamics .
the born infeld electromagnetic lagrangian ( 21 ) is a well - motivated choice which , in turn , allows one to find exact analytical solutions .
one then finds that the global qualitative picture provided by maxwell s theory is preserved but with relevant quantitative differences .
in particular , following the same procedure as in the maxwell case , the mass spectrum ( 24 ) turns now into26\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } m_0^{bi}=\left ( \frac{4\gamma } { 1 + 4\gamma } \right ) ^{1/4}m_0 , \end{aligned}$$\end{document}m0bi=41 + 41/4m0,where \documentclass[12pt]{minimal }
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\begin{document}$$\gamma = ( l_\epsilon /l_\beta ) ^2$$\end{document}=(l/l)2 . if the quantum effects of the matter manifest themselves much earlier than the quantum effects of gravity , i.e. , if \documentclass[12pt]{minimal }
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\begin{document}$$l_\beta \gg l_\epsilon $ $ \end{document}ll , then \documentclass[12pt]{minimal }
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\begin{document}$$m_0^{bi}$$\end{document}m0bi may become much smaller than \documentclass[12pt]{minimal }
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\begin{document}$$m_0$$\end{document}m0 .
in particular , if \documentclass[12pt]{minimal }
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\begin{document}$$l_\epsilon = l_p$$\end{document}l=lp and \documentclass[12pt]{minimal }
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\begin{document}$$\beta $ $ \end{document} is pushed to the limits of validity of the effective lagrangians scheme of quantum electrodynamics , \documentclass[12pt]{minimal }
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\begin{document}$$\beta \sim 10^{18}$$\end{document}1018 , then \documentclass[12pt]{minimal }
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\begin{document}$$\gamma \sim ( 10^{-17})^4$$\end{document}(10 - 17)4 and the mass spectrum for \documentclass[12pt]{minimal }
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\begin{document}$$m_0^{bi}$$\end{document}m0bi becomes \documentclass[12pt]{minimal }
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\begin{document}$$17$$\end{document}17 orders of magnitude smaller than the original \documentclass[12pt]{minimal }
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\begin{document}$$m_0$$\end{document}m0 .
this means that the mass spectrum may drop from \documentclass[12pt]{minimal }
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\begin{document}$$m_0\sim m_p\sim 10^{19}$$\end{document}m0mp1019 gev down to \documentclass[12pt]{minimal }
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\begin{document}$$m_0^{bi}\sim 10 ^ 2 $ $ \end{document}m0bi102 gev .
though more accurate descriptions of the matter sector might alter these numbers , the fact is that new quantum gravity phenomenology within the reach of current particle accelerators arises within a purely four - dimensional scenario .
we have shown that for spherically symmetric charged systems eibi theory recovers the gr predictions for \documentclass[12pt]{minimal }
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\begin{document}$$r\gg r_c$$\end{document}rrc .
the theory , however , changes the microstructure of the space - time replacing the gr singularity by a wormhole .
though curvature divergences may exist at \documentclass[12pt]{minimal }
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\begin{document}$$r = r_c$$\end{document}r = rc , their role is uncertain , since they affect neither the properties of the flux through the wormhole nor the finiteness of the total electric energy .
the theory makes definite predictions as regards the existence of black hole remnants and their mass spectrum , with non - trivial implications for the hawking evaporation process , the information loss problem , and potentially new dark matter candidates . | we show that electrically charged solutions within the eddington - inspired born
infeld theory of gravity replace the central singularity by a wormhole supported by the electric field . as a result ,
the total energy associated with the electric field is finite and similar to that found in the born infeld electromagnetic theory . when a certain charge - to - mass ratio is satisfied , in the lowest part of the mass and charge spectrum the event horizon disappears , yielding stable remnants .
we argue that quantum effects in the matter sector can lower the mass of these remnants from the planck scale down to the tev scale . | Introduction
Theory and field equations
Electrovacuum solutions
EiBI as quadratic gravity
Charge without charges and mass without masses
Horizons and remnants
Coupling of BI matter
Conclusions and outlook | recovering the idea of the determinantal form of the gravitational action suggested by eddington [ 6 , 7 ] , an eddington - inspired born
infeld action ( eibi ) for the gravitational field has been introduced recently [ 8 , 9 ] . we take advantage of this property to explore in detail the internal structure of the electrovacuum solutions of the theory and find that the central singularity is generically replaced by a wormhole supported by the electric field . the wormhole structure turns out to be crucial to regularize the total energy stored in the electric field , which occurs in a way that resembles the original born infeld electromagnetic theory . among the solutions of the theory ,
there exist a family ( characterized by a certain charge - to - mass ratio ) for which curvature invariants are finite everywhere . these solutions , whose mass exactly coincides with the energy contained in the electric field , lose the event horizon when the number of charges drops below a critical value , \documentclass[12pt]{minimal }
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\begin{document}$$n_q^c\sim 16$$\end{document}nqc16 , yielding remnants which are not affected by hawking s quantum instability . the mass spectrum of these remnants can be lowered from the planck scale ( \documentclass[12pt]{minimal }
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\begin{document}$$\sim 10^{19}$$\end{document}1019 gev ) down to the tev scale if quantum matter corrections are considered . the connection equation ( 4 ) is formally identical to that found in the palatini version of gr and implies that \documentclass[12pt]{minimal }
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\begin{document}$$g(r)$$\end{document}g(r ) encodes the electric field contribution . one then finds that , in general , there exist curvature divergences at \documentclass[12pt]{minimal }
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\begin{document}$$*f$$\end{document}f is the two - form dual to faraday s tensor . remarkably , this result is finite , regardless of the value of \documentclass[12pt]{minimal }
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\begin{document}$$\delta _ 1$$\end{document}1 , which implies that the total energy is insensitive to the presence of curvature divergences . we note that these objects , with their charge having a topological origin and their mass being generated by the electric field , naturally realize the idea of geon ( self - consistent solutions of the sourceless gravito - electromagnetic field equations ) originally introduced by wheeler . this means that black holes , understood as objects with an event horizon , can be continuously connected with horizonless configurations lying in the lowest part of the charge and mass spectrum . in order to test the robustness of the results obtained so far against quantum corrections in the matter sector , one can work within the effective lagrangians approach and consider the coupling of the eibi gravity model to some nonlinear theory of electrodynamics . the born infeld electromagnetic lagrangian ( 21 ) is a well - motivated choice which , in turn , allows one to find exact analytical solutions . though more accurate descriptions of the matter sector might alter these numbers , the fact is that new quantum gravity phenomenology within the reach of current particle accelerators arises within a purely four - dimensional scenario . the theory , however , changes the microstructure of the space - time replacing the gr singularity by a wormhole . | [
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ceramide , the initial product of the sphingomyelin cycle , functions as a key component in the regulation of various cellular functions like differentiation , proliferation , apoptosis , and inflammation [ 1 , 2 ] . in the past years , ceramide-1-phosphate ( cer-1-p ) , a metabolite of ceramide and sphingomyelin , also gained more attention and turned out to be a powerful bioactive sphingolipid .
cer-1-p has been implicated as a regulator of different cellular processes , like mitosis , apoptosis , phagocytosis , and inflammation [ 3 , 4 ] . in mice lacking cerk ,
until now , cerk is the only mammalian enzyme known to phosphorylate ceramide , but the residual cer-1-p levels in tissues of the cerk - deficient mice , indicate the existence of alternative pathways to generate cer-1-p .
cerk was first described in 1989 as a ca - dependent lipid kinase and cloned in 2002 by sugiura et al .
cerk appears to associate with ( endo)membranes via a pleckstrin domain [ 9 , 10 ] and is highly selective for the d - erythro configuration of ceramides [ 8 , 11 ] .
cerk activity is commonly determined using a radioactivity - based assay [ 711 ] based on [ -p]atp .
after extraction of lipids , the amount of radiolabeled ceramide-1-phosphate in the organic phase is determined by directly counting or by tlc followed by autoradiography and quantitation [ 7 , 8 ] . to avoid the use of radioactivity , we intended to develop a fluorescence - based cerk assay , sensitive enough to be employed for cellular work and whose format would be suitable or could be adapted to search for cerk inhibitors via hts .
based on previous work , showing that truncated ceramides such as c2-ceramide ( n - acetyl - sphingenine ) and c6-ceramide ( n - hexanoyl - sphingenine ) , when presented bound to albumin , are well recognized by human cerk [ 8 , 13 ] , we tested a fluorescent analogue , namely , n-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl - sphingenine ( nbd - c6-ceramide , nbd - c6-cer ) , in which nbd is coupled to sphingenine via an 6-aminohexanoic acid linker ( see figure 1 ) .
this lipid was introduced several years ago by pagano and coworkers for the study of sphingolipid metabolism and shown to be metabolized in a similar way as ceramide , being incorporated into nbd - sphingomyelin and nbd - cerebrosides . during these and
subsequent studies its phosphorylation , as far as we are aware of , was never detected or described , likely due to the very low activity of cerk compared to the other ceramide utilizing pathways .
also when using ceramides containing a shorter n - acyl chain ( truncated ceramides ) , phosphorylation by intact cells is difficult to reveal , requiring p - uploading of the cells as reported for neutrophils [ 15 , 16 ] , cerebellar granule cells , and hela cells .
upon overexpression of cerk , detection of intracellular formed truncated ceramide-1-p is facilitated [ 8 , 13 ] .
here , we studied the kinetics , revealing that nbd - c6-cer is a good substrate for cerk , both in vitro and in vivo , and developed a simple solid phase extraction scheme to measure cerk activity ( this work was presented in a preliminary form at the lki oncoforum meeting , february 15 , 2008 , leuven ( belgium ) ( h. van overloop and p. p.
van veldhoven , development of hts - assays for enzymes acting on bioactive sphingolipids , new players in chemotherapy resistance .
part i. ceramide kinase ) ) . at the start of this work , graf et al . reported that nbd - c6-cer is phosphorylated when given to cerk - expressing cos-1 cells , as revealed by tlc of cellular extracts and scanning . independently , don and rosen have described in the meantime a cerk assay using the same substrate but based on liquid / liquid extraction , phase separation , and transfer of the upper phase for analysis .
recombinant hscerk was expressed in top10f ' e. coli cells transformed with plasmid ppvv072 , coding for a ( his)6-tagged fusion of hscerk , as described before .
the harvested bacteria , resuspended in pbs ( 25 ml/100 ml culture ) containing a mix of protease inhibitors , were sonicated on ice ( branson sonifier b115 , microtip ) , followed by a clearing step ( 10,000 g for 10 min ) .
aliquots of the supernatant were frozen in liquid nitrogen , stored at 80c and diluted 1/15 in pbs containing protease inhibitors before use . compared to the pelleted fraction , the specific activity of the soluble lysate fraction is three fold lower ( 9.3 nmol / minmg protein at 100 m c6-cer/40 m bsa for the batch used in these experiments ) , but it was considered to more compatible with spe work up .
recombinant human sphingosine kinase 1 ( hssphk1 ) was obtained from top10f ' cells transformed with plasmid psg003 as described before .
n-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoic acid ( 33 mol , molecular probes ) , dissolved in 3 ml anhydrous dimethylformamide and activated with carbonyldiimidazole ( 40 mol , fluka ) , was mixed with d , erythro - sphingenine ( 33 mol ; acros organics ) , dissolved in 2 ml dimethylformamide , and stirred overnight at room temperature . after drying the reaction mixture , the amides were phase - separated in chloroform / methanol / water ( 1/1/0.9 ) , the lower phase was dried , dissolved in 0.5 ml 33% methylamine in ethanol / water ( 7/3 ) , and heated to 70c for 90 min to remove any formed o - acylated products . after evaporating the hydrolysis mixture ,
nbd - c6-cer was extracted and further purified by preparative tlc ( silica g60 ; merck ) in solvent a ( chloroform / methanol / acetic acid , 93/7/1 , v / v ) .
stock solutions were standardized by nitrogen determination ( yield 63% ) and purity , based on fluoroscanning ( storm 840 with blue led ( 450 nm ) , ge healthcare ) after tlc separation in solvent b ( chloroform / acetone / methanol / acetic acid / water , 10/4/3/2/1 , v / v ) was 89% , substantially better than that of commercially obtained nbd - c6-cer ( sigma ; avanti polar lipids ) .
nbd - c6-ceramide-1-phosphate ( nbd - c6-cer-1-p ) was prepared by phosphorylation of homemade nbd - cer using bacterially expressed hscerk under the conditions described before .
briefly , the reaction mixture containing 100 m nbd - c6-cer , solubilized with ethanol / bsa , was incubated with excess recombinant hscerk for 1 h at 37c . after acidic phase separation
, the lower phase was dried and the phosphorylated nbd - c6-cer was further purified by preparative tlc ( silica g60 ; merck ) in solvent b , followed by elution with chloroform / methanol / water ( 5/5/1 , v / v ) .
stock solutions were standardized by organic phosphate content ( yield 85% ) . based on fluoroscanning of the phosphate ester , tlc - separated in solvent b ,
purity was estimated at > 82% ( based on main spot ; due some streaking , actual purity is higher ) , being slightly better compared to the phosphate ester prepared from commercially obtained nbd - c6-ceramide ( avanti polar lipids , > 75% ) . to document the separation of nbd - labeled cerk substrate and product , 100 l cerk assay mixture containing nbd - c6-cer and nbd - c6-cer-1-p ( both at 5 m final concentration ) was mixed with 300 l methanol and applied on a 25 mg nh2-spe column ( varian ) , which had been conditioned with methanol and water .
total fluorescence of the flow - through and eluted fractions was measured by fluorimetry ( ex 465 nm ; em 535 nm ; tecan infinite 200 ) .
the amount of nbd - c6-cer and nbd - c6-cer-1-p in the different fractions was estimated by drying them , redissolving the residu in chloroform / methanol ( 1/1 , v / v ) , followed by separation on silica g tlc ( solvent b ) , and scanning of the fluorescent spots . to prepare the reaction mixture for the fluorescent cerk assay ,
largely based on previous work , nbd - c6-cer was dissolved in ethanol and mixed with 4 volumes of bsa ( resulting in a molar ceramide / bsa ratio of 2.5 ) , followed by addition of reaction mixture up to 75 l and a 25 l aliquot of recombinant cerk or cell lysate .
final concentrations were 5 m nbd - c6-cer1 mm atp50 mm mops / naoh ph 7.23 mm mgcl240 mm naf1 mm dithriothreitol100 m orthovanadate .
after 10 min at 37c , the reaction was stopped by addition of 300 l methanol .
the mixture was applied to an nh2-column ( 25 mg , chromabond multi-96 , macherey - nagel ) , which had been activated with 0.5 ml methanol followed by 0.5 ml water .
after washing the column with 800 l methanol containing 2% formic acid , followed by 100 l methanol containing 0.5 m trifluoroacetic acid ( tfa ) , nbd - c6-cer-1-p was eluted into a black fia 96-well plate ( greiner ) with 250 l methanol containing 3 m tfa .
fluorescence was measured in a multi - reader ( ex 465 nm , em 535 nm ; tecan infinite 200 ) .
the amount of nbd - c6-cer-1-p in the eluate was calculated based on a calibration curve with nbd - c6-cer-1-p at concentrations of 0 to 5 m treated simultaneously and equivalently with the samples .
fluorescence measurements from calibration curves fitted to a linear equation ( r = 0.998 ; n = 12 ) .
cerk activity based on radioactivity was measured as described before , but using 1 mm [ -p]-atp ( ge healthcare ) and reducing the volumes to obtain similar conditions as in the fluorescent assay described above .
as substrates c6-ceramide ( 40 m ) or nbd - c6-cer ( 5 m ) , both bound to bsa ( molar ceramide / bsa ratio = 2.5 ) , were used .
the substrate specificity of cerk , documented by different groups [ 7 , 8 , 11 ] , reveals that the n - acyl chain can be shortened up to two carbons [ 7 , 8 , 13 ] and that the presence of a bulky group in this chain is tolerated ( unpublished data ) .
previously , we documented that natural ceramides ( with a long n - acyl chain ) are better recognized by cerk when presented in a micellar form , whereas less hydrophobic ceramide analogues , either with a shortened base or a truncated acyl chain , display better activity in the presence of bsa [ 8 , 13 ] . in agreement with this observation , nbd - c6-cer , containing the polar nbd moiety ,
was well phosphorylated when bound to bsa , but substantially less when incorporated in octylglucoside / cardiolipin micelles , certainly at low substrate concentration ( 30 fold , less at 25 m ) . similarly ,
use of triton x-100 or chaps micelles resulted in low activities ( data not shown ) .
using bacterially expressed human cerk in a radiometric assay and separation of the products by tlc , a km of 6 m was obtained for nbd - c6-cer , bound to bsa ( data not shown ) . despite the bulky nbd - group ,
this value is about 2.5-fold lower than the km for the nonfluorescent c6-ceramide ( 14 m ) , obtained under the same assay conditions . a similar km , but based on fluorimetry , was obtained by rosen and don ( 1 m in presence of 7 m bsa ) , who also documented the inhibitory action of triton x-100 .
given the low km , it appeared justified to evaluate some procedures to separate nbd - c6-cer from nbd - c6-cer-1-p in order to develop a nonradioactive cerk assay .
in addition , we attempted to avoid tedious liquid extraction steps and aimed for a procedure compatible with hts .
hence , to separate the fluorescent substrate and product , after some trials on reversed phase ( nbd - c6-cer and its phosphate ester were both retained on c18-spe ( varian ) at 50% methanol , without or with addition of acid ( 0.5% ( v / v ) tfa ) or base ( 0.5% ( v / v ) triethylamine ) , but coeluted with pure methanol ; similar findings were obtained with oasis - hlb ( waters ) columns , except that nbd - c6-cer-1-p was recovered in the flow through under alkaline conditions . ) and ion exchange - spe , we focused finally on nh2-spe columns .
these systems have been employed to separate phospholipids , and lipids containing a primary phosphate group , such as phosphatidate and phosphorylated phosphoinositides , do bind strongly [ 16 , 17 ] .
elution of these phosphate esters is achieved by increasing the polarity of the eluting solvent and addition of strong acids such as phosphoric acid or hcl .
given the reported dependence of the fluorescence of nbd - derivatives with regard to the lipid environment , solvent composition , polarity and ph [ 2527 ] and low quantum yield in water , in a first step the fluorescence of nbd - c6-cer and its phosphate ester was evaluated in solvents and in the presence of acids . for solvent we focused on the use of methanol , being suitable for dissolution of these lipids , compatible with plastic ( e.g. , polystyrene ) , and having a low toxicity and moderate cost . as shown in figure 2(a ) , increasing the amount of water in a methanolic nbd - c6-cer-1-p solution , strongly reduced the fluorescence .
compared to pure methanol as solvent , fluorescence dropped to 3% in a solution containing 50% of water .
the influence of different acids , present at 0.5 n final concentration , on the fluorescence of nbd - c6-cer-1-p , dissolved in methanol , is shown in figure 2(b ) .
both hcl and h2so4 caused a severe drop in fluorescence ( less than 20% compared to the neutral methanolic solution ) .
the influence of tfa and h3po4 was less drastic , since more than 90% of fluorescent signal remained ( figure 2(b ) ) .
this effect seems to be related to the strength of the acid , although it is not strictly linearly related to the acid dissociation constant .
when applied in pure methanol or methanol containing up to 25% water , nbd - c6-cer-1-p was quantitatively retained on nh2-spe systems .
both tfa and h3po4 displaced nbd - c6-cer-1-p from the nh2-phase , but tfa was chosen for further optimisation .
less volume was required to elute the phosphate ester compared to methanol containing h3po4 in equal normality , and the decrease in nbd fluorescence at increasing tfa concentrations was rather small ( figure 2(c ) ) , both factors improving the sensitivity of the assay . by increasing the tfa concentration to 3 n
, it was possible to elute the bound lipid in a small volume , 250 l when using 25 mg spe . finally , we analyzed how well nbd - c6-cer and its phosphate ester could be separated using small spe systems ( 25 mg nh2-spe , column format or contained in a 96-well format ) .
hereto , a mixture containing nbd - c6-cer and nbd - c6-cer-1-p and with a similar composition as the cerk assay mixture , was diluted with methanol ( 75% final concentration ) , and applied to the nh2-spe column , which had been conditioned with methanol and water .
after washing the column with methanol containing 2% formic acid ( fa ) ( 800 l ) and 0.5 n tfa ( 100 l ) , nbd - c6-cer-1-p was eluted from the column using methanol containing 3 n tfa ( 250 l ) .
this resulted in a good separation between nbd - c6-cer and nbd - c6-cer-1-p ( if employing a larger spe format , adjust volumes accordingly ; e.g. for 100 mg nh2-spe columns , nbd - c6-cer - p is eluted with a similar yield with 1 ml 3 n tfa in methanol ( data not shown ) ) .
when loading equal amounts of both lipids , more than 99% of nbd - c6-cer was present in the flow - through and wash fractions ( 99.6 0.01% , mean sem , n = 5 ) , and less than 0.4% was found in the acidic eluate ( 0.36 0.02% ) , based upon fluorescence - scanning of the tlc - separated fractions ( data not shown ) .
fluorescence in the tfa - eluate was almost completely ( 99.4 0.08% ) associated with nbd - c6-cer-1-p ( figure 3(a ) ) .
apparently , these numbers do not change when varying the relative amounts of both fluorescent lipids initially present ( figure 3(b ) ) : about 96% of the total amount of nbd - c6-cer-1-p is present in the eluted fraction ( 95.6 0.42% ) .
the use of tfa at lower normality than 3 n resulted in a lower recovery for nbd - c6-cer-1-p ( data not shown ) .
higher normalities did not improve the recoveries , but resulted in lower sensitivity because of increased quenching . having established optimum spe - separation conditions , the kinetics of cerk were reevaluated with the new assay .
the assay conditions were similar to the traditional radiometric assay , but assay volume was reduced to 100 l and atp concentration was fixed at 1 mm .
in addition , soluble cerk was used to avoid potential spe clogging . to halt the reaction ,
3 volumes of methanol were added , followed by transfer of the mixture to the 96-well spe - plate .
a km value of 4 m was obtained ( figure 4(a ) ) , comparable to the km of 6 m obtained for nbd - c6-cer with the radiometric assay ( see above ) . for economical use of the substrate ,
its concentration was fixed at 5 m ( standard assay conditions ) . at this concentration , although close to the km , phosphorylation continued at a linear rate till about 50% of the substrate was converted ( figure 4(b ) ) .
likewise , when varying the amounts of cerk , production of nbd - c6-cer-1-p proceeded linearly till about 50% of nbd-
hence , the amount of nbd - c6-cer converted under the standard conditions is a good measure for cerk activity . as documented in figure 5 , the fluorescent assay is very convenient to document cerk activity in cultured cells .
upon overexpression of cerk , a 50-fold increase in kinase activity ( 1.40 versus 0.029 nmol / minmg protein ) was measured .
similar values were obtained using the radiometric assay ( 1.57 versus 0.030 nmol / minmg protein ) , supporting the use of the fluorescence assay as a valuable alternative .
the detection limit of the assay is estimated at 10 pmol nbd - c6-cer-1-p , meaning that cerk activities corresponding to 1 pmol / min can be measured ( or less if incubation time is prolonged ) .
when relying on a [ -p]-atp based assay , and labelling of the produced ceramide-1-p to 1001000 dpm , this would require an input of 0.55 ci / assay at 1 mm atp and 100 l assay volume .
since cerk activity in most tissues and cells is quite low ( < 40 pmol / minmg protein ) , our assay will facilitate further work on cerk and its regulation .
in addition , the low km implicates that nbd - c6-cer might be a handy substrate for in vivo cerk measurements .
indeed , when added to cerk - expressing cultured cells , formation of nbd - c6-cer-1-p can be followed by tlc analysis of the cellular lipid extracts ( data not shown ) , in full agreement with data reported by bornancin and coworkers . by comparing the scanned intensities against the fluorescence of nbd - c6-cer-1-p standards , tlc analysis of cell extracts
is another means to calculate cerk activity ( data not shown ) . during our attempts to publish this work ,
don and rosen reported on the same ceramide analogue as a substrate but their assay was based on either tlc spotting for the micellar assay or for the bsa - based assay , extraction , followed by phase separation and transfer of the upper phase for analysis ; the latter was done in a 96-well format .
omission of a liquid - liquid extraction step clearly speeds up the assay and allows for other formats like multiwell plates used in hts . to simulate an hts ,
a commercial library was tested in a 96 well format . to increase the chance to get some positive hits , we selected hereto a protein kinase inhibitor library given that their targets rely on the same cofactor as cerk . to show specificity ,
the same library was also tested on another lipid kinase , human sphingosine kinase 1 .
cerk activity was blocked ( more than 95% inhibition ) by ag-494 , ag-825 , bay11 - 7082 , 2-hydroxy-5-(2,5-dihydroxybenzylamino)benzoic acid , hypericin , indirubin-3-monoxime ( and its 6-bromo - derivative ) , piceatannol , quercetin , ro31 - 8220 , rottlerin , d - erythro - sphingosine ( and d - threo - sphinganine ; not shown ) , u-0126 , staurosporine , and zm449829 ( at 500 m ) .
sphk1 was clearly inhibited by fewer compounds , the most potent being ag-494 , piceatannol , and quercetin . for the most potent cerk - inhibitors ,
ic50 values were determined : u-0126 ( 4 m ) , followed by 6-bromo - indirubin-3-oxime ( 9 m ) and hypericin and rottlerin ( both 19 m ) .
u-0126 is considered to be a selective map kinase kinase inhibitor and ic50 values are indeed lower ( 72 nm for mek1 ; 58 nm for mek2 ) .
a few ceramide analogues and lipophilic amides , partly commercially obtained , partly homemade , were also tested as substrate and/or inhibitor ( full list available upon request ) .
compounds that were not phosphorylated but strong inhibitory were further evaluated . from this screen ,
we retained fenretinide { ( n-4-hydroxyphenyl)retinamide ) ; ic50 1.1 m } en amg-9810 { ( e)-3-(4-t - butylphenyl)-n-(2,3-dihydrobenzo [ b]dioxin-6-yl)acrylamide ; ic50 1.4 m}. these compounds are known to influence other biological processes .
the retinoic acid receptor , slows the growth of transformed cells , and induces apoptosis in cultured cells ( effective concentrations 110 m ) , the latter likely via increasing dihydroceramide levels .
amg-9810 is known as an antagonist of the vanilloid / tprv1 receptor ; its endogenous ligand , anandamide , is also a fatty amide . summarizing , by further analyzing the substrate spectrum of cerk
, it was shown that nbd - c6-ceramide is a suitable substrate , allowing for a fluorescence based cerk measurement . by combining this substrate with the use of nh2-spe to isolate the product ,
a straightforward assay has been developed , useful for basic research ( 100 mg spe ) and adaptable to hts for cerk inhibitors / activators ( 25 mg spe-96 well format ) .
, which can be miniaturized to 1,536 well plates . however , this assay is based on chemiluminescent detection of the disappearing atp and c12-ceramide as substrate .
hence , an extra control is required for the effect of compounds on the coupling reaction / enzymes . moreover ,
this assay is not applicable to crude cell / tissue lysates given the interfering presence of atpases and other phosphatase activities and the low cerk activity . | ceramide kinase ( cerk ) has been implicated in important cellular processes such as inflammation and apoptosis .
its activity is usually measured using radiolabeled ceramide or [ -32p]-atp , followed by extraction , thin - layer chromatography , and detection of the formed labeled ceramide-1-phosphate . to eliminate the use of radioactivity
, we developed similarly but independently from the approach by don and rosen ( 2008 ) , a fluorescence - based ceramide kinase assay , using n-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl - sphingenine ( nbd - c6-ceramide ) as substrate .
its km value ( 4 m ) was comparable to that of n - hexanoyl - sphingenine ( c6-ceramide ) .
the produced fluorescent nbd - c6-ceramide-1-phosphate was captured by means of solid - phase extraction on an aminopropyl phase , resulting in a fast and sensitive cerk measurement . by performing this assay in a 96-well format ,
it is also suitable for high - throughput screening ( hts ) to search for cerk modulators .
a limited screen revealed that some protein kinase inhibitors ( e.g. , u-0126 ; ic50 4 m ) and ceramide analogues ( e.g. , fenretinide , amg-9810 ; ic50 1.1 m ) affect cerk in vitro . | 1. Introduction
2. Materials and Methods
3. Results and Discussion | ceramide , the initial product of the sphingomyelin cycle , functions as a key component in the regulation of various cellular functions like differentiation , proliferation , apoptosis , and inflammation [ 1 , 2 ] . in the past years , ceramide-1-phosphate ( cer-1-p ) , a metabolite of ceramide and sphingomyelin , also gained more attention and turned out to be a powerful bioactive sphingolipid . cer-1-p has been implicated as a regulator of different cellular processes , like mitosis , apoptosis , phagocytosis , and inflammation [ 3 , 4 ] . cerk activity is commonly determined using a radioactivity - based assay [ 711 ] based on [ -p]atp . to avoid the use of radioactivity , we intended to develop a fluorescence - based cerk assay , sensitive enough to be employed for cellular work and whose format would be suitable or could be adapted to search for cerk inhibitors via hts . based on previous work , showing that truncated ceramides such as c2-ceramide ( n - acetyl - sphingenine ) and c6-ceramide ( n - hexanoyl - sphingenine ) , when presented bound to albumin , are well recognized by human cerk [ 8 , 13 ] , we tested a fluorescent analogue , namely , n-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl - sphingenine ( nbd - c6-ceramide , nbd - c6-cer ) , in which nbd is coupled to sphingenine via an 6-aminohexanoic acid linker ( see figure 1 ) . this lipid was introduced several years ago by pagano and coworkers for the study of sphingolipid metabolism and shown to be metabolized in a similar way as ceramide , being incorporated into nbd - sphingomyelin and nbd - cerebrosides . also when using ceramides containing a shorter n - acyl chain ( truncated ceramides ) , phosphorylation by intact cells is difficult to reveal , requiring p - uploading of the cells as reported for neutrophils [ 15 , 16 ] , cerebellar granule cells , and hela cells . here , we studied the kinetics , revealing that nbd - c6-cer is a good substrate for cerk , both in vitro and in vivo , and developed a simple solid phase extraction scheme to measure cerk activity ( this work was presented in a preliminary form at the lki oncoforum meeting , february 15 , 2008 , leuven ( belgium ) ( h. van overloop and p. p.
van veldhoven , development of hts - assays for enzymes acting on bioactive sphingolipids , new players in chemotherapy resistance . independently , don and rosen have described in the meantime a cerk assay using the same substrate but based on liquid / liquid extraction , phase separation , and transfer of the upper phase for analysis . the harvested bacteria , resuspended in pbs ( 25 ml/100 ml culture ) containing a mix of protease inhibitors , were sonicated on ice ( branson sonifier b115 , microtip ) , followed by a clearing step ( 10,000 g for 10 min ) . compared to the pelleted fraction , the specific activity of the soluble lysate fraction is three fold lower ( 9.3 nmol / minmg protein at 100 m c6-cer/40 m bsa for the batch used in these experiments ) , but it was considered to more compatible with spe work up . n-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoic acid ( 33 mol , molecular probes ) , dissolved in 3 ml anhydrous dimethylformamide and activated with carbonyldiimidazole ( 40 mol , fluka ) , was mixed with d , erythro - sphingenine ( 33 mol ; acros organics ) , dissolved in 2 ml dimethylformamide , and stirred overnight at room temperature . after drying the reaction mixture , the amides were phase - separated in chloroform / methanol / water ( 1/1/0.9 ) , the lower phase was dried , dissolved in 0.5 ml 33% methylamine in ethanol / water ( 7/3 ) , and heated to 70c for 90 min to remove any formed o - acylated products . stock solutions were standardized by nitrogen determination ( yield 63% ) and purity , based on fluoroscanning ( storm 840 with blue led ( 450 nm ) , ge healthcare ) after tlc separation in solvent b ( chloroform / acetone / methanol / acetic acid / water , 10/4/3/2/1 , v / v ) was 89% , substantially better than that of commercially obtained nbd - c6-cer ( sigma ; avanti polar lipids ) . nbd - c6-ceramide-1-phosphate ( nbd - c6-cer-1-p ) was prepared by phosphorylation of homemade nbd - cer using bacterially expressed hscerk under the conditions described before . after acidic phase separation
, the lower phase was dried and the phosphorylated nbd - c6-cer was further purified by preparative tlc ( silica g60 ; merck ) in solvent b , followed by elution with chloroform / methanol / water ( 5/5/1 , v / v ) . based on fluoroscanning of the phosphate ester , tlc - separated in solvent b ,
purity was estimated at > 82% ( based on main spot ; due some streaking , actual purity is higher ) , being slightly better compared to the phosphate ester prepared from commercially obtained nbd - c6-ceramide ( avanti polar lipids , > 75% ) . to document the separation of nbd - labeled cerk substrate and product , 100 l cerk assay mixture containing nbd - c6-cer and nbd - c6-cer-1-p ( both at 5 m final concentration ) was mixed with 300 l methanol and applied on a 25 mg nh2-spe column ( varian ) , which had been conditioned with methanol and water . the amount of nbd - c6-cer and nbd - c6-cer-1-p in the different fractions was estimated by drying them , redissolving the residu in chloroform / methanol ( 1/1 , v / v ) , followed by separation on silica g tlc ( solvent b ) , and scanning of the fluorescent spots . to prepare the reaction mixture for the fluorescent cerk assay ,
largely based on previous work , nbd - c6-cer was dissolved in ethanol and mixed with 4 volumes of bsa ( resulting in a molar ceramide / bsa ratio of 2.5 ) , followed by addition of reaction mixture up to 75 l and a 25 l aliquot of recombinant cerk or cell lysate . the mixture was applied to an nh2-column ( 25 mg , chromabond multi-96 , macherey - nagel ) , which had been activated with 0.5 ml methanol followed by 0.5 ml water . after washing the column with 800 l methanol containing 2% formic acid , followed by 100 l methanol containing 0.5 m trifluoroacetic acid ( tfa ) , nbd - c6-cer-1-p was eluted into a black fia 96-well plate ( greiner ) with 250 l methanol containing 3 m tfa . as substrates c6-ceramide ( 40 m ) or nbd - c6-cer ( 5 m ) , both bound to bsa ( molar ceramide / bsa ratio = 2.5 ) , were used . previously , we documented that natural ceramides ( with a long n - acyl chain ) are better recognized by cerk when presented in a micellar form , whereas less hydrophobic ceramide analogues , either with a shortened base or a truncated acyl chain , display better activity in the presence of bsa [ 8 , 13 ] . in agreement with this observation , nbd - c6-cer , containing the polar nbd moiety ,
was well phosphorylated when bound to bsa , but substantially less when incorporated in octylglucoside / cardiolipin micelles , certainly at low substrate concentration ( 30 fold , less at 25 m ) . using bacterially expressed human cerk in a radiometric assay and separation of the products by tlc , a km of 6 m was obtained for nbd - c6-cer , bound to bsa ( data not shown ) . despite the bulky nbd - group ,
this value is about 2.5-fold lower than the km for the nonfluorescent c6-ceramide ( 14 m ) , obtained under the same assay conditions . a similar km , but based on fluorimetry , was obtained by rosen and don ( 1 m in presence of 7 m bsa ) , who also documented the inhibitory action of triton x-100 . given the low km , it appeared justified to evaluate some procedures to separate nbd - c6-cer from nbd - c6-cer-1-p in order to develop a nonradioactive cerk assay . hence , to separate the fluorescent substrate and product , after some trials on reversed phase ( nbd - c6-cer and its phosphate ester were both retained on c18-spe ( varian ) at 50% methanol , without or with addition of acid ( 0.5% ( v / v ) tfa ) or base ( 0.5% ( v / v ) triethylamine ) , but coeluted with pure methanol ; similar findings were obtained with oasis - hlb ( waters ) columns , except that nbd - c6-cer-1-p was recovered in the flow through under alkaline conditions . ) these systems have been employed to separate phospholipids , and lipids containing a primary phosphate group , such as phosphatidate and phosphorylated phosphoinositides , do bind strongly [ 16 , 17 ] . elution of these phosphate esters is achieved by increasing the polarity of the eluting solvent and addition of strong acids such as phosphoric acid or hcl . given the reported dependence of the fluorescence of nbd - derivatives with regard to the lipid environment , solvent composition , polarity and ph [ 2527 ] and low quantum yield in water , in a first step the fluorescence of nbd - c6-cer and its phosphate ester was evaluated in solvents and in the presence of acids . for solvent we focused on the use of methanol , being suitable for dissolution of these lipids , compatible with plastic ( e.g. , polystyrene ) , and having a low toxicity and moderate cost . as shown in figure 2(a ) , increasing the amount of water in a methanolic nbd - c6-cer-1-p solution , strongly reduced the fluorescence . this effect seems to be related to the strength of the acid , although it is not strictly linearly related to the acid dissociation constant . both tfa and h3po4 displaced nbd - c6-cer-1-p from the nh2-phase , but tfa was chosen for further optimisation . less volume was required to elute the phosphate ester compared to methanol containing h3po4 in equal normality , and the decrease in nbd fluorescence at increasing tfa concentrations was rather small ( figure 2(c ) ) , both factors improving the sensitivity of the assay . by increasing the tfa concentration to 3 n
, it was possible to elute the bound lipid in a small volume , 250 l when using 25 mg spe . finally , we analyzed how well nbd - c6-cer and its phosphate ester could be separated using small spe systems ( 25 mg nh2-spe , column format or contained in a 96-well format ) . hereto , a mixture containing nbd - c6-cer and nbd - c6-cer-1-p and with a similar composition as the cerk assay mixture , was diluted with methanol ( 75% final concentration ) , and applied to the nh2-spe column , which had been conditioned with methanol and water . after washing the column with methanol containing 2% formic acid ( fa ) ( 800 l ) and 0.5 n tfa ( 100 l ) , nbd - c6-cer-1-p was eluted from the column using methanol containing 3 n tfa ( 250 l ) . this resulted in a good separation between nbd - c6-cer and nbd - c6-cer-1-p ( if employing a larger spe format , adjust volumes accordingly ; e.g. when loading equal amounts of both lipids , more than 99% of nbd - c6-cer was present in the flow - through and wash fractions ( 99.6 0.01% , mean sem , n = 5 ) , and less than 0.4% was found in the acidic eluate ( 0.36 0.02% ) , based upon fluorescence - scanning of the tlc - separated fractions ( data not shown ) . apparently , these numbers do not change when varying the relative amounts of both fluorescent lipids initially present ( figure 3(b ) ) : about 96% of the total amount of nbd - c6-cer-1-p is present in the eluted fraction ( 95.6 0.42% ) . the use of tfa at lower normality than 3 n resulted in a lower recovery for nbd - c6-cer-1-p ( data not shown ) . to halt the reaction ,
3 volumes of methanol were added , followed by transfer of the mixture to the 96-well spe - plate . a km value of 4 m was obtained ( figure 4(a ) ) , comparable to the km of 6 m obtained for nbd - c6-cer with the radiometric assay ( see above ) . for economical use of the substrate ,
its concentration was fixed at 5 m ( standard assay conditions ) . likewise , when varying the amounts of cerk , production of nbd - c6-cer-1-p proceeded linearly till about 50% of nbd-
hence , the amount of nbd - c6-cer converted under the standard conditions is a good measure for cerk activity . upon overexpression of cerk , a 50-fold increase in kinase activity ( 1.40 versus 0.029 nmol / minmg protein ) was measured . similar values were obtained using the radiometric assay ( 1.57 versus 0.030 nmol / minmg protein ) , supporting the use of the fluorescence assay as a valuable alternative . the detection limit of the assay is estimated at 10 pmol nbd - c6-cer-1-p , meaning that cerk activities corresponding to 1 pmol / min can be measured ( or less if incubation time is prolonged ) . when relying on a [ -p]-atp based assay , and labelling of the produced ceramide-1-p to 1001000 dpm , this would require an input of 0.55 ci / assay at 1 mm atp and 100 l assay volume . indeed , when added to cerk - expressing cultured cells , formation of nbd - c6-cer-1-p can be followed by tlc analysis of the cellular lipid extracts ( data not shown ) , in full agreement with data reported by bornancin and coworkers . during our attempts to publish this work ,
don and rosen reported on the same ceramide analogue as a substrate but their assay was based on either tlc spotting for the micellar assay or for the bsa - based assay , extraction , followed by phase separation and transfer of the upper phase for analysis ; the latter was done in a 96-well format . to simulate an hts ,
a commercial library was tested in a 96 well format . to increase the chance to get some positive hits , we selected hereto a protein kinase inhibitor library given that their targets rely on the same cofactor as cerk . cerk activity was blocked ( more than 95% inhibition ) by ag-494 , ag-825 , bay11 - 7082 , 2-hydroxy-5-(2,5-dihydroxybenzylamino)benzoic acid , hypericin , indirubin-3-monoxime ( and its 6-bromo - derivative ) , piceatannol , quercetin , ro31 - 8220 , rottlerin , d - erythro - sphingosine ( and d - threo - sphinganine ; not shown ) , u-0126 , staurosporine , and zm449829 ( at 500 m ) . for the most potent cerk - inhibitors ,
ic50 values were determined : u-0126 ( 4 m ) , followed by 6-bromo - indirubin-3-oxime ( 9 m ) and hypericin and rottlerin ( both 19 m ) . a few ceramide analogues and lipophilic amides , partly commercially obtained , partly homemade , were also tested as substrate and/or inhibitor ( full list available upon request ) . from this screen ,
we retained fenretinide { ( n-4-hydroxyphenyl)retinamide ) ; ic50 1.1 m } en amg-9810 { ( e)-3-(4-t - butylphenyl)-n-(2,3-dihydrobenzo [ b]dioxin-6-yl)acrylamide ; ic50 1.4 m}. the retinoic acid receptor , slows the growth of transformed cells , and induces apoptosis in cultured cells ( effective concentrations 110 m ) , the latter likely via increasing dihydroceramide levels . amg-9810 is known as an antagonist of the vanilloid / tprv1 receptor ; its endogenous ligand , anandamide , is also a fatty amide . summarizing , by further analyzing the substrate spectrum of cerk
, it was shown that nbd - c6-ceramide is a suitable substrate , allowing for a fluorescence based cerk measurement . by combining this substrate with the use of nh2-spe to isolate the product ,
a straightforward assay has been developed , useful for basic research ( 100 mg spe ) and adaptable to hts for cerk inhibitors / activators ( 25 mg spe-96 well format ) . however , this assay is based on chemiluminescent detection of the disappearing atp and c12-ceramide as substrate . | [
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alzheimer 's disease ( ad ) is one of the biggest challenges of current research for new drugs [ 1 - 3 ] .
in addition to cancer , diabetes , rheumatoid arthritis and other chronic inflammatory and neurodegenerative diseases , ad is characterized by multiple factors involving physiological , biochemical , chemical mediators operating concurrently with , caused by the same or different pathways [ 4 , 5 ] . in recent years
, advances in biochemistry , neuro - pharmacology and other biological fields have been responsible for new insights in the high complexity multifactorial pathophysiological hallmarks of ad .
thus , the development of innovative and effective multi - target directed drugs for ad represents a new paradigm in the discovery and drug design . until then
, the question that guided the planning of a new drug was what is the best target for the treatment or prevention of a disease ? which supported , since the decade of 90 , the origin of the reductionist strategy . by this concept
, a drug should have its action limited to individual genes , a single protein or enzyme and a specific ligand to a target , following the current paradigm
[ 1 , 4 , 6 ] . the limitations imposed by the low therapeutic efficacy against most of these diseases , coupled with the apparent reduction in the approval of new bioactive chemical entities , despite significant investments in the pharmaceutical industry , have reinforced the need to look up new strategies for planning and discovery of more effective and safer drugs . in this context ,
three therapeutic strategies are possible : the first would be the use in combination of multiple active ingredients , like the associations of natural ingredients used for centuries in traditional chinese medicine ; or cocktails of drugs , such as used in the treatment of the acquired immunodeficiency syndrome ( aids ) and cancer .
another alternative would be a combination of more than one active ingredient in the same formulation . in a third and more recent
approach , it would be a single molecule assembling structural requirements to allow molecular recognition of more than one target simultaneously , featuring a multi - target drug [ 4 , 5 ] .
when multiple targets related to the same pathology are associated with distinct biochemical cascades , the active ligand can be also called as a symbiotic compound .
with the increase in average life expectancy worldwide , neurodegenerative diseases such as ad have attracted great attention , especially in more developed countries . as a progressive ,
disabling and incurable disease , patients die within average of 10 years after its installation .
the age is its main risk factor , with a prevalence of 0.7% among individuals 60 - 65 years of age and about 40% in the age groups above 90 years [ 8 , 9 ] . in 2012 , about 5.4 million of american people were carriers of ad .
despite the very imprecise estimates available , it is believed that by 2050 , over 16 million americans will have ad , with an annual cost that could exceeds u$ 1.1 trillion [ 11 , 12 ] .
other estimates point to a global epidemic of ad , reaching 26 million people by 2050 , if a cure is not yet discovered and available [ 10 , 14 ] .
the increase in life quality , as a natural consequence of recent advances in medicine and technological development , has been reflected directly in the increased longevity worldwide .
the united nations ( un ) believes that the age of aging began in 1975 and this trend is expected to extend until 2025 [ 14 , 15 ] . according to the brazilian institute of geography and statistics
, europe occupies the 1 position in the proportion of the population above 60 years ( 19.8% ) , followed by north america ( 16.3% ) .
ad is a degenerative disorder of the central nervous system ( cns ) and is one of the most common dementia among the population above age 65 .
ad is characterized by a progressive loss of memory , cognition , motor and functional capacity , gradually undermining social behavior and ability to perform routine tasks such as feeding , personal hygiene and interpersonal relationships .
although the etiology of ad is not completely understood , it is well established that operation of multiple interconnected factors are related to the installation , development and evolution of the disease .
apparently , physiological deregulation in some brain regions , which the origin remains not entirely understood , is responsible for an overproduction of amyloid peptide ( a ) . then , - e -secretase enzymes abnormally cleave a , producing insoluble fragments of 39 - 43 amino acid residues . fragments a1 - 42 , even at low concentrations , seem to be more prone to oligomerization and formation of insoluble neurotoxic aggregates , the so - called amyloid plaques , and are the main therapeutic target advocated by the amyloid hypothesis [ 5 , 6 , 17 ] .
a secondary event due to neurotoxicity of amyloid plaques is the hyperphosphorylation of the neuronal microtubule constitutive tau protein .
this abnormal phosphorylation of tau leads to structural collapse of microtubules and the consequent release of tau protein fragments , which take the form of insoluble fibrillar coiled , depositing intracellular as neurofibrillary tangles [ 6 , 12 , 18 - 20 ] .
these two biochemical factors acting together lead to accentuate changes in the cns homeostasis and function , causing numerous cellular processes and organelles activation , acting as starting points of a complex process of interconnected physiological damage , also leading to neuronal death .
although most studies have emphasized the neurotoxicity of amyloid plaques , recent evidences indicate that increasing the concentration of soluble amyloid oligomers generated by overproduction of a can be also responsible for increasing neuronal injury [ 6 , 21 ] .
mitochondria are the major intracellular targets of soluble a oligomers ( sa ) . in excess ,
sa eventually interfere with normal function of mitochondria , causing overproduction of reactive oxygen species ( ros ) , inhibition of cellular respiration , atp production and damage in mitochondrial structure [ 21 , 22 ] .
it seems that , the deleterious effects of sa the mitochondria are the result of changes in homeostasis and intracellular ca signaling , as evidenced by the induction of massive influx of ca in cultured neurons , causing its concentration increasing in mitochondria and neuronal apoptosis .
accumulation of calcium in mitochondria causes opening of the mitochondrial permeability transition pore ( mptp ) , a wide mitochondrial membrane channel that allows the passage of large molecules bidirectional uncontrolled , resulting in disintegration of organelles and functional structure .
recent studies have shown that the dephosphorylation of neurofibrillary tangles of tau protein was able to restore its ability to bind to microtubules neurons , indicating that the kinetic mechanisms that regulate the phosphorylation / dephosphorylation process are altered in ad .
the nature of the protein kinases , phosphatases and tau sites involved in these lesions was recently unveiled , suggesting that activation of protein phosphatase fosfoseril or fosfotreonil ( pp-2a ) or inhibition of glycogen synthase - kinase 3 ( gsk-3 ) protein as cyclin - dependent kinase 5 ( cdk5 ) may be required for inhibiting the degeneration caused by neurofibrils in ad . in recent years
, many other hypotheses have been proposed to explain the complexity and multifactorial pathogenesis of ad , including oxidative stress , disruption of homeostasis by metal ions and neuroinflammation [ 19 , 24 , 25 ] .
currently , oxidative stress is considered one of the major causative factors of ad , unifying a number of other sequential or individual pathophysiologic events .
oxidative damage in the brain of ad patients is a result of excessive production of free radicals induced by a , functional alteration in mitochondria , inadequacy in energy supplement , production of inflammatory mediators and alteration of antioxidant defenses [ 26 , 28 ] .
modulation of cellular oxidative processes is closely related to the redox properties of some metals .
change in concentration of such ions can lead to oxidative stress and increased ros production .
cupper ( cu ) and zinc ( zn ) and other metal ions influence the processes of protein aggregation , a critical step in many neurodegenerative diseases . in the case of ad ,
amyloid precursor protein ( app ) and a are able to form complexes with and reduce cu , which forms a high affinity complex with a , promoting their aggregation .
furthermore , in vitro studies showed that a neuro - toxicity is dependent on the catalytic generation of h2o2 , which is enhanced by the presence of a-cu complexes .
in addition , cu , zn and fe are present in amyloid plaques in brains with ad , which can be dissolved by the action of metal chelating substances [ 6 , 20 , 29 ] .
the production of a also depends on the bioavailability of cholesterol in nerve cells , since the activity balance of the - and -secretases is related to the lipid composition of cells .
high concentrations of cholesterol into the cells lead to an increase in amyloidogenic app process by -secretase , whereas at lower levels , cholesterol metabolism stimulates the increasing the physiological -secretase action on app .
the hypothesis that the control of plasmatic cholesterol levels would be beneficial for treating ad has been demonstrated by using anticholesterolemic drugs , such as statins , which act as hmg - coa reductase inhibitors .
finally , deposition of a fragments and neurofibrils , coupled with the uncontrolled production of ros , are crucial for the installation of a neuroinflammatory process , with the same complexity observed in peripheral tissues .
the scope and relevance of this process in the establishment and development of chronic da have been demonstrated in several recent studies in the literature [ 30 - 33 ] . among all the brain cells , microglia appears to have fundamental importance in cns inflammation associated with ad .
these cells could be activated by a and modulate the production of cytokines , chemokines and neurotoxins that are highly neurotoxic , contributing to neuronal degeneration [ 19 , 30 - 33 ] .
given the variety of factors associated with the onset , progress and severity of ad , increasing their degree of pathophysiological complexity , and associated to the inefficiency of the current therapeutic arsenal available , it becomes unavoidable to adopt a new concept for the rational design of new drugs against da . in this context ,
drug candidate prototypes with dual mode of action were the first attempts to look up ligands recognized by more than one molecular target or more than one site on the same macromolecular target .
currently , a new strategy of multi - target directed ligands is gaining special attention in the scientific community , which has been seeking in molecular hybridization , a tool for designing new molecular patterns . these molecular hybrids could lead to the identification of new bioactive chemical entities with selectively affinity for multiple targets , preferably in different biochemical cascades .
therefore , these innovative ligands could play a singular role in the advance of a broadly and more efficient therapy , and perhaps , in the cure of ad .
this new approach , considering multifunctional drugs or ligands directed at multiple targets associated with the same disease ( symbiotic drugs ) , has gained special importance and introducing a new thinking approach in the design of new drug candidates for ad .
molecular hybridization of pharmacophoric subunits of different bioactive molecules , is the main tool for structural planning , and have provided the discovery of numerous ligands with multiple properties , including antioxidant , neuroprotective , metal chelation , anti - inflammatory , anti - a aggregation , and cholinesterase and secretase inhibitory activities .
therefore , a set of other potential therapeutic targets have been studied for simultaneous intervention , seeking for a more efficacy in relieve the symptoms and slowing the progression of ad , and why not , its definitive control and cure [ 34 - 38 ] .
since 2005 , the literature has shown several results from applying this innovative approach of drug design .
drugs such as donepezil , tacrine and rivastigmine [ 39 , 40 ] have been used as structural models for molecular hybridization with bioactive synthetic and natural products such as curcumine [ 41 , 42 ] , berberine [ 43 , 44 ] , 8-hydroxyquinolines , among others in the search for new chemical entities with multiple bioactive properties useful for the treatment of ad .
as one example of this strategy , berberine ( 1 ) was used as a basic skeleton for the construction of a series of hybrid compounds with molecular subunits that include melatonin ( 2 ) and ferulic acid ( 3 , fig .
1 ) . the aiming was to obtain new derivatives with antioxidant properties and anti - a aggregation , but with reduced inhibitory potency of acetylcholinesterase ( ache ) .
the hybrid compound hidroquine 4 demonstrated the best ability for suppression of a aggregation , plus excellent antioxidant effect and reasonable ability to inhibit acetylcholinesterase ( ache ) and butirylcholinesterase ( buche ) .
1 ) was identified as a potent inhibitor of ache , with strong antioxidant properties , confirming the multi - target potential of these substances in comparison to berberine , the original prototype .
2 ) , a 1,4-benzoquinone - poliamine hybrid compound , was reported by cavalli and co - workers as a promising drug prototype for ad .
the structural design of compound 8 was based on a polyamine core , derived from coproctamine ( 9 ) , a dimeric agent with anticholinesterasic and antimuscarinic . aiming to add to these properties , the ability to neutralize ros ( reactive oxygen species ) and neuroprotection , authors proposed the insertion of a 1,4-benzoquinone , from coenzyme q10 ( coq10 ) , a fragment with a recognized potent mitochondrial antioxidant property and a protector of the hippocampus neurons against a(140)induced neurotoxicity .
the insertion of the benzoquinone nucleus contributes to increase the neuroprotective activity , because a hydrophobic and planar system is generated , which is able in principle to bind a [ 42 , 47 , 48 , 50 ] .
memoquin ( 8) exhibited ability to inhibit ache ( ic50= 1.55 nm ) , almost 15-fold more potent than the reference compound ( coproctamine , 9 ) .
memoquin ( 8) also inhibited ache - induced a1 - 40 aggregation with an ic50 of 28.3 m and the self - induced a 142 aggregation with an ic50 of 5.93 m .
in addition , compound 8 is also capable to inhibit bace-1 ( secretase ) activity in range nanomolar concentrations ( ic50= 108 nm ) and showed antioxidant properties , reducing the formation of free radicals by 44.1% .
it was demonstrated that the 1,4-benzoquinone moiety can be reduced in vivo to the 1,4-dihydroquinone form , increasing its antioxidant potential and scavenging properties .
finally , in vivo assays showed the potential of memoquin to restore the cholinergic deficit and successfully reduce a expression and accumulation , also reducing hyperphosphorylation of tau protein [ 48 , 49 ] . considering all these multi - target profile of memoquin ( 8) , bolognesi and co - workers reported in 2009 the design of new hybrid lipoic acid - memoquin ( 11 ) derivatives inspired on the memoquin structure and lipoic acid structure which is a potent antioxidant .
in addition to the observed biological properties of memoquin , compound 12 also exhibited a good neuroprotective effect against oxidative stress , greater than the reference control lipoic acid ( 10 ) .
3 ) is the major constituent of curcuma longa , a plant species used in indian traditional medicine , with a-aggregation inhibitory properties have been used as a structural model for molecular to generate new analogs of memoquin with enhanced neuroprotective ache and buche inhibitory properties . among the novel memoquin hybrid derivatives , compounds 16 and 17 ( fig .
3 ) showed the best multiple - target action profile with selective inhibitory activities of ache and buche , with modulatory effects on the amyloid aggregation and inhibition of neurofibrils formation .
4 ) is the first approved ches inhibitor by the fda for the treatment of ad .
however , due to some undesirable side effects this drug have its therapeutic use restricted and , therefore , the search for more effective and secure tacrine derivatives is still of interest . in recent years
, many studies have focused on the combining the potent ches inhibitory properties of tacrine with additional biological properties to search for new mtdls .
based on this strategy , contelles and co - workers synthesized a new series of 4h - pyrano [ 2,3-b ] quinolone derivatives ( 20 ) , planned by molecular hybridization of tacrine fused to a benzylamino-3-pyridyl ring moiety , aiming to obtain new acheis with an innovative molecular scaffold and to explore its pharmacophoric contributions to additional neuroprotective properties and modulatory effects on voltage - dependent ca channels .
in general , the new tacrine - hybrid compounds showed to be better selective for ache inhibition than tacrine .
it seems that the substitution at c-20 position of the 3-pyridyl ring system ( compounds 22 , 23 , 24 ) was deleterious for ache inhibition as observed for derivative 21 ( x= h ) .
in contrast , compounds 22 - 24 showed a good neuroprotective profile , as the derivative 23 exhibiting the best neuroprotective effect , with only 46% of cell - death suppression .
5 ) , was one the first homodimers reported in the literature with increased ache affinity , exhibiting a 1000-fold higher inhibitory potency than tacrine .
this best inhibitory profile is due to a dual simultaneously interaction with active and peripheral sites of ache .
further studies , demonstrated that this compound was also capable to inhibit the ache - induced a aggregation with an ic50 of 41.7 m .
because of this symbiotic profile , compound 25 has been described as an important structural model for rational design of novel mtdls . in 2007 ,
bolognesi and co - workers proposed modifications on the structure of bis-(7)-tacrine ( 25 ) with an insertion of a spacer subunit with functional groups capable to of chelate metals , which are involved in the degenerative process .
for this purpose , compounds 26 ( bw284c51 ) and ambenonium ( 27 ) were selected as model prototypes due to their cholinesterase inhibitory activity and their singular structural feature , carrying carbonyl and oxalamide functionalities with could lead to a the desired metal chelation property ( fig .
the multifunctional compounds maintained a potent acheis in the nanomolar range , showed additional inhibitory effect of ache - induced amyloid- aggregation .
the results showed the ability for chelation of cu and fe ions and suggest that these compounds might act against ad by a chelation mechanism .
minarini and co - workers also explored the structure of bis-(7)-tacrine ( 25 ) , in an attempt to obtain the hybrid derivative cystamine bis-(7)-tacrine ( 31 ) , with an insertion of the cystamine moiety 32 as a linker between two acrydine subunits of the bis - tacrine framework of 25 ( fig .
this study revealed the hybrid dimer cystamine - tacrine 31 was able to inhibit ache ( ic50= 5.04 nm ) , buche ( ic50= 4.23 nm ) , self - a aggregation ( ic50= 24.2 m ) and ache - induced a aggregation ( 52,6% ) in the same range of the reference compound , with additional neuroprotective effect on sh - sy5y cell line against h2o2-induced oxidative injury , with low toxicity .
fang and co - workers reported in 2008 the synthesis and pharmacologic evaluation of a new series of tacrine - ferulic acid hybrid derivatives ( 33 ) ( fig .
they planned a new structural pattern by the connection of ferulic acid ( 34 ) to the structure of tacrine ( 18 ) via an alkylenediamine as a linker side chain .
this approach was used aiming to obtain new multi - target hybrid compounds which could conjugate the ache inhibitory activity originating from the tacrine template and the antioxidant activity from the ferulic acid moiety .
the compounds showed lower antioxidant activity than ferulic acid , but all hybrids tested showed moderate to good antioxidant activity .
particularly , compounds 35 showed a 10-fold higher ache inhibitory activity ( ic50= 4.4 nm ) than tacrine .
enzymatic kinetics studies suggest that this compound possess high affinity binding to the peripheral anionic site ( pas ) of ache and , therefore , could inhibit a-peptide fibril formation . in another approach , rosini and
co - workers also used tacrine as a model for the planning of a new series of hybrid compounds ( 36 - 39 ) based on the structural feature of carvedilol ( 35 ) .
this compound had been identified by endows a significant neuroprotective efficacy , probably related to its modulatory action as a low - affinity antagonist of n - methyl - d - aspartate ( nmdar ) .
thus , carbazole moiety of 35 was elected as a probable structural pharmacophoric subunit responsible by its antioxidant properties and efficient inhibitory activity of a-fibril formation ( fig .
biological evaluation revealed that all compounds showed effective ache - inhibiting activity in a nanomolar range , being more potent than tacrine .
particularly , compounds 36 - 39 showed an adequate biological profile for multi - target drugs , inhibiting ache activity and also capable to block in vitro a self - aggregation , ache - mediated a-aggregation , nmdars antagonistic effect and to reduce cerebral oxidative stress . in another approach , tacripyrines ( 40 )
were designed by combining tacrine with a calcium antagonist such as nimodipine ( 41 ) ( fig .
besides acting as an inhibitor of ache , these compounds also showed significant neuroprotective effects .
in particular , compound 42 ( ic50= 105 15 nm ) , proved to be moderately active in models ache - induced and a-self aggregation models ( 30 and 35% inhibition , respectively ) .
further kinetic studies and molecular modeling data suggest that compound 42 could interact with the pas of the ache , exerting its inhibitory properties by noncompetitive mode .
additionally , the most of the compounds showed moderate ca channel blocking effect and therefore the compounds are neuroprotective agents . a novel series of tacrine caffeic acid hybrids ( 43 ) were designed by combining the structure of caffeic acid ( 44 ) and tacrine ( 19 ) ( fig . 10 ) as multitarget - directed ligands against alzheimer s disease .
in vitro studies showed that most of the target molecules exhibited significant antioxidant activities on oxygen radical absorbance capacity method ( orac ) .
the hybrid molecules showed to be good metal chelators and also exhibited higher antioxidant capacity than the caffeic acid with values of 2.759.37 m .
in particular , compound 45 exhibited the greatest ability in inhibition of self- or ache - induced -amyloid140 aggregation , as well as showed potent neuroprotective effects against h2o2- and glutamate- induced cell death , with low toxicity in ht22 cells .
further molecular modeling and enzymatic kinetics studies clarified that these inhibitors may act in two binding sites of ache , which could explain the inhibitory effect exerted over a-aggregation . in another work ,
wang and collaborators reported a series of tacrine - based hybrid compounds , containing an additional pharmacophoric subunit 4-phenyl-2-aminothiazole with different spacer subunits .
the pharmacophoric groupment 4-phenyl-2-aminothiazole was elected based on data form literature that suggest this functionality as responsible for inhibitory effects on tau protein aggregation and on a self - aggregation , neuroprotective and anti - inflammatory .
11 ) were potent inhibitors of cholinesterases with ic50 values ranging from 5.78 0.05 to 7.14 0.01 nm for ache , and from 5.75 0.03 to 10.35 0.15 nm for buche .
moreover , a structure - activity relationship study was conducted to study the influence on the type of middle linker and substitutions at 40-position of 4-phenyl-2-aminothiazole .
the results were indicative that the length of middle linker affected the ache inhibitory potency . compound 48 ,
with the largest linker subunit exhibited showed the greater potency in ache inhibition ( ic50= 7.14 0.01 nm ) .
data from kinetic studies have shown that this compound is a mixed - type inhibitor and could bind simultaneously at the catalytic and the peripheral sites of ache . most of phenylthiazole
tacrine hybrids showed a good inhibitory potency on a142 self - aggregation , although the activity was lower when compared with subunit 4-phenyl-2-aminothiazole .
additionally , compound 48 displayed a blockade effect on ca overload in the primary cultured cortical neurons . according to the studies of simoni and co - workers , a series of hybrid derivatives of galanthamine ( 49 ) , an ache inhibitors and memantine ( 50 ) , a noncompetitive nmda receptor antagonist , showed potent ache inhibitory activities with multiple neuroprotective effects in vitro ( fig .
some of these hybrid compounds were also capable to prevent glutamate - induced neurotoxicity by moderately blocking glutamate receptor nmda subtype and neuronal cell death via nmda receptor - dependent effect at nanomolar concentrations ( e.g. , memagal ( 52 ) , ic50 = 0.28 nm ) .
these molecules were the first examples reported of dual ache / nr2b drug prototype candidates .
a new series of 30 bivalent -carboline derivatives ( 53 ) was synthesized by rook and co - workers based on previous studies that disclosed monovalent -carbolines as potent dual ache / buche inhibitors ( fig .
13 ) . during an investigative study for the ability of these -carboline derivatives to interact with multiple targets
, the researchers also found that several compounds were also potent nmda receptor blockers , in addition to the potent inhibitory properties of both ache / buche enzymes .
a structure - activity study revealed that the inhibitory activity of quaternary n9-bivalent -carbolines was increased compared to their monovalent analogues .
compound 55 showed the higher ache inhibitory activity ( ic50= 0.5 nm ) and proved to be a potent inhibitor of the transient induced - glutamate ca with an ic50= 1.4 m , which is 4-fold more potent than memantine ( ic50= 5 5.6 m ) in the same assay conditions . in another approach for drug design of mdtls ,
rizzo and co - workers reported a new series of hybrid molecules based on the structure of the ache inhibitor n - methyl - n - benzylamine ( 56 ) , and compound skf-64346 ( 57 ) , a benzofuran derivative with good inhibitory properties of a-fibril formation , using a heptyloxy moiety as a linker subunit .
all compounds evaluated have shown to be good ache and a-fibril formation inhibitors , reducing the a neurotoxicity .
compound 58 showed an ic50 of 1.82 m in buche inhibition , being 5.7-fold more selective toward buche than ache ( ic50 = 10.5 m ) .
furthermore , this compound was also able to inhibit a self - aggregation with an ic50 of 13 m and showed a neuroprotective effect of 63% at 30 mm [ 60 , 61 ] .
some years later , aiming to improve its pharmacological profile the researchers modified the original structure of compound 58 by variation of the spacer subunit between the 2-arylbenzofuran and the n - methyl - n - benzylamine moieties .
they proposed a variation in the length of the spacer subunit , modification in the substituents in 3 position of the benzofuran scaffold and change of the n - methyl - n - benzylamine heptyloxy side chain from the para to meta position ( fig .
these structural modifications led to a new series 59 that exhibited an increased in activity for most of the modified compounds . among the most active compounds of the series 59 , compounds
60 and 61 exhibited the best buche inhibitory properties , with carbamate 62 also showing higher dual inhibitory activity of ache and buche in comparison to rivastigmine .
in addition , compounds 63 and 64 also showed significant in vitro inhibitory activities on a-fibril formation up to 81% ( fig .
piazza and co - workers also searching for multifunctional drug candidates for ad planned a series dual of inhibitors of human ache ( hache ) and -secretase ( bace-1 ) .
the molecular design was based in a dual binding site ache inhibitor , ap2238 ( 65 ) , which is able to simultaneously interact with both the central and the peripheral anionic sites of ache .
it was synthesized a series of compound 65 derivatives by replacing the methoxy substituents of the coumarin moiety by an amidic chain subunit aiming to extend the activity to bace-1 ( fig .
the positions 6 or 7 of the coumarin moiety were also replaced by a dihalophenyl acid subunit , considering that this moiety emerged as a leitmotif in different bace-1 inhibitors reported in the literature .
all compounds were potent inhibitors , particularly compounds 67 ( ic50
hache = 0.551 m , ( ic50
bace-1= 0.149 m ) , and 65 ( ic50
hache= 0.181 m , ic50
bace-1= 0.150 m ) that exhibited the best inhibitory profile in the target series . biological data clearly indicated that the introduction of a halophenyl alkylamidic subunit on the scaffold of 65 allowed obtaining more potent bace-1 inhibitors , and the presence of substituents in the positions 6 or 7 on the coumarin nucleus seems to equally contribute to the relative binding affinity of this molecular series .
a new family of multitarget molecules able to interact with both ache and buche , as well as with monoamine oxidase ( mao ) a and b , has been synthesized by samadi and collaborators .
rational structural pattern of these compounds considered conjunctive approach by combination of benzylpiperidine and n - propargylamine moieties present in the ache inhibitor donepezil ( 70 ) and the mao inhibitor pf9601n ( 71 ) , respectively , connected by a central pyridine or naphthyridine ring system ( fig .
the most promising derivative 72 showed a potent inhibitory activity of ache ( ic50= 37 nm ) , but less potent than donepezil .
this compound also showed a moderate inhibitory potency of mao - a ( ic50= 41 m ) .
moreover , molecular modeling showed that these inhibitors probably act in two binding sites of ache , and that the length of the spacer subunit is of particular relevance in the modulation of ache inhibition , being crucial for the dual interaction of these molecules with both cas and pas sites the enzyme . in 2011 , another recent publication by bolea and collaborators also reported the synthesis and pharmacological evaluation of a new family of multifunctional molecules capable to interact simultaneously with the ad - related enzymes ache , buche , mao - a and mao - b .
18 ) was based on the molecular hybridization of the benzylpiperidine moiety of donepezil ( 70 ) and the indolyl - propapylamino subunit present in the structure of the mao inhibitor n-(5benzyloxy-1-methyl-1h - indol-2-yl)methyl - n - methylprop-2-yn-1-amine ( 73 ) , connected a central oxy - methylene chain .
compound 75 was identified from series 74 as the most promising ligand , showing a good selectivity and high potency in the inhibition of mao - a with an ic50 = 5.2 nm ( ic50 mao - b = 43.0 nm ) , but less potent and with poor selectivity for the ache ( ic50 = 0.35 m ) and buche ( ic50 = 0.46 m ) .
further molecular modeling and enzymatic kinetics studies clarified that these inhibitors act in two binding sites of ache , which could explain the inhibitory effect over a-self - induced ( 47.8 2.1 % ) and ache - dependent aggregation ( 32.4 7 % ) exerted compound 75 . based on the remarkable capacity of tacrine to interact in the active site of ache , martins and collaborators proposed a new series of tacrine - derived analogues by the insertion of a furo [ 2,3-b ] quinolin-4-amine ( 76 ) and pyrrolo [ 2,3-b ] quinolin-4-amine ( 77 ) subunits ( fig .
pharmacological results revealed that the furanotacrines and pyrrolotacrines showed selective inhibition of buche , in a micromolar range , but these compounds have lower potency than tacrine .
the lead - compound 79 , exhibited the best inhibitory profile with ic50= 0.61 nm and ic50
buche= 0.074 nm , for ache from electrophorus electricus and buche from equine serum , with significant neuroprotective effects against a-induced toxicity in concentrations below 300 nm .
molecular modelling studies have corroborate that the hybrid derivatives containing pyrrole or furan ring subunits interacts with ache and buche and the presence of a phenyl ring at the position 1 of the pyrrole ring is beneficial for both ache and buche inhibition .
taking the structure of donepezil ( 70 ) as a model , huang and co - workers reported in 2012 a new family of derivatives based on the structure of benzylidene - indanone ( 80 ) pharma - cophoric subunit of 70 with the insertion of set of substituted benzene ring systems ( 81 ) ( fig .
20 ) . in vitro evaluation showed that most of the compounds were potent inhibitors of mao - b ( ic50 of 7.540.5 m ) , besides antioxidant ( orac - fl value of 2.759.37 m ) and metal chelator properties .
in addition , they showed a great ability for inhibition self - induced -amyloid aggregation ( 10.580.1% , 20 m ) .
compound 82 , was identified as the most potent inhibitor agent of a142 aggregation ( 80.1% ) and mao - b ( ic50 = 7.5 m ) . moreover ,
compound 82 proved to be an excellent antioxidant and metal chelator agent , being able to inhibit cu - induced a142 aggregation and disassembling the a fibrils .
thus compound 82 is one of the most current example from literature of a genuine mdtl drug prototype candidate for ad treatment .
resveratrol ( 83 ) , a potent phenolic natural plant meta - bolite , with remarkable antioxidant and anti - inflammatory properties , have been recently described as an inhibitor of a aggregation .
thus , its molecular scaffold has been also explored for assembly of innovative mtdls with antioxidant , anti - inflammatory and inhibitory of a aggregation properties .
compounds 86 and 87 were the lead - compounds identified among a series of resveratrol - derived molecular hybrids ( 85 ) , planned by the combination of the catechol pharmacophore of 83 with the well - known metal chelator clioquinol ( 84 ) ( fig .
these two hybrid - compounds showed high potency in the inhibition of self - induced a aggregation ( 86 , ic50 = 7.56 m and 87 , ic50 = 6.51 m ) in comparison to resveratrol ( ic50 = 15.11 m ) . furthermore
, antioxidant assays performed by the orac method , showed the ability of compounds 86 and 87 in decreasing the generation of ros species in 4.72 and 4.70 trolox equivalent , respectively .
these compounds were also capable to disassembling the highly structured a fibrils generated by self- and cu ( ii)-induced a aggregation and exhibited significant inhibitory effect on mao - a and mao - b with a moderate ache inhibition and low neurotoxicity .
the development of drugs for a more effective treatment , and hopefully curing , ad is still a challenge for medicinal chemistry .
the more recent insights about the pathophysiology of ad have proven a complex inter - connected variety of deleterious events , with multiple pathways involving in its etiology and a variety of factors that are associated with the installation , progress and severity of ad .
this complex multifactorial aspect of ad and other neuronal disorders have supported the new paradigm of mtdls ( or one - compound - multi - targets strategy ) as a more rational strategy approach to the development of new drug candidates , than the one - target - one drug concept .
the low efficacy of the therapeutic approach currently employed could be , in part attributed to this reductionist concept , and it becomes inevitable to adopt a new concept for the rational design of new drugs against ad .
therefore , a drug for the treatment of ad will only be effective if modulate multiple targets simultaneously involved in the disease . in the last decade efforts
have being employed in the design and search for new biological chemical entities capable to direct simultaneously with different molecular targets involved in the pathogenesis of ad and many new significantly active and promise molecules have been discovered .
consequently , new drug prototype candidates have being identified and they are the starting point of a revolutionary new way of thinking drug design and some drug candidates are under pre - clinical evaluation . despite none of these mtdl drug candidates have reached clinical phase of development , we hope that in the next few years the system biology approach could effectively contribute to the medicinal chemists in the discovery of innovative chemical entities able to understand and effectively modify the course of this devastating neurodegenerative disorder .
= acetylcholinesterase = butirylcholinesterase = central nervous system = doena de alzheimer = acetilcolinesterase inhibitor = minimum inhibitory concentration of 50% = monoamine oxidase a = monoamine oxidase b = multitarget - directed ligand = n - methyl - d - aspartate = peripheral anionic site = reactive oxygen species | alzheimer s disease ( ad ) is a complex neurodegenerative disorder with a multi - faceted pathogenesis . so far ,
the therapeutic paradigm
one - compound - one - target has failed and despite enormous efforts to elucidate the
pathophysiology of ad , the disease is still incurable .
the multiple factors involved in ad include amyloid aggregation to form insoluble neurotoxic plaques of a ,
hyperphosphorylation of tau protein , oxidative stress , calcium imbalance , mitochondrial dysfunction and deterioration of
synaptic transmission .
these factors together , accentuate changes in the cns homeostasis , starting a complex process of
interconnected physiological damage , leading to cognitive and memory impairment and neuronal death .
a recent approach for the rational design of new drug candidates , also called multitarget - directed ligand ( mtdl )
approach , has gained increasing attention by many research groups , which have developed a variety of hybrid compounds
acting simultaneously on diverse biological targets .
this review aims to show some recent advances and examples of the
exploitation of mtdl approach in the rational design of novel drug candidate prototypes for the treatment of ad . | INTRODUCTION
SOCIO-ECONOMIC IMPACT AND PREVALENCE OF ALZHEIMER'S DISEASE
MULTIFACTORIAL ASPECTS OF ALZHEIMER'S DISEASE
RECENT MULTI-TARGET DIRECTED DRUG CANDIDATES DESIGNED FOR THE TREATMENT OF AD
CONCLUSION
CONFLICT OF INTEREST
ABBREVIATIONS | alzheimer 's disease ( ad ) is one of the biggest challenges of current research for new drugs [ 1 - 3 ] . in addition to cancer , diabetes , rheumatoid arthritis and other chronic inflammatory and neurodegenerative diseases , ad is characterized by multiple factors involving physiological , biochemical , chemical mediators operating concurrently with , caused by the same or different pathways [ 4 , 5 ] . in recent years
, advances in biochemistry , neuro - pharmacology and other biological fields have been responsible for new insights in the high complexity multifactorial pathophysiological hallmarks of ad . thus , the development of innovative and effective multi - target directed drugs for ad represents a new paradigm in the discovery and drug design . until then
, the question that guided the planning of a new drug was what is the best target for the treatment or prevention of a disease ? which supported , since the decade of 90 , the origin of the reductionist strategy . the limitations imposed by the low therapeutic efficacy against most of these diseases , coupled with the apparent reduction in the approval of new bioactive chemical entities , despite significant investments in the pharmaceutical industry , have reinforced the need to look up new strategies for planning and discovery of more effective and safer drugs . in this context ,
three therapeutic strategies are possible : the first would be the use in combination of multiple active ingredients , like the associations of natural ingredients used for centuries in traditional chinese medicine ; or cocktails of drugs , such as used in the treatment of the acquired immunodeficiency syndrome ( aids ) and cancer . in a third and more recent
approach , it would be a single molecule assembling structural requirements to allow molecular recognition of more than one target simultaneously , featuring a multi - target drug [ 4 , 5 ] . when multiple targets related to the same pathology are associated with distinct biochemical cascades , the active ligand can be also called as a symbiotic compound . the age is its main risk factor , with a prevalence of 0.7% among individuals 60 - 65 years of age and about 40% in the age groups above 90 years [ 8 , 9 ] . other estimates point to a global epidemic of ad , reaching 26 million people by 2050 , if a cure is not yet discovered and available [ 10 , 14 ] . the increase in life quality , as a natural consequence of recent advances in medicine and technological development , has been reflected directly in the increased longevity worldwide . according to the brazilian institute of geography and statistics
, europe occupies the 1 position in the proportion of the population above 60 years ( 19.8% ) , followed by north america ( 16.3% ) . ad is a degenerative disorder of the central nervous system ( cns ) and is one of the most common dementia among the population above age 65 . although the etiology of ad is not completely understood , it is well established that operation of multiple interconnected factors are related to the installation , development and evolution of the disease . fragments a1 - 42 , even at low concentrations , seem to be more prone to oligomerization and formation of insoluble neurotoxic aggregates , the so - called amyloid plaques , and are the main therapeutic target advocated by the amyloid hypothesis [ 5 , 6 , 17 ] . a secondary event due to neurotoxicity of amyloid plaques is the hyperphosphorylation of the neuronal microtubule constitutive tau protein . this abnormal phosphorylation of tau leads to structural collapse of microtubules and the consequent release of tau protein fragments , which take the form of insoluble fibrillar coiled , depositing intracellular as neurofibrillary tangles [ 6 , 12 , 18 - 20 ] . these two biochemical factors acting together lead to accentuate changes in the cns homeostasis and function , causing numerous cellular processes and organelles activation , acting as starting points of a complex process of interconnected physiological damage , also leading to neuronal death . it seems that , the deleterious effects of sa the mitochondria are the result of changes in homeostasis and intracellular ca signaling , as evidenced by the induction of massive influx of ca in cultured neurons , causing its concentration increasing in mitochondria and neuronal apoptosis . recent studies have shown that the dephosphorylation of neurofibrillary tangles of tau protein was able to restore its ability to bind to microtubules neurons , indicating that the kinetic mechanisms that regulate the phosphorylation / dephosphorylation process are altered in ad . the nature of the protein kinases , phosphatases and tau sites involved in these lesions was recently unveiled , suggesting that activation of protein phosphatase fosfoseril or fosfotreonil ( pp-2a ) or inhibition of glycogen synthase - kinase 3 ( gsk-3 ) protein as cyclin - dependent kinase 5 ( cdk5 ) may be required for inhibiting the degeneration caused by neurofibrils in ad . in recent years
, many other hypotheses have been proposed to explain the complexity and multifactorial pathogenesis of ad , including oxidative stress , disruption of homeostasis by metal ions and neuroinflammation [ 19 , 24 , 25 ] . currently , oxidative stress is considered one of the major causative factors of ad , unifying a number of other sequential or individual pathophysiologic events . oxidative damage in the brain of ad patients is a result of excessive production of free radicals induced by a , functional alteration in mitochondria , inadequacy in energy supplement , production of inflammatory mediators and alteration of antioxidant defenses [ 26 , 28 ] . in the case of ad ,
amyloid precursor protein ( app ) and a are able to form complexes with and reduce cu , which forms a high affinity complex with a , promoting their aggregation . in addition , cu , zn and fe are present in amyloid plaques in brains with ad , which can be dissolved by the action of metal chelating substances [ 6 , 20 , 29 ] . the production of a also depends on the bioavailability of cholesterol in nerve cells , since the activity balance of the - and -secretases is related to the lipid composition of cells . finally , deposition of a fragments and neurofibrils , coupled with the uncontrolled production of ros , are crucial for the installation of a neuroinflammatory process , with the same complexity observed in peripheral tissues . given the variety of factors associated with the onset , progress and severity of ad , increasing their degree of pathophysiological complexity , and associated to the inefficiency of the current therapeutic arsenal available , it becomes unavoidable to adopt a new concept for the rational design of new drugs against da . in this context ,
drug candidate prototypes with dual mode of action were the first attempts to look up ligands recognized by more than one molecular target or more than one site on the same macromolecular target . currently , a new strategy of multi - target directed ligands is gaining special attention in the scientific community , which has been seeking in molecular hybridization , a tool for designing new molecular patterns . therefore , these innovative ligands could play a singular role in the advance of a broadly and more efficient therapy , and perhaps , in the cure of ad . this new approach , considering multifunctional drugs or ligands directed at multiple targets associated with the same disease ( symbiotic drugs ) , has gained special importance and introducing a new thinking approach in the design of new drug candidates for ad . therefore , a set of other potential therapeutic targets have been studied for simultaneous intervention , seeking for a more efficacy in relieve the symptoms and slowing the progression of ad , and why not , its definitive control and cure [ 34 - 38 ] . drugs such as donepezil , tacrine and rivastigmine [ 39 , 40 ] have been used as structural models for molecular hybridization with bioactive synthetic and natural products such as curcumine [ 41 , 42 ] , berberine [ 43 , 44 ] , 8-hydroxyquinolines , among others in the search for new chemical entities with multiple bioactive properties useful for the treatment of ad . as one example of this strategy , berberine ( 1 ) was used as a basic skeleton for the construction of a series of hybrid compounds with molecular subunits that include melatonin ( 2 ) and ferulic acid ( 3 , fig . 1 ) was identified as a potent inhibitor of ache , with strong antioxidant properties , confirming the multi - target potential of these substances in comparison to berberine , the original prototype . aiming to add to these properties , the ability to neutralize ros ( reactive oxygen species ) and neuroprotection , authors proposed the insertion of a 1,4-benzoquinone , from coenzyme q10 ( coq10 ) , a fragment with a recognized potent mitochondrial antioxidant property and a protector of the hippocampus neurons against a(140)induced neurotoxicity . the insertion of the benzoquinone nucleus contributes to increase the neuroprotective activity , because a hydrophobic and planar system is generated , which is able in principle to bind a [ 42 , 47 , 48 , 50 ] . finally , in vivo assays showed the potential of memoquin to restore the cholinergic deficit and successfully reduce a expression and accumulation , also reducing hyperphosphorylation of tau protein [ 48 , 49 ] . considering all these multi - target profile of memoquin ( 8) , bolognesi and co - workers reported in 2009 the design of new hybrid lipoic acid - memoquin ( 11 ) derivatives inspired on the memoquin structure and lipoic acid structure which is a potent antioxidant . in addition to the observed biological properties of memoquin , compound 12 also exhibited a good neuroprotective effect against oxidative stress , greater than the reference control lipoic acid ( 10 ) . 3 ) showed the best multiple - target action profile with selective inhibitory activities of ache and buche , with modulatory effects on the amyloid aggregation and inhibition of neurofibrils formation . 4 ) is the first approved ches inhibitor by the fda for the treatment of ad . however , due to some undesirable side effects this drug have its therapeutic use restricted and , therefore , the search for more effective and secure tacrine derivatives is still of interest . in general , the new tacrine - hybrid compounds showed to be better selective for ache inhibition than tacrine . because of this symbiotic profile , compound 25 has been described as an important structural model for rational design of novel mtdls . in 2007 ,
bolognesi and co - workers proposed modifications on the structure of bis-(7)-tacrine ( 25 ) with an insertion of a spacer subunit with functional groups capable to of chelate metals , which are involved in the degenerative process . this study revealed the hybrid dimer cystamine - tacrine 31 was able to inhibit ache ( ic50= 5.04 nm ) , buche ( ic50= 4.23 nm ) , self - a aggregation ( ic50= 24.2 m ) and ache - induced a aggregation ( 52,6% ) in the same range of the reference compound , with additional neuroprotective effect on sh - sy5y cell line against h2o2-induced oxidative injury , with low toxicity . fang and co - workers reported in 2008 the synthesis and pharmacologic evaluation of a new series of tacrine - ferulic acid hybrid derivatives ( 33 ) ( fig . this approach was used aiming to obtain new multi - target hybrid compounds which could conjugate the ache inhibitory activity originating from the tacrine template and the antioxidant activity from the ferulic acid moiety . in another approach , rosini and
co - workers also used tacrine as a model for the planning of a new series of hybrid compounds ( 36 - 39 ) based on the structural feature of carvedilol ( 35 ) . particularly , compounds 36 - 39 showed an adequate biological profile for multi - target drugs , inhibiting ache activity and also capable to block in vitro a self - aggregation , ache - mediated a-aggregation , nmdars antagonistic effect and to reduce cerebral oxidative stress . in another approach , tacripyrines ( 40 )
were designed by combining tacrine with a calcium antagonist such as nimodipine ( 41 ) ( fig . additionally , the most of the compounds showed moderate ca channel blocking effect and therefore the compounds are neuroprotective agents . 10 ) as multitarget - directed ligands against alzheimer s disease . in another work ,
wang and collaborators reported a series of tacrine - based hybrid compounds , containing an additional pharmacophoric subunit 4-phenyl-2-aminothiazole with different spacer subunits . some of these hybrid compounds were also capable to prevent glutamate - induced neurotoxicity by moderately blocking glutamate receptor nmda subtype and neuronal cell death via nmda receptor - dependent effect at nanomolar concentrations ( e.g. during an investigative study for the ability of these -carboline derivatives to interact with multiple targets
, the researchers also found that several compounds were also potent nmda receptor blockers , in addition to the potent inhibitory properties of both ache / buche enzymes . compound 55 showed the higher ache inhibitory activity ( ic50= 0.5 nm ) and proved to be a potent inhibitor of the transient induced - glutamate ca with an ic50= 1.4 m , which is 4-fold more potent than memantine ( ic50= 5 5.6 m ) in the same assay conditions . in another approach for drug design of mdtls ,
rizzo and co - workers reported a new series of hybrid molecules based on the structure of the ache inhibitor n - methyl - n - benzylamine ( 56 ) , and compound skf-64346 ( 57 ) , a benzofuran derivative with good inhibitory properties of a-fibril formation , using a heptyloxy moiety as a linker subunit . they proposed a variation in the length of the spacer subunit , modification in the substituents in 3 position of the benzofuran scaffold and change of the n - methyl - n - benzylamine heptyloxy side chain from the para to meta position ( fig . among the most active compounds of the series 59 , compounds
60 and 61 exhibited the best buche inhibitory properties , with carbamate 62 also showing higher dual inhibitory activity of ache and buche in comparison to rivastigmine . the positions 6 or 7 of the coumarin moiety were also replaced by a dihalophenyl acid subunit , considering that this moiety emerged as a leitmotif in different bace-1 inhibitors reported in the literature . biological data clearly indicated that the introduction of a halophenyl alkylamidic subunit on the scaffold of 65 allowed obtaining more potent bace-1 inhibitors , and the presence of substituents in the positions 6 or 7 on the coumarin nucleus seems to equally contribute to the relative binding affinity of this molecular series . moreover , molecular modeling showed that these inhibitors probably act in two binding sites of ache , and that the length of the spacer subunit is of particular relevance in the modulation of ache inhibition , being crucial for the dual interaction of these molecules with both cas and pas sites the enzyme . 18 ) was based on the molecular hybridization of the benzylpiperidine moiety of donepezil ( 70 ) and the indolyl - propapylamino subunit present in the structure of the mao inhibitor n-(5benzyloxy-1-methyl-1h - indol-2-yl)methyl - n - methylprop-2-yn-1-amine ( 73 ) , connected a central oxy - methylene chain . compound 75 was identified from series 74 as the most promising ligand , showing a good selectivity and high potency in the inhibition of mao - a with an ic50 = 5.2 nm ( ic50 mao - b = 43.0 nm ) , but less potent and with poor selectivity for the ache ( ic50 = 0.35 m ) and buche ( ic50 = 0.46 m ) . based on the remarkable capacity of tacrine to interact in the active site of ache , martins and collaborators proposed a new series of tacrine - derived analogues by the insertion of a furo [ 2,3-b ] quinolin-4-amine ( 76 ) and pyrrolo [ 2,3-b ] quinolin-4-amine ( 77 ) subunits ( fig . molecular modelling studies have corroborate that the hybrid derivatives containing pyrrole or furan ring subunits interacts with ache and buche and the presence of a phenyl ring at the position 1 of the pyrrole ring is beneficial for both ache and buche inhibition . in vitro evaluation showed that most of the compounds were potent inhibitors of mao - b ( ic50 of 7.540.5 m ) , besides antioxidant ( orac - fl value of 2.759.37 m ) and metal chelator properties . thus compound 82 is one of the most current example from literature of a genuine mdtl drug prototype candidate for ad treatment . compounds 86 and 87 were the lead - compounds identified among a series of resveratrol - derived molecular hybrids ( 85 ) , planned by the combination of the catechol pharmacophore of 83 with the well - known metal chelator clioquinol ( 84 ) ( fig . these two hybrid - compounds showed high potency in the inhibition of self - induced a aggregation ( 86 , ic50 = 7.56 m and 87 , ic50 = 6.51 m ) in comparison to resveratrol ( ic50 = 15.11 m ) . the more recent insights about the pathophysiology of ad have proven a complex inter - connected variety of deleterious events , with multiple pathways involving in its etiology and a variety of factors that are associated with the installation , progress and severity of ad . this complex multifactorial aspect of ad and other neuronal disorders have supported the new paradigm of mtdls ( or one - compound - multi - targets strategy ) as a more rational strategy approach to the development of new drug candidates , than the one - target - one drug concept . the low efficacy of the therapeutic approach currently employed could be , in part attributed to this reductionist concept , and it becomes inevitable to adopt a new concept for the rational design of new drugs against ad . therefore , a drug for the treatment of ad will only be effective if modulate multiple targets simultaneously involved in the disease . in the last decade efforts
have being employed in the design and search for new biological chemical entities capable to direct simultaneously with different molecular targets involved in the pathogenesis of ad and many new significantly active and promise molecules have been discovered . consequently , new drug prototype candidates have being identified and they are the starting point of a revolutionary new way of thinking drug design and some drug candidates are under pre - clinical evaluation . despite none of these mtdl drug candidates have reached clinical phase of development , we hope that in the next few years the system biology approach could effectively contribute to the medicinal chemists in the discovery of innovative chemical entities able to understand and effectively modify the course of this devastating neurodegenerative disorder . = acetylcholinesterase = butirylcholinesterase = central nervous system = doena de alzheimer = acetilcolinesterase inhibitor = minimum inhibitory concentration of 50% = monoamine oxidase a = monoamine oxidase b = multitarget - directed ligand = n - methyl - d - aspartate = peripheral anionic site = reactive oxygen species | [
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] |
the gold standard technique for glenoid replacement in total anatomical shoulder prosthesis is still the use of full polyethylene cemented implant like neer concept 40 years ago .
the frequency of radio lucent lines ( rll ) is high , 70 % in the most recent literature with an increasing number of glenoid loosenings , 40 % at 10 years [ 24 ] .
however , the number of revisions is low , around 5 % [ 5 , 6 ] .
cementless glenoid devices have been tried , as much as the success of reverse prosthesis has obliged to develop metal back screwed glenoid trays , able to resist to the shear forces induced by a glenosphere .
these new uncemented implants in anatomical shoulder replacement have been greatly criticised , accused to be responsible of loosening , dissociation , and early pe wear .
furthermore , it has been advocated that the increased thickness induced by the metal tray [ 8 , 9 ] could be a risk for the rotator cuff .
the excellent primary fixation of these screwed implants in reverse arthroplasty has led us to extend our indications to anatomical replacements , in order to see whether with this kind of design we are able to decrease the frequency of rll observed with the cemented glenoids .
the metal back glenoid implant ( mb ) of the universal shoulder arthroplastic system arrow ( fh orthopedics , 3 rue de la fort 68990-heimsbrunn - france ) is 6.5 mm thick , 3.5 for the pe and 3 for the metal tray .
whatever the size of the humeral head , there is a systematic mismatch between the radius of curvature of the glenoid and of the humeral head with an average of 4 mm ( between 1 and 6 ) .
the ancillary system allows a precise preparation of the glenoid with a reaming of the bone surface and a press fit preparation of the keel grove , in order to insure a perfect contact between hydroxyapatite and bone .
the primary fixation is insured by 2 axial screws and can be enhanced by a third sagittal screw .
it can be useful in case of osteoporotic patient and glenoid bone loss , allowing an easy bone graft fixation .
on the humeral side , noncemented press fit stems were preferentially used , with grafting of the metaphysis using some cancellous bone from the humeral head . in case of osteoporotic bone , a classical cemented technique was recommended .
the clinical analysis included a pre- and postoperative evaluation of the score of constant and murley , of the active and passive range of motion and of the simple shoulder test from matsen .
radiographic preoperative assessment consisted of plain anteroposterior radiographs with medial , neutral and lateral rotation , axillary and outlet view under fluoroscopic guidance .
a systematic ct scan completed the preoperative radiographic analysis to evaluate the status of the cuff and the glenoid bone stock according to walch classification .
postoperative radiological study included an ap view with a standardised fluoroscopic technique and the x - ray beam perpendicular to the plane of the joint space .
the study included 2 parts : a radiological study done on the first postoperative x - rays in order to check if the increased thickness of the mb component induced a measurable lateralisation .
the lateral offset was measured between the centre of the glenoid bone and the lateral border of the great tuberosity ( fig . 1 ) .
1technique of the measurement of the lateral offsetthe secund part consisted of analysing the final clinical and radiological results on the patients with more than 24 months of follow - up .
a radiological study done on the first postoperative x - rays in order to check if the increased thickness of the mb component induced a measurable lateralisation .
the lateral offset was measured between the centre of the glenoid bone and the lateral border of the great tuberosity ( fig . 1 ) .
1technique of the measurement of the lateral offset technique of the measurement of the lateral offset the secund part consisted of analysing the final clinical and radiological results on the patients with more than 24 months of follow - up .
the student s t test was used for statistical analysis when two groups had to be compared . when the comparison involved more than two groups , a variance analysis was applied . the chosen level of significance ( p )
from november 2003 to december 2011 , 143 total anatomical shoulder arthroplasties have been performed .
aetiology is summarised in table 1 , dominated by primary osteoarthritis with 90 % of normal cuff.table 1aetiology for the whole seriesarthritis without teararthritis with tearposttrauma arthritisrevisionchronic dislocationsecondary necrosismalunionmb ( n = 143)116 ( 81.1 % ) 16 ( 11.2 % ) 44111 aetiology for the whole series delto pectoral approach was performed except in 2 cases .
the common size for the mb was 44 in 3/4 of the cases , and 46 for the remaining .
25 % of the humeral heads were 44 , 25 % for 46 and 48 .
65 % were excentric heads with a height of 16 for half of them .
radiological analysis : the results of the first part of this work are summarised in the table 2 .
the postoperative immediate radiological study showed a lateralisation of 0.36 cm between the operated and the contralateral normal side ( p = 0.02).table 2study of the lateralisation , only for unilateral pathologic involvement with normal contralateral side and excluding the bad x - raylateralisationcontrol sidedifferencemb ( n = 48)6.09 ( 3.17 , mean 6.3)5.73 ( 2.27 , mean 6)0.36 ( p = 0.02 ) study of the lateralisation , only for unilateral pathologic involvement with normal contralateral side and excluding the bad x - ray with more than 2 years of follow - up 2 cases presented a well - tolerated superior migration . one , with a follow - up of 24 months , had no pain , an active flexion of 90 and a constant score of 53 ( 68 % ) .
the other , at 44 months , presented a slight pain , an active elevation of 100 and a score of 54 ( 74 % ) . in the whole series of 143 mb prosthesis
part of the cohort allowed a study of the joint narrowing . at 3 months ,
the difference between operated and contralateral normal side was 0.47 cm ( n = 30 , 01 ) , at 1 year , we found a 0.44 of average ( 1 to 1 ) , at 2 years 0.42 ( 01 ) and finally 0.39 ( 0.1 to 1 ) with no statistical significance .
11 complications were recorded ( 7.7 % ) : three dissociations occured in the beginning of the experiment .
a first design did not allow to precisely centre the pe tray before impaction . in 2007 , the addition of a pe central peg allowed to insure a good alignment .
two of these cases were revised , one with a cemented glenoid with an excellent final constant score of 85 ( 121 % ) and an active flexion of 160. the other one is 60 months of follow - up of a simple reimpaction of a new pe tray with also an excellent result , a constant score of 90 ( 103 % ) and an active elevation of 170. the third case of dissociation did not accept any revision.three dislocations occured , all with b2 and c glenoid types .
two were revised with a conversion in reverse . in one case with a type b2 glenoid , the conversion consisted of simply changing the pe tray to a glenosphere and the humeral head to a cup .
this patient with 18 months of follow - up had an active flexion of 120 , no pain and a constant score of 54 ( 76 % ) ( fig . 2 ) .
for the 2 cases with a glenoid type c , because of the necessity of grafting the glenoid it was necessary to take out the metal tray despite a good integration .
conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 monthstwo secondary rotator cuff tears were converted to a reverse .
the first one , at 17 months of the revision , had no pain but a fair active and passive mobility with a constant score of 44 ( 68 % ) .
the other one was a b2 glenoid and had also a contralateral mb glenoid with an excellent result . during revision performed at 2 years a posterosuperior ,
pe wear was found with a contact between the metal tray and the humeral head . at 36 months of the revision
, the result was excellent with a constant score of 64 ( 100 % ) , an active elevation of 140 ( fig .
no problem for the right side . on the left side a secondary cuff tear occured .
a conversion for a reverse was realised with a good clinical result a c clinical results at 36 months , d x - ray on the right side e x - ray on the left side after the revisionone case sustained a superior migration due to a bad initial choice of the size of the glenoid with a too low implantation .
this allowed a quick superior migration of the humeral head above the glenoid implant but without cuff tear .
the revision at 1 month consisted on a higher implantation of a bigger metal tray .
this patient was 60 months of follow - up , and this case of revision was included in the final review.one patient had a postoperative painful stiffness because of a complex regional pain syndrome.another case of painful shoulder was reoperated at 6 months .
a first design did not allow to precisely centre the pe tray before impaction . in 2007 , the addition of a pe central peg allowed to insure a good alignment .
two of these cases were revised , one with a cemented glenoid with an excellent final constant score of 85 ( 121 % ) and an active flexion of 160. the other one is 60 months of follow - up of a simple reimpaction of a new pe tray with also an excellent result , a constant score of 90 ( 103 % ) and an active elevation of 170. the third case of dissociation did not accept any revision .
two were revised with a conversion in reverse . in one case with a type b2 glenoid , the conversion consisted of simply changing the pe tray to a glenosphere and the humeral head to a cup .
this patient with 18 months of follow - up had an active flexion of 120 , no pain and a constant score of 54 ( 76 % ) ( fig . 2 ) .
for the 2 cases with a glenoid type c , because of the necessity of grafting the glenoid it was necessary to take out the metal tray despite a good integration .
conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 months mb glenoid implanted on a type b2 .
conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 months two secondary rotator cuff tears were converted to a reverse .
the first one , at 17 months of the revision , had no pain but a fair active and passive mobility with a constant score of 44 ( 68 % ) .
the other one was a b2 glenoid and had also a contralateral mb glenoid with an excellent result . during revision performed at 2 years a posterosuperior ,
pe wear was found with a contact between the metal tray and the humeral head . at 36 months of the revision ,
the result was excellent with a constant score of 64 ( 100 % ) , an active elevation of 140 ( fig .
no problem for the right side . on the left side a secondary cuff tear occured .
c clinical results at 36 months , d x - ray on the right side e x - ray on the left side after the revision patient operated on both sides with a metal back glenoid implant .
no problem for the right side . on the left side a secondary cuff tear occured .
a conversion for a reverse was realised with a good clinical result a c clinical results at 36 months , d x - ray on the right side e x - ray on the left side after the revision one case sustained a superior migration due to a bad initial choice of the size of the glenoid with a too low implantation .
this allowed a quick superior migration of the humeral head above the glenoid implant but without cuff tear .
the revision at 1 month consisted on a higher implantation of a bigger metal tray .
this patient was 60 months of follow - up , and this case of revision was included in the final review .
but if we do not take account of the dissociation cases , the percentage decreases to 4.19 % ( 6/143 ) .
thirty - seven cases in 36 patients were 2 years of follow - up or more , 38.3 months in average ( 2475 , mean 32 ) , 28 female ( one bilateral ) 8 men , 21 right side , and 32 right handed .
the first one was this already mentioned patient with a too small mb glenoid implanted too low and revised at 3 months .
the second one sustained previously a total anatomical arthroplasty with a cemented glenoid , revised with a mb glenoid and a grafting because of a glenoid loosening.table 3aetiologies for the cases with more than 24 monthsarthritis without teararthritis with tearposttrauma arthritisrevisionchronic dislocationmb ( n
= 37)321121 aetiologies for the cases with more than 24 months two presented a superior migration due to a rotator cuff tear .
as already mentioned , these 2 cases were converted to a reverse , simple and rapid procedure thanks to the universality of the arrow system , consisting of a simple change of the intermediate devices .
these 2 cases were excluded from the final review.one patient presented an early dissociation revised at 2 months . a simple change of the pe tray allowed an excellent result at 39 months with an active flexion of 170 and a constant score of 85 ( 102 % ) .one patient died from a medical cause in january 2012 after the final review in november 2011 .
her result was excellent , 150 of flexion and a score of 72 ( 101 % ) .
as already mentioned , these 2 cases were converted to a reverse , simple and rapid procedure thanks to the universality of the arrow system , consisting of a simple change of the intermediate devices .
a simple change of the pe tray allowed an excellent result at 39 months with an active flexion of 170 and a constant score of 85 ( 102 % ) .
one patient died from a medical cause in january 2012 after the final review in november 2011 .
her result was excellent , 150 of flexion and a score of 72 ( 101 % ) .
finally , 35 cases were available for the final results which are summarised in table 4 .
pain increased from 1.6 to 13.4 , flexion from 92 to 146 and constant score from 27 ( 36 % ) to 70 ( 95 % ) .
the statistical difference between pre- and postoperative values was greatly significant.table 4pre- and postoperative constant score : the pre- and postoperative comparison is highly significantmb ( n = 35)painadlstrengthconstant scoreactive elevationre1re2sstpreop1.6 ( 05)8.8 ( 218)2.8 ( 010 , med 2.5)27 ( 1256.36 % ) 92 ( 40160 mean
90)12.5 ( 2070)27.6 ( 080 , mean 30)2.6 yespostop13.4 ( 515 , mean 15)17.9 ( 920 , mean 20)7 ( 017 , mean 6)70 ( 3590.95 % ) 146 ( 80180
, mean 150)44 ( 1070 mean 45)65 ( 1095 mean 70)9.8 yes pre- and postoperative constant score : the pre- and postoperative comparison is highly significant we analysed the results according to the preoperative type of glenoid : between types a1 and a2 the preoperative clinical values were systematically inferior for the most used glenoids with no statistical significance . the same differences were noted postoperatively excepted for the activities of daily living.we found the same differences between a1 and b1 and between type a and type b.if we study the types a1 and b1 versus a2 , b2 and c , the values were systematically lower pre- and postoperatively for the most used glenoids . between types a1 and a2 the preoperative clinical values
we found the same differences between a1 and b1 and between type a and type b. if we study the types a1 and b1 versus a2 , b2 and c , the values were systematically lower pre- and postoperatively for the most used glenoids .
the results of the first part of this work are summarised in the table 2 .
the postoperative immediate radiological study showed a lateralisation of 0.36 cm between the operated and the contralateral normal side ( p = 0.02).table 2study of the lateralisation , only for unilateral pathologic involvement with normal contralateral side and excluding the bad x - raylateralisationcontrol sidedifferencemb ( n = 48)6.09 ( 3.17 , mean 6.3)5.73 ( 2.27 , mean 6)0.36 ( p = 0.02 ) study of the lateralisation , only for unilateral pathologic involvement with normal contralateral side and excluding the bad x - ray with more than 2 years of follow - up 2 cases presented a well - tolerated superior migration .
one , with a follow - up of 24 months , had no pain , an active flexion of 90 and a constant score of 53 ( 68 % ) .
the other , at 44 months , presented a slight pain , an active elevation of 100 and a score of 54 ( 74 % ) . in the whole series of 143 mb prosthesis
part of the cohort allowed a study of the joint narrowing . at 3 months ,
the difference between operated and contralateral normal side was 0.47 cm ( n = 30 , 01 ) , at 1 year , we found a 0.44 of average ( 1 to 1 ) , at 2 years 0.42 ( 01 ) and finally 0.39 ( 0.1 to 1 ) with no statistical significance .
among the 143 cases , 11 complications were recorded ( 7.7 % ) : three dissociations occured in the beginning of the experiment .
a first design did not allow to precisely centre the pe tray before impaction . in 2007 , the addition of a pe central peg allowed to insure a good alignment .
two of these cases were revised , one with a cemented glenoid with an excellent final constant score of 85 ( 121 % ) and an active flexion of 160. the other one is 60 months of follow - up of a simple reimpaction of a new pe tray with also an excellent result , a constant score of 90 ( 103 % ) and an active elevation of 170. the third case of dissociation did not accept any revision.three dislocations occured , all with b2 and c glenoid types .
two were revised with a conversion in reverse . in one case with a type b2 glenoid , the conversion consisted of simply changing the pe tray to a glenosphere and the humeral head to a cup .
this patient with 18 months of follow - up had an active flexion of 120 , no pain and a constant score of 54 ( 76 % ) ( fig . 2 ) .
for the 2 cases with a glenoid type c , because of the necessity of grafting the glenoid it was necessary to take out the metal tray despite a good integration .
conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 monthstwo secondary rotator cuff tears were converted to a reverse .
the first one , at 17 months of the revision , had no pain but a fair active and passive mobility with a constant score of 44 ( 68 % ) .
the other one was a b2 glenoid and had also a contralateral mb glenoid with an excellent result . during revision performed at 2 years a posterosuperior ,
pe wear was found with a contact between the metal tray and the humeral head . at 36 months of the revision ,
the result was excellent with a constant score of 64 ( 100 % ) , an active elevation of 140 ( fig .
no problem for the right side . on the left side a secondary cuff tear occured .
a conversion for a reverse was realised with a good clinical result a c clinical results at 36 months , d x - ray on the right side e x - ray on the left side after the revisionone case sustained a superior migration due to a bad initial choice of the size of the glenoid with a too low implantation .
this allowed a quick superior migration of the humeral head above the glenoid implant but without cuff tear .
the revision at 1 month consisted on a higher implantation of a bigger metal tray .
this patient was 60 months of follow - up , and this case of revision was included in the final review.one patient had a postoperative painful stiffness because of a complex regional pain syndrome.another case of painful shoulder was reoperated at 6 months .
a first design did not allow to precisely centre the pe tray before impaction . in 2007 , the addition of a pe central peg allowed to insure a good alignment .
two of these cases were revised , one with a cemented glenoid with an excellent final constant score of 85 ( 121 % ) and an active flexion of 160. the other one is 60 months of follow - up of a simple reimpaction of a new pe tray with also an excellent result , a constant score of 90 ( 103 % ) and an active elevation of 170. the third case of dissociation did not accept any revision .
two were revised with a conversion in reverse . in one case with a type b2 glenoid , the conversion consisted of simply changing the pe tray to a glenosphere and the humeral head to a cup .
this patient with 18 months of follow - up had an active flexion of 120 , no pain and a constant score of 54 ( 76 % ) ( fig . 2 ) .
for the 2 cases with a glenoid type c , because of the necessity of grafting the glenoid it was necessary to take out the metal tray despite a good integration .
conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 months mb glenoid implanted on a type b2 .
conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 months two secondary rotator cuff tears were converted to a reverse .
the first one , at 17 months of the revision , had no pain but a fair active and passive mobility with a constant score of 44 ( 68 % ) .
the other one was a b2 glenoid and had also a contralateral mb glenoid with an excellent result . during revision performed at 2 years a posterosuperior ,
pe wear was found with a contact between the metal tray and the humeral head . at 36 months of the revision ,
the result was excellent with a constant score of 64 ( 100 % ) , an active elevation of 140 ( fig .
no problem for the right side . on the left side a secondary cuff tear occured .
c clinical results at 36 months , d x - ray on the right side e x - ray on the left side after the revision patient operated on both sides with a metal back glenoid implant .
no problem for the right side . on the left side a secondary cuff tear occured .
c clinical results at 36 months , d x - ray on the right side e x - ray on the left side after the revision one case sustained a superior migration due to a bad initial choice of the size of the glenoid with a too low implantation .
this allowed a quick superior migration of the humeral head above the glenoid implant but without cuff tear .
the revision at 1 month consisted on a higher implantation of a bigger metal tray .
this patient was 60 months of follow - up , and this case of revision was included in the final review .
but if we do not take account of the dissociation cases , the percentage decreases to 4.19 % ( 6/143 ) .
thirty - seven cases in 36 patients were 2 years of follow - up or more , 38.3 months in average ( 2475 , mean 32 ) , 28 female ( one bilateral ) 8 men , 21 right side , and 32 right handed .
the first one was this already mentioned patient with a too small mb glenoid implanted too low and revised at 3 months .
the second one sustained previously a total anatomical arthroplasty with a cemented glenoid , revised with a mb glenoid and a grafting because of a glenoid loosening.table 3aetiologies for the cases with more than 24 monthsarthritis without teararthritis with tearposttrauma arthritisrevisionchronic dislocationmb ( n = 37)321121 aetiologies for the cases with more than 24 months
as already mentioned , these 2 cases were converted to a reverse , simple and rapid procedure thanks to the universality of the arrow system , consisting of a simple change of the intermediate devices .
these 2 cases were excluded from the final review.one patient presented an early dissociation revised at 2 months . a simple change of the pe tray allowed an excellent result at 39 months with an active flexion of 170 and a constant score of 85 ( 102 % ) .one patient died from a medical cause in january 2012 after the final review in november 2011 .
her result was excellent , 150 of flexion and a score of 72 ( 101 % ) .
as already mentioned , these 2 cases were converted to a reverse , simple and rapid procedure thanks to the universality of the arrow system , consisting of a simple change of the intermediate devices .
one patient presented an early dissociation revised at 2 months . a simple change of the pe tray allowed an excellent result at 39 months with an active flexion of 170 and a constant score of 85 ( 102 % )
. one patient died from a medical cause in january 2012 after the final review in november 2011 .
her result was excellent , 150 of flexion and a score of 72 ( 101 % ) .
finally , 35 cases were available for the final results which are summarised in table 4 .
pain increased from 1.6 to 13.4 , flexion from 92 to 146 and constant score from 27 ( 36 % ) to 70 ( 95 % ) .
the statistical difference between pre- and postoperative values was greatly significant.table 4pre- and postoperative constant score : the pre- and postoperative comparison is highly significantmb ( n = 35)painadlstrengthconstant scoreactive elevationre1re2sstpreop1.6 ( 05)8.8 ( 218)2.8 ( 010 , med 2.5)27 ( 1256.36 % ) 92 ( 40160 mean
90)12.5 ( 2070)27.6 ( 080 , mean 30)2.6 yespostop13.4 ( 515 , mean 15)17.9 ( 920 , mean 20)7 ( 017 , mean 6)70 ( 3590.95 % ) 146 ( 80180 , mean 150)44 ( 1070 mean 45)65 ( 1095 mean 70)9.8 yes pre- and postoperative constant score : the pre- and postoperative comparison is highly significant we analysed the results according to the preoperative type of glenoid : between types a1 and a2 the preoperative clinical values were systematically inferior for the most used glenoids with no statistical significance . the same differences were noted postoperatively excepted for the activities of daily living.we found the same differences between a1 and b1 and between type a and type b.if we study the types a1 and b1 versus a2 , b2 and c , the values were systematically lower pre- and postoperatively for the most used glenoids . between types a1 and a2 the preoperative clinical values
we found the same differences between a1 and b1 and between type a and type b. if we study the types a1 and b1 versus a2 , b2 and c , the values were systematically lower pre- and postoperatively for the most used glenoids .
in total shoulder anatomical arthroplasty using a cemented glenoid , the percentage of radio lucent lines is high despite a good and stable clinical results whatever the model , keeled or pegged , at 10 years , 76 % presented a rll and 40 % a glenoid loosening .
the modular second or third generation tried to give a better adaptation to the patient s anatomy but did not solve the problem [ 15 , 16 ] .
one of the most recent publication , with more than 5 years of follow - up , showed 18.9 % of loosenings , among them 23 % were progressive with a functional repercussion .
parallely , the feasibility of the metal back trays , able to sustain without failure the shear forces induced by a glenosphere in reverse shoulder arthroplasty , led to experiment these devices in anatomical arthroplasty . with this kind of design , the literature evoqued early polyethylene wear , number of dissociation between pe and metal tray , loosenings and superior migration due to rotator cuff tears . the first referred cause of those failures was the thickness of the implant [ 4 , 19 , 20 ] .
however , most of the articles referred to old conceptions [ 2123 ] or designs which did not insure a good primary fixation .
however , some more recent publications presented some encouraging results [ 25 , 26 ] . in 1992 , we started to work on a new implant which was available for clinical use in human since 2003 .
the convexity was preferred to a flat back tray , being widely recognised as insuring a good bone - implant contact and transforming the shear forces in compressive forces [ 1 , 23 , 2730 ] .
iannotti , like neer previously , insisted in the better easiness of well positioning the convexe implant , which parallely decreases the frequency of lucent lines .
the principle of a mismatch was also adopted [ 15 , 22 , 24 , 25 ] . in our experience ,
the fit induced by a precise ancillary system and the frontal and the eventual saggital screwing avoided any primary fixation problems and enhanced the indications to the cases necessitating a glenoid bone graft . the hydroxyapatite coverage on all the parts in contact with the glenoid bone insured a good secondary fixation with no migrations , loosenings or even rll .
a modification of the design , consisting of adding a small central peg on the pe allowed to well centre the pe before impaction on the metal tray .
our results showed an increasing in the lateral offset in comparison with a normal contralateral side .
another work ( accepted as a free paper in the sofcot 2012 meeting ) comparing the results between mb and cemented glenoid showed that this induced lateralisation did not influence the clinical results .
radiologically , no loosening , no lucent lines , no narrowing of the joint space , witness of a polyethylene wear were found . however , our tendency is actually to increase the pe thickness .
the rigidity of the metal back device was suspected to induce stress shielding and osteolysis under the metal [ 24 , 32 ] .
these experimental publications , in our knowledge , were never confirmed by clinical studies and not confirmed in our work .
some rotator cuff tears happened in our series , but with the same frequency than the most recent publications on cemented glenoid designs . despite the fact that , in the contrary than the literature [ 35 , 36 ] , the results were not statistically different between types a and b , the type of preoperative glenoid bone wear influenced our choice , as the only cases of dislocations were on posteriorly used glenoid bones , type b2 or c. in those cases where the posterior wear is not too important
, we recommend to increase the anterior reaming , to add a posterior bone graft and eventually to utilise the long keeled implant , designed for revisions . in old patients with a thin cuff
however , we prefer the noncemented glenoid , as , in case of secondary superior migration , the conversion in a reverse is facilitated , the same metal back tray been the support of the glenosphere .
moreover , this new design , thanks to the anterior plate and the 2 available directions of screwing , allows to extend the indications to big glenoid bone loss necessiting a bone graft .
however , this study presented some limitations : this is a medium - term study with a mean follow - up of 3 years.the measurement of the radiological lateral offset is technician dependent , and no scoring of the rll has been used.this is a multicentric not randomised study , and the choice of either a noncemented or a cemented prosthesis was often subjective and surgeon dependent .
this is a medium - term study with a mean follow - up of 3 years .
the measurement of the radiological lateral offset is technician dependent , and no scoring of the rll has been used .
this is a multicentric not randomised study , and the choice of either a noncemented or a cemented prosthesis was often subjective and surgeon dependent .
however , in the view of our good and predictable results , our indications of using a mb glenoid in total anatomical shoulder arthroplasty are increasing ( fig .
4diagram showing the growing number of mb glenoid implantations diagram showing the growing number of mb glenoid implantations in conclusion , we did not find in this medium - term clinical and radiological review of a noncemented metal back glenoid implant , the classical complications pointed out in the literature for those uncemented glenoid implants . with this new design , despite a radiological increase in the lateral offset , there is no proved risk for the cuff , no early polyethylene wear , no dissociation and the clinical results seem to be similar to the cemented glenoids but avoiding their frequent troubles such as evolutive lucent lines and loosenings . | the uncemented glenoid implants in total anatomical shoulder arthroplasty are likely to be accused of problems like dissociations , secondary rotator cuff tear , and wear of polyethylene ( pe ) .
this work is a clinical and radiological prospective review of 143 cases of anatomical total shoulder arthroplasty using a new metal back uncemented glenoid implant ( mb ) in order to see if this new implant induces those complications .
a total of 143 cases were operated between 2003 and 2011 . in a first part
, the whole series of 143 cases was radiologically studied in order to quantify the lateralisation induced by the mb implant . in a second study ,
37 cases had a mean follow - up of 38 months ( 2475 , mean 32 ) and served for the clinical and radiological final study .
pre- and postoperative clinical evaluation was done using the constant murley score and the simple shoulder test from matsen .
the final x - rays served to detect an eventual secondary narrowing of the joint space and to analyse the frequency of radio lucent lines ( rll ) and loosenings . despite a small radiological lateralisation in comparison with the normal contralateral side ( 0.36 cm , p = 0.02 ) , the clinical results after 2 years were similar to the published cemented glenoid implants series but without any rll , glenoid loosening or joint narrowing .
some dissociations occured in the beginning and definitely eliminated by a design modification of the pe tray .
the discussion tried to show that , despite a still short follow - up , this series is encouraging to continue to use this new mb implant
. different applications of the concept of universality and conversion are discussed , this tray been also the support of a glenosphere in reverse arthroplasty . | Introduction
Materials and methods
Results
1. Radiological results
2. Clinical results
3. Results after 24months or more
4. Three revisions were recorded
Discussion | the frequency of radio lucent lines ( rll ) is high , 70 % in the most recent literature with an increasing number of glenoid loosenings , 40 % at 10 years [ 24 ] . cementless glenoid devices have been tried , as much as the success of reverse prosthesis has obliged to develop metal back screwed glenoid trays , able to resist to the shear forces induced by a glenosphere . these new uncemented implants in anatomical shoulder replacement have been greatly criticised , accused to be responsible of loosening , dissociation , and early pe wear . furthermore , it has been advocated that the increased thickness induced by the metal tray [ 8 , 9 ] could be a risk for the rotator cuff . the excellent primary fixation of these screwed implants in reverse arthroplasty has led us to extend our indications to anatomical replacements , in order to see whether with this kind of design we are able to decrease the frequency of rll observed with the cemented glenoids . the metal back glenoid implant ( mb ) of the universal shoulder arthroplastic system arrow ( fh orthopedics , 3 rue de la fort 68990-heimsbrunn - france ) is 6.5 mm thick , 3.5 for the pe and 3 for the metal tray . the clinical analysis included a pre- and postoperative evaluation of the score of constant and murley , of the active and passive range of motion and of the simple shoulder test from matsen . postoperative radiological study included an ap view with a standardised fluoroscopic technique and the x - ray beam perpendicular to the plane of the joint space . the study included 2 parts : a radiological study done on the first postoperative x - rays in order to check if the increased thickness of the mb component induced a measurable lateralisation . 1technique of the measurement of the lateral offsetthe secund part consisted of analysing the final clinical and radiological results on the patients with more than 24 months of follow - up . a radiological study done on the first postoperative x - rays in order to check if the increased thickness of the mb component induced a measurable lateralisation . 1technique of the measurement of the lateral offset technique of the measurement of the lateral offset the secund part consisted of analysing the final clinical and radiological results on the patients with more than 24 months of follow - up . the postoperative immediate radiological study showed a lateralisation of 0.36 cm between the operated and the contralateral normal side ( p = 0.02).table 2study of the lateralisation , only for unilateral pathologic involvement with normal contralateral side and excluding the bad x - raylateralisationcontrol sidedifferencemb ( n = 48)6.09 ( 3.17 , mean 6.3)5.73 ( 2.27 , mean 6)0.36 ( p = 0.02 ) study of the lateralisation , only for unilateral pathologic involvement with normal contralateral side and excluding the bad x - ray with more than 2 years of follow - up 2 cases presented a well - tolerated superior migration . in the whole series of 143 mb prosthesis
part of the cohort allowed a study of the joint narrowing . at 3 months ,
the difference between operated and contralateral normal side was 0.47 cm ( n = 30 , 01 ) , at 1 year , we found a 0.44 of average ( 1 to 1 ) , at 2 years 0.42 ( 01 ) and finally 0.39 ( 0.1 to 1 ) with no statistical significance . 11 complications were recorded ( 7.7 % ) : three dissociations occured in the beginning of the experiment . two of these cases were revised , one with a cemented glenoid with an excellent final constant score of 85 ( 121 % ) and an active flexion of 160. the other one is 60 months of follow - up of a simple reimpaction of a new pe tray with also an excellent result , a constant score of 90 ( 103 % ) and an active elevation of 170. the third case of dissociation did not accept any revision.three dislocations occured , all with b2 and c glenoid types . in one case with a type b2 glenoid , the conversion consisted of simply changing the pe tray to a glenosphere and the humeral head to a cup . conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 monthstwo secondary rotator cuff tears were converted to a reverse . this allowed a quick superior migration of the humeral head above the glenoid implant but without cuff tear . this patient was 60 months of follow - up , and this case of revision was included in the final review.one patient had a postoperative painful stiffness because of a complex regional pain syndrome.another case of painful shoulder was reoperated at 6 months . two of these cases were revised , one with a cemented glenoid with an excellent final constant score of 85 ( 121 % ) and an active flexion of 160. the other one is 60 months of follow - up of a simple reimpaction of a new pe tray with also an excellent result , a constant score of 90 ( 103 % ) and an active elevation of 170. the third case of dissociation did not accept any revision . in one case with a type b2 glenoid , the conversion consisted of simply changing the pe tray to a glenosphere and the humeral head to a cup . conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 months two secondary rotator cuff tears were converted to a reverse . c clinical results at 36 months , d x - ray on the right side e x - ray on the left side after the revision patient operated on both sides with a metal back glenoid implant . this allowed a quick superior migration of the humeral head above the glenoid implant but without cuff tear . this patient was 60 months of follow - up , and this case of revision was included in the final review . thirty - seven cases in 36 patients were 2 years of follow - up or more , 38.3 months in average ( 2475 , mean 32 ) , 28 female ( one bilateral ) 8 men , 21 right side , and 32 right handed . the second one sustained previously a total anatomical arthroplasty with a cemented glenoid , revised with a mb glenoid and a grafting because of a glenoid loosening.table 3aetiologies for the cases with more than 24 monthsarthritis without teararthritis with tearposttrauma arthritisrevisionchronic dislocationmb ( n
= 37)321121 aetiologies for the cases with more than 24 months two presented a superior migration due to a rotator cuff tear . as already mentioned , these 2 cases were converted to a reverse , simple and rapid procedure thanks to the universality of the arrow system , consisting of a simple change of the intermediate devices . a simple change of the pe tray allowed an excellent result at 39 months with an active flexion of 170 and a constant score of 85 ( 102 % ) .one patient died from a medical cause in january 2012 after the final review in november 2011 . as already mentioned , these 2 cases were converted to a reverse , simple and rapid procedure thanks to the universality of the arrow system , consisting of a simple change of the intermediate devices . the statistical difference between pre- and postoperative values was greatly significant.table 4pre- and postoperative constant score : the pre- and postoperative comparison is highly significantmb ( n = 35)painadlstrengthconstant scoreactive elevationre1re2sstpreop1.6 ( 05)8.8 ( 218)2.8 ( 010 , med 2.5)27 ( 1256.36 % ) 92 ( 40160 mean
90)12.5 ( 2070)27.6 ( 080 , mean 30)2.6 yespostop13.4 ( 515 , mean 15)17.9 ( 920 , mean 20)7 ( 017 , mean 6)70 ( 3590.95 % ) 146 ( 80180
, mean 150)44 ( 1070 mean 45)65 ( 1095 mean 70)9.8 yes pre- and postoperative constant score : the pre- and postoperative comparison is highly significant we analysed the results according to the preoperative type of glenoid : between types a1 and a2 the preoperative clinical values were systematically inferior for the most used glenoids with no statistical significance . the results of the first part of this work are summarised in the table 2 . the postoperative immediate radiological study showed a lateralisation of 0.36 cm between the operated and the contralateral normal side ( p = 0.02).table 2study of the lateralisation , only for unilateral pathologic involvement with normal contralateral side and excluding the bad x - raylateralisationcontrol sidedifferencemb ( n = 48)6.09 ( 3.17 , mean 6.3)5.73 ( 2.27 , mean 6)0.36 ( p = 0.02 ) study of the lateralisation , only for unilateral pathologic involvement with normal contralateral side and excluding the bad x - ray with more than 2 years of follow - up 2 cases presented a well - tolerated superior migration . in the whole series of 143 mb prosthesis
part of the cohort allowed a study of the joint narrowing . at 3 months ,
the difference between operated and contralateral normal side was 0.47 cm ( n = 30 , 01 ) , at 1 year , we found a 0.44 of average ( 1 to 1 ) , at 2 years 0.42 ( 01 ) and finally 0.39 ( 0.1 to 1 ) with no statistical significance . among the 143 cases , 11 complications were recorded ( 7.7 % ) : three dissociations occured in the beginning of the experiment . two of these cases were revised , one with a cemented glenoid with an excellent final constant score of 85 ( 121 % ) and an active flexion of 160. the other one is 60 months of follow - up of a simple reimpaction of a new pe tray with also an excellent result , a constant score of 90 ( 103 % ) and an active elevation of 170. the third case of dissociation did not accept any revision.three dislocations occured , all with b2 and c glenoid types . in one case with a type b2 glenoid , the conversion consisted of simply changing the pe tray to a glenosphere and the humeral head to a cup . conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 monthstwo secondary rotator cuff tears were converted to a reverse . this allowed a quick superior migration of the humeral head above the glenoid implant but without cuff tear . this patient was 60 months of follow - up , and this case of revision was included in the final review.one patient had a postoperative painful stiffness because of a complex regional pain syndrome.another case of painful shoulder was reoperated at 6 months . two of these cases were revised , one with a cemented glenoid with an excellent final constant score of 85 ( 121 % ) and an active flexion of 160. the other one is 60 months of follow - up of a simple reimpaction of a new pe tray with also an excellent result , a constant score of 90 ( 103 % ) and an active elevation of 170. the third case of dissociation did not accept any revision . in one case with a type b2 glenoid , the conversion consisted of simply changing the pe tray to a glenosphere and the humeral head to a cup . conversion for a reverse shoulder arthroplasty : a preoperative x - ray , b preoperative ct scan showing the posterior subluxation , c immediate postop x - ray , d posterior dislocation at 6 months two secondary rotator cuff tears were converted to a reverse . c clinical results at 36 months , d x - ray on the right side e x - ray on the left side after the revision patient operated on both sides with a metal back glenoid implant . this allowed a quick superior migration of the humeral head above the glenoid implant but without cuff tear . this patient was 60 months of follow - up , and this case of revision was included in the final review . thirty - seven cases in 36 patients were 2 years of follow - up or more , 38.3 months in average ( 2475 , mean 32 ) , 28 female ( one bilateral ) 8 men , 21 right side , and 32 right handed . the second one sustained previously a total anatomical arthroplasty with a cemented glenoid , revised with a mb glenoid and a grafting because of a glenoid loosening.table 3aetiologies for the cases with more than 24 monthsarthritis without teararthritis with tearposttrauma arthritisrevisionchronic dislocationmb ( n = 37)321121 aetiologies for the cases with more than 24 months
as already mentioned , these 2 cases were converted to a reverse , simple and rapid procedure thanks to the universality of the arrow system , consisting of a simple change of the intermediate devices . a simple change of the pe tray allowed an excellent result at 39 months with an active flexion of 170 and a constant score of 85 ( 102 % ) .one patient died from a medical cause in january 2012 after the final review in november 2011 . as already mentioned , these 2 cases were converted to a reverse , simple and rapid procedure thanks to the universality of the arrow system , consisting of a simple change of the intermediate devices . the statistical difference between pre- and postoperative values was greatly significant.table 4pre- and postoperative constant score : the pre- and postoperative comparison is highly significantmb ( n = 35)painadlstrengthconstant scoreactive elevationre1re2sstpreop1.6 ( 05)8.8 ( 218)2.8 ( 010 , med 2.5)27 ( 1256.36 % ) 92 ( 40160 mean
90)12.5 ( 2070)27.6 ( 080 , mean 30)2.6 yespostop13.4 ( 515 , mean 15)17.9 ( 920 , mean 20)7 ( 017 , mean 6)70 ( 3590.95 % ) 146 ( 80180 , mean 150)44 ( 1070 mean 45)65 ( 1095 mean 70)9.8 yes pre- and postoperative constant score : the pre- and postoperative comparison is highly significant we analysed the results according to the preoperative type of glenoid : between types a1 and a2 the preoperative clinical values were systematically inferior for the most used glenoids with no statistical significance . in total shoulder anatomical arthroplasty using a cemented glenoid , the percentage of radio lucent lines is high despite a good and stable clinical results whatever the model , keeled or pegged , at 10 years , 76 % presented a rll and 40 % a glenoid loosening . one of the most recent publication , with more than 5 years of follow - up , showed 18.9 % of loosenings , among them 23 % were progressive with a functional repercussion . parallely , the feasibility of the metal back trays , able to sustain without failure the shear forces induced by a glenosphere in reverse shoulder arthroplasty , led to experiment these devices in anatomical arthroplasty . in our experience ,
the fit induced by a precise ancillary system and the frontal and the eventual saggital screwing avoided any primary fixation problems and enhanced the indications to the cases necessitating a glenoid bone graft . a modification of the design , consisting of adding a small central peg on the pe allowed to well centre the pe before impaction on the metal tray . our results showed an increasing in the lateral offset in comparison with a normal contralateral side . another work ( accepted as a free paper in the sofcot 2012 meeting ) comparing the results between mb and cemented glenoid showed that this induced lateralisation did not influence the clinical results . radiologically , no loosening , no lucent lines , no narrowing of the joint space , witness of a polyethylene wear were found . despite the fact that , in the contrary than the literature [ 35 , 36 ] , the results were not statistically different between types a and b , the type of preoperative glenoid bone wear influenced our choice , as the only cases of dislocations were on posteriorly used glenoid bones , type b2 or c. in those cases where the posterior wear is not too important
, we recommend to increase the anterior reaming , to add a posterior bone graft and eventually to utilise the long keeled implant , designed for revisions . in old patients with a thin cuff
however , we prefer the noncemented glenoid , as , in case of secondary superior migration , the conversion in a reverse is facilitated , the same metal back tray been the support of the glenosphere . moreover , this new design , thanks to the anterior plate and the 2 available directions of screwing , allows to extend the indications to big glenoid bone loss necessiting a bone graft . however , this study presented some limitations : this is a medium - term study with a mean follow - up of 3 years.the measurement of the radiological lateral offset is technician dependent , and no scoring of the rll has been used.this is a multicentric not randomised study , and the choice of either a noncemented or a cemented prosthesis was often subjective and surgeon dependent . this is a medium - term study with a mean follow - up of 3 years . this is a multicentric not randomised study , and the choice of either a noncemented or a cemented prosthesis was often subjective and surgeon dependent . however , in the view of our good and predictable results , our indications of using a mb glenoid in total anatomical shoulder arthroplasty are increasing ( fig . 4diagram showing the growing number of mb glenoid implantations diagram showing the growing number of mb glenoid implantations in conclusion , we did not find in this medium - term clinical and radiological review of a noncemented metal back glenoid implant , the classical complications pointed out in the literature for those uncemented glenoid implants . with this new design , despite a radiological increase in the lateral offset , there is no proved risk for the cuff , no early polyethylene wear , no dissociation and the clinical results seem to be similar to the cemented glenoids but avoiding their frequent troubles such as evolutive lucent lines and loosenings . | [
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] |
each origin generates a pair of sister replication forks that subsequently move along parental dna in a bidirectional manner to undergo dna replication .
replication forks then terminate when they encounter forks from the adjacent replication origins moving in the opposite direction .
thus , replication initiated at each origin leads to duplication of a discrete dna region , which is called replicon . in budding yeast saccharomyces
cerevisiae , dna replication origins are defined by a 200-bp dna sequence called an autonomously replicating sequence , which was originally identified based on its ability to support the replication of plasmid dna ( newlon and theis 1993 ) .
the budding yeast genome ( about 14 mb ) contains 300 replication origins at average intervals of 50 kb ( raghuraman et al .
2001 ; yabuki et al . 2002 ; feng et al . 2006 ; nieduszynski et al . 2006 ) . in fission
yeast schizosaccharomyces pombe , replication origins lack a consensus dna sequence but consist of at - rich sequences ( robinson and bell 2005 ) .
it is estimated that at least half of the approximately 2,500 intergenic regions have potential origin activity ( dai et al . 2005 ) , and 460 of these are actually licensed for replication ( hayashi et al .
metazoan cells also lack any dna consensus sequence for replication origins ( robinson and bell 2005 ) , but intriguingly , the initiation points of replication at the nucleotide level show very similar distribution patterns within the origin regions in budding yeast , fission yeast , and humans ( bielinsky and gerbi 2001 ) . despite the difference in the dna sequences of replication origins between yeast and metazoa , the protein components assembling at replication origins and replication forks
the prereplicative complex ( prerc ) is a large protein complex , comprised of the origin recognition complex ( orc ) , cdc6 , cdt1 , and mcm27 ( blow and dutta 2005 ) .
the prerc is formed at replication origins from telophase and throughout g1 phase to license the origins for dna replication initiation . at the onset of s phase ,
more proteins such as dna polymerases and a sliding clamp called proliferating cell nuclear antigen ( pcna ) are loaded at origins , establishing a protein complex called the replisome , which subsequently moves with a replication fork to undergo dna replication ( johnson and o'donnell 2005 ) .
dna replication initiation at various origins ( origin firing ) occurs by a coordinated temporal program ; some origins fire early and others late during s phase . inside the nuclei ,
duplication of chromosomal dna is physically organized into replication factories , consisting of dna polymerases and other replication proteins . in this review article , we examine the spatial organization and regulation of dna replication within the nucleus and discuss how this spatial organization is linked to temporal regulation .
we focus on dna replication in budding yeast and fission yeast and , in chosen topics , compare yeast dna replication with that in bacteria and metazoans . in this context
, we briefly touch upon spatial regulation of dna damage and replication checkpoints , which are , however , reviewed in more detail in herrick and bensimon ( 2008 ) and branzei and foiani ( 2009 ) .
when replication origins are isolated and placed on minichromosomes , they normally replicate in early s phase in budding yeast ( ferguson and fangman 1992 ) .
however , in their normal chromosomal context , some origins show delayed firing within s phase .
this delay is due to proximal cis - acting chromosomal elements , telomeres , and other dna sequences for subtelomeric and nontelomeric late - firing origins , respectively ( ferguson and fangman 1992 ; friedman et al .
so far , among such cis - acting chromosomal elements , no consensus dna sequences , apart from telomeres , have been identified .
it has been shown that both subtelomeric and nontelomeric late - firing origins localize preferentially in the nuclear periphery during g1 phase ( heun et al .
does this nuclear periphery localization have a causative role in the late firing of replication origins during s phase ?
indeed , in various conditions , the nuclear periphery localization of origins is correlated with their delayed replication .
for example , cis - acting chromosomal elements , which determine the late firing of the origins , are also required for nuclear periphery localization ( friedman et al .
1996 ; heun et al . 2001 ) . moreover , after a subtelomeric late - firing origin was excised from its chromosome locus prior to g1 phase ( in g1 , telomeres localize preferentially at the nuclear periphery ) ; the origin advanced the timing of its firing to early s phase ( raghuraman et al .
1997 ) . however , in some circumstances , late firing of replication origins is not correlated with their nuclear periphery localization during g1 .
for example , after a normally early - firing origin was tethered to the nuclear periphery by targeted interaction with an integral membrane protein , the origin did not show late firing ( zappulla et al . 2002 ) .
moreover , genetic screening identified mutants that disrupt telomere localization at the nuclear periphery but still maintain late firing of subtelomeric origins ( hiraga et al .
therefore , nuclear periphery localization of replication origins is neither sufficient nor required for their late firing .
it seems that chromatin states and structures , such as silencing by sir proteins and chromosome - end binding of the ku complex , affect more directly the initiation timing of subtelomeric origins ( stevenson and gottschling 1999 ; cosgrove et al .
sir proteins and the ku complex also regulate the nuclear periphery localization of telomeres ( hediger et al . 2002 ; taddei and gasser 2004 ) ; however , the nuclear periphery localization is probably not a direct determinant of their replication timing .
perhaps a similar argument can be also applied for nontelomeric late - firing origins , although regulators other than sir and ku proteins may be involved in delaying their replication .
for example , it was shown that histone deacetylase rpd3 is important for delaying their replication ( vogelauer et al .
2009 ) ; it is known that rpd3 is targeted to promoters and coding regions and regulates their transcription ( kadosh and struhl 1997 ; carrozza et al .
, it does not seem that the subnuclear localization of replication origins per se determines their timing of replication initiation in yeast ; however , underlying chromatin states and structures probably regulate both their localization and initiation timing .
nonetheless , it is still possible that the subnuclear localization assists maintenance of underlying chromatin states and structures in a feedback and thereby affects replication timing moderately even if it is not an essential determinant .
for example , euchromatin and heterochromatin undergo dna replication in early and late s phase , respectively ( gilbert 2002 ) .
replication timing of a chromosomal region is correlated with its subnuclear localization and with chromatin states such as histone modifications ( hiratani et al .
upon replication initiation , dna polymerases and other replication proteins such as pcna and replication factor c assemble at a licensed replication origin , forming a replisome , which subsequently moves together with a replications fork to undergo dna replication ( johnson and o'donnell 2005 ) .
a range of evidence suggests that each replisome synthesizes both leading and lagging strands of dna simultaneously ( baker and bell 1998 ; waga and stillman 1998 ; johnson and o'donnell 2005 ) . in bacteria , one type of dna polymerase ( e.g. , dna polymerase iii in escherichia coli ) synthesizes both leading and lagging strands .
in contrast , in eukaryotes , the identity of dna polymerases that synthesize each strand had been unclear until recently .
the mutation rates were evaluated using polymerase mutants with reduced replication fidelity in budding yeast , and it was discovered that dna polymerase and primarily synthesize lagging and leading strands , respectively ( pursell et al . 2007 ; nick mcelhinny et al . 2008 ) .
it was originally thought that the two replisomes at sister forks ( i.e. , initiated from the same origin ) would behave separately since they travel in opposite directions along template dna .
however , it was found that on bacterial circular chromosomes where dna replication starts from a single defined origin , sister forks move along dna and normally complete dna replication with similar timing at a defined region on the chromosome ( bussiere and bastia 1999 ) . to explain this coordinated termination of dna replication
, it was proposed that two replisomes at sister forks ( sister replisomes ) remain attached during dna replication ( dingman 1974 ; falaschi 2000 ) .
this model predicts that template dna moves into two associated replisomes , and newly replicated sister dna strands are extruded as replication proceeds . such dna motion relative to centrally located stationary replisomes ( lemon and grossman 1998 ) was indeed confirmed in bacteria bacillus subtilis and caulobacter crescentus ( lemon and grossman 2000 ; jensen et al .
furthermore , electron microscopy of large tumor antigen ( t antigen ) in simian virus 40 , which functions as a dna helicase at replication forks ( herendeen and kelly 1996 ) , showed that unwound dna from viral replication origins forms two loops which are pinched by the same pair of associated t - antigen hexamers ( wessel et al .
on the other hand , in e. coli , sister replisomes separate shortly after dna replication initiation and undergo dna replication independently ( bates and kleckner 2005 ; reyes - lamothe et al .
in contrast to bacteria and viruses , it remained unknown until recently whether sister replisomes are associated together in eukaryotes . in budding yeast
, live - cell imaging was used to analyze the replication timing of chromosome loci ( fig . 1 ) ( kitamura et al .
2006 ) , at which bacteria - derived teto and laco arrays were integrated ( straight et al . 1996 ; michaelis et al .
these arrays bound tetr and laci proteins , fused with fluorescent proteins , and were thus visualized as small fluorescent dots .
the fluorescent dots increased their intensity upon their dna replication when the number of teto and laco arrays was doubled , which defined their replication timing by microscopy ( kitamura et al .
two loci were selected and visualized within a single replicon so that they locate at the opposite sides of the relevant replication origin and show similar replication timing ( based on a genome - wide replication timing data : raghuraman et al .
remarkably , these two loci came close to each other , increased their intensity , and subsequently diverged from each other during s phase ( kitamura et al .
such behavior of the two loci suggests that sister replisomes are associated together during replication of the replicon .
furthermore , in a separate study , nascent dna segments were pulse - labeled and observed by electron microscopy .
this study suggested that human sister replisomes are also associated with each other during dna replication ( ligasov et al .
1sister replisomes are associated with each other during replication in budding yeast . a model of a closely associated double replisome and expected behavior of two chromosomal loci , teto , and laco , which bound tetr-3cfp and gfp - laci , respectively ( top ) .
b two loci come close to each other upon dna replication . cfp ( red ) , gfp ( green ) , and bright field images of a representative cell are shown .
the teto and laco are visualized as small fluorescent of dots of cfp and gfp , respectively .
two loci came close to each other , increased their intensity ( 3 to 1 min ) and subsequently diverged from each other during s phase .
scale bar represents 1 m . the figure is adapted from kitamura et al .
( 2006 ) with permission ( copyright elsevier 2006 ) sister replisomes are associated with each other during replication in budding yeast .
a model of a closely associated double replisome and expected behavior of two chromosomal loci , teto , and laco , which bound tetr-3cfp and gfp - laci , respectively ( top ) .
b two loci come close to each other upon dna replication . cfp ( red ) , gfp ( green ) , and bright field images of a representative cell are shown .
the teto and laco are visualized as small fluorescent of dots of cfp and gfp , respectively .
two loci came close to each other , increased their intensity ( 3 to 1 min ) and subsequently diverged from each other during s phase .
scale bar represents 1 m . the figure is adapted from kitamura et al .
why do cells keep sister replisomes closely associated during replication ? what benefits can cells reap from it ?
one possibility is that the close association enables temporal coordination of dna replication between sister replisomes .
indeed , such temporal coordination was revealed in human cells : by labeling nascent dna on single - molecule dna fibers ( michalet et al .
1997 ; herrick et al . 2000 ) ; it was possible to measure the velocity of replication fork movements along template dna , and it was found that the majority pairs of sister forks showed very similar velocity ( conti et al .
if one fork changed its speed , its sister also changed its speed in a similar way .
given that replication forks in the adjacent replicon also shows similar velocity ( conti et al .
2007 ) , this temporal coordination may help replication forks in the same and neighboring replicons change their speed collaboratively and promptly , responding to replication stress such as the reduced amount of deoxy - nucleotides available in the nucleus .
the velocity of sister replication forks also show significant correlation in budding yeast ( fig .
2 ) ; thus , the temporal coordination seems to be conserved in evolution .
we used the genome - wide replication profile ( black line ; yabuki et al .
2002 ) , which represents the time ( minutes ) after release from factor arrest at which 50% of cells complete dna replication , along the chromosomes ( 1-kb intervals ) .
peaks and valleys ( rectangles pointing down and up , respectively ) of the profile represent replication origins and termini , respectively . to measure the velocity ,
first , we excluded a 5-kb region on each side of peaks and valleys in order to avoid errors due to smoothing when drawing the replication profile in that region .
second , the regions were selected for measurement of the velocity of the leftward and rightward forks ( red lines ) so that they end with the same replication timing ; for example , if the right valley goes deeper than the left , the selected region for the right terminated when the left one ended .
third , we chose replicons for the analysis only when their defined regions for measurement span more than 8 kb along a chromosome both at left and right sides , as smaller ones may give larger errors .
the replicon , locating at 508 kb on chromosome viii ( from the left telomere ) , was excluded from the analysis as it showed much larger fork velocity ( 1117 kb / min ) than others .
b as described in a , we chose 67 replicons out of 260 identified in yabuki et al .
the graph indicates that the velocity of replication fork movements shows significant correlation between sister forks ( pearson s correlation , r = 0.4725 , p < 0.0001 , n = 67 ) the velocity of replication fork movements is correlated between sister forks in budding yeast .
we used the genome - wide replication profile ( black line ; yabuki et al .
2002 ) , which represents the time ( minutes ) after release from factor arrest at which 50% of cells complete dna replication , along the chromosomes ( 1-kb intervals ) . peaks and valleys ( rectangles pointing down and up , respectively ) of the profile represent replication origins and termini , respectively . to measure the velocity , first , we excluded a 5-kb region on each side of peaks and valleys in order to avoid errors due to smoothing when drawing the replication profile in that region .
second , the regions were selected for measurement of the velocity of the leftward and rightward forks ( red lines ) so that they end with the same replication timing ; for example , if the right valley goes deeper than the left , the selected region for the right terminated when the left one ended .
third , we chose replicons for the analysis only when their defined regions for measurement span more than 8 kb along a chromosome both at left and right sides , as smaller ones may give larger errors .
the replicon , locating at 508 kb on chromosome viii ( from the left telomere ) , was excluded from the analysis as it showed much larger fork velocity ( 1117 kb / min ) than others .
b as described in a , we chose 67 replicons out of 260 identified in yabuki et al .
the graph indicates that the velocity of replication fork movements shows significant correlation between sister forks ( pearson s correlation , r = 0.4725 , p < 0.0001 , n = 67 ) the temporal coordination between associated sister replisomes would be indeed useful for replisomes to respond promptly to replication stress if this stress affects the whole genome .
on the other hand , it may be rather harmful if the replication stress is imposed locally on particular chromosome loci .
for example , when dna damage on a chromosomal region halts or terminates the motion of a fork ( branzei and foiani 2005 ) , the behavior of its sister fork would be also affected , widening the adverse effects of the dna damage . intriguingly , however , it was shown that in yeast cells , a replication fork continues to move while its sister fork is halted or terminated due to a dna double - strand break ( doksani et al .
similarly , within yeast rdna regions , halting of a replication fork by a replication - fork barrier did not stop or slow down the progression of its sister fork ( brewer and fangman 1988 ; linskens and huberman 1988 ) .
taken together , when a replication fork is stalled upon the encounter on a local replication obstacle , its sister can behave independently .
thus , there might be a mechanism that senses a stalled replication fork and uncouples it functionally from its sister fork ( herrick and bensimon 2008 ) . are there any other functional consequences or benefits of the association of sister replisomes ?
once a replication origin is unwound and replication forks are generated , the origin loses its ability to initiate replication , which requires prerc formation at the origin in eukaryotes ( see introduction ) and the origin methylation on both dna strands in bacteria ( boye et al . 2000 ) .
therefore , a half replicon might fail to replicate if one replisome could initiate without waiting for the other replisome to be loaded onto the origin . if avoidance of this problem is a major benefit of associated sister replisomes , this association might not be necessary once both of them start dna replication from an origin .
indeed , at least in bacterium e. coli , sister replisomes separate shortly after initiation and undergo dna replication independently ( bates and kleckner 2005 ; reyes - lamothe et al .
nonetheless , in other bacteria such as b. subtilis and c. crescentus , or in eukaryotes such as budding yeast and humans , sister replisomes seem to be associated for a longer time , perhaps throughout replication of the whole replicon ( see above ) .
the associated sister replisomes may coordinate the dna polymerase operation for two leading and two lagging strands to avoid chromosome entanglement and to facilitate smooth reeling in and out of unreplicated and replicated dna strands
. this spatial coordination might be particularly important in eukaryotic cells , in which more complex spatial regulation may be required as their multiple replicons are processed for dna replication in a single replication factory ( see below ) .
when mammalian cells are pulse - labeled with nucleoside analogs ( such as bromodeoxyuridine ( brdu ) ) or tagged nucleotides during s phase , dna replication appears to start at several discrete sites called replication foci ( nakamura et al .
studies with different mammalian cell lines showed that 1001,000 foci are observed in early s - phase nuclei ( berezney et al .
it is estimated that each focus contains 10100 replicons , which together represent a chromatin territory , a stable unit maintained until the next cell cycle ( jackson and pombo 1998 ) .
the average replication focus is estimated to contain 1 mbp of genomic dna in mouse cells ( ma et al .
similar replication foci were also observed in budding yeast nuclei . in vitro experiments using isolated yeast nuclei showed that a tagged nucleotide was incorporated as 1520 discrete foci in an orc - dependent and origin - specific manner ( pasero et al .
because yeast cells lack a thymidine kinase ( tk ) , they can not utilize brdu or isotope - labeled thymidine , which is widely used to visualize sites of dna replication in intact mammalian cells . however , introduction of heterogeneous tk enabled yeast cells to incorporate brdu in vivo ( mcneil and friesen 1981 ; lengronne et al .
, several studies have shown that brdu is incorporated as discrete foci into nuclei using immunostaining ( lengronne et al .
, however , it is unlikely that replication foci represent stable chromatin units maintained to the next cell cycle , in contrast to mammalian cells ( see above ) .
in fact , a chromosome arm locus can move vigorously covering a wide area of the yeast nucleus in a single cell cycle ( berger et al . 2008 ; our unpublished results ) .
this is presumably due to the small size of the yeast nucleus ( see fig .
3 ) and may also reflect potentially different chromatin organization between yeast and mammalian cells .
fig . 3comparing the size of replication factories and the nucleus between budding yeast and mammalian cells .
the subnuclear localization of pcna fused with gfp during s phase in a mouse cell ( top left ; scale bar 1 m ; adapted from leonhardt et al .
( 2000 ) with permission ) and in budding yeast ( top right , asterisks ; scale bar 1 m ) . a magnified image of the yeast nucleus is also shown ( bottom right ) .
the nuclei of yeast and mouse cells are outlined in yellow for comparison of their sizes .
note that a large factory is composed of several small ones in a mouse cell ( leonhardt et al .
2000 ; z series , bottom left ) comparing the size of replication factories and the nucleus between budding yeast and mammalian cells .
the subnuclear localization of pcna fused with gfp during s phase in a mouse cell ( top left ; scale bar 1 m ; adapted from leonhardt et al .
( 2000 ) with permission ) and in budding yeast ( top right , asterisks ; scale bar 1 m ) . a magnified image of the yeast nucleus is also shown ( bottom right ) .
the nuclei of yeast and mouse cells are outlined in yellow for comparison of their sizes .
note that a large factory is composed of several small ones in a mouse cell ( leonhardt et al .
2000 ; z series , bottom left ) when replisome components such as dna polymerase a and pcna are visualized by immunolabeling in mammalian cells , they show discrete punctate signals in the nucleus during s phase ( frouin et al . 2003 ) .
these punctate signals are called replication factories as they colocalize with replication foci , i.e. , the sites of ongoing dna replication ; thus , replisome components are concentrated into discrete foci , in which multiple replicons are processed for replication ( hozk et al . 1993 ) .
the organization and dynamics of replication factories were also examined in live mammalian cells that expressed pcna , fused with a fluorescent protein , ( leonhardt et al .
live - cell imaging revealed that replication factories show dynamic assembly and disassembly throughout s phase .
replication factories are also formed in the nucleus of budding yeast , as revealed by immunostaining and live - cell imaging ( ohya et al .
for example , when pcna or dna polymerases and were visualized with fluorescent proteins , yeast cells showed 1015 globular signals in their nuclei during s phase ( kitamura et al .
the size of each globular signal , i.e. , replication factory , was up to 200 nm in diameter , which is smaller than the 0.52-mm diameter replication factories of mammalian cells ( leonhardt et al .
2000 ; fig . 3 ) . however , given that large factories are composed of several small ones in mammalian cells ( leonhardt et al .
2000 ) , yeast factories may correspond to the small units of mammalian factories in terms of the size and mode of function .
replication factories in yeast change their shapes and show dynamic assembly and reassembly , similarly to mammalian cells .
these replication factories at least partially colocalize with replication foci , visualized with pulse - labeled brdu , in fixed cells ( hiraga et al . 2005 ; kitamura et al .
, when a teto array ( bound by tetr fusion with a fluorescent protein ) was visualized as a small fluorescent dot on a chromosome locus , the dot increased its intensity specifically upon colocalization with a replication factory , thus , confirming de novo dna replication at factories in live cells ( kitamura et al .
fission yeast nuclei also show globular signals of pcna and dna polymerase during s phase ( meister et al .
is a replication factory a preformed complex , inside of which replication is initiated ? alternatively , only after replication initiation , is the factory formed as a result of assembly of replisomes undergoing replication ?
a number of evidences suggest that the factory is formed only after dna replication initiation .
for example , the factory formation is dependent on the activity of cyclin - dependent kinase ( cdk ) 2 that triggers dna replication initiation in vertebrate cells ( cardoso et al . 1993
; jackson et al . 1995 ; yan and newport 1995 ) . on the other hand , punctate signals of replication protein a ( rpa ) appear prior to dna replication in xenopus egg extract system ( adachi and laemmli 1992 , 1994 ) . however , it turns out that rpa , which binds single - strand dna with dependence on prerc ( and therefore , directly relevant to dna replication ) , forms factories only after replication initiation in s phase ( jackson et al .
replication factories are also formed after replication initiation in yeast cells , where the factory formation is delayed if the activation of s - phase cdk is retarded ( kitamura et al .
if the origin licensing becomes defective in yeast cells by depletion of cdc6 , the factory formation is abolished even if other s - phase events such as s - phase cdk activation takes place normally .
these results suggest that in cells ranging from yeast to vertebrates , the assembly of active replisomes undergoing dna replication leads to the formation of replication factories . as discussed above
, replication factories show dynamic assembly and disassembly during s phase . as a result ,
in mammalian cells , a large number of factories are distributed throughout the nucleus , except for the nucleolus , during early s phase . during mid s phase
then , in late s phase , large factories , composed of several independent small ones ( see fig . 3 ) , are formed inside the nucleus ( leonhardt et al .
the change in the distribution of replication factories was also examined in fission yeast ( meister et al .
2007 ) . after the onset of s phase , factories appear throughout the nucleus except for the nucleolus . later in s phase ,
interestingly , this temporal pattern is regulated by cds1 ( chk2 ) kinase , a regulator of s - phase checkpoint , even in the absence of replication stress ( meister et al .
, it was shown that another checkpoint kinase chk1 is involved in temporal pattern of origin firing during unperturbed s phase ( maya - mendoza et al .
when dna replication is halted due to replication stress , the replication checkpoint pathway is also required to maintain the organization of replication factories ( dimitrova and gilbert 2000 ) . in mammalian cells ,
a replication focus is considered to represent a cluster of multiple replicons ( 10100 ) that synchronously fire in s phase , although the number of replicons per focus and its synchrony seem to be highly heterogeneous ( berezney et al . 2000 ) .
what group of replicons forms a replication focus that is processed for replication in a single replication factory ?
intriguingly , as s phase proceeds , a replication focus appears in close proximity to a focus replicating earlier , suggesting that replication may proceed to neighboring regions by a domino effect involving local changes of chromatin states ( sporbert et al .
2004 ) . in budding yeast , neighboring replicons along a chromosome region can be grouped into clusters , each of which comprises several origins that initiate replication with similar timing and behave similarly after deletion of an s - phase cyclin ( yabuki et al .
the origins in the same cluster might be processed in the same replication factory . on the other hand , replicons on different chromosomes , such as those at centromere or telomere regions ( see below ) , might be processed in the same factory due to their proximity in the nucleus . are there any benefits of forming replication factories and undergoing replication at discrete sites ?
one possible benefit might be that by concentrating replisome components and dna - building materials such as deoxy - nucleotides , cells may increase the efficiency of dna replication .
in addition , a group of replicons processed in each replication factory may form a unit that responds coordinately to a replication stress or dna damage .
for example , it is suggested that under a replication stress , the replication initiation from dormant origins is promoted within the factories that have been already formed while replication initiation is suppressed outside of these factories ( ge et al .
moreover , when the speed of replication forks changes , this affects the programming of origin firing in the next cell cycle ( courbet et al .
2008 ) , in which replication factories may signal a change of the fork speed .
in this section , we briefly discuss dna replication at centromeres and telomeres as examples of spatial regulation of replication in particular chromosome contexts . in budding yeast ,
spindle pole bodies ( spbs ; microtubule - organizing centers in yeast ) are embedded in the nuclear envelope , which remains intact throughout the cell cycle ( closed mitosis ; heath 1980 ) , and kinetochores are tethered to spbs by microtubules during most of the cell cycle .
however , it was revealed that , upon centromere dna replication , kinetochores are transiently disassembled , causing centromere detachment from microtubules for 12 min ( kitamura et al .
subsequently kinetochores are reassembled and interact with microtubules again . because centromeres are replicated in early s phase in budding yeast ( mccarroll and fangman 1988 ; raghuraman et al .
the timing of these events is presumably crucial to make a time window sufficient ( even in the absence of g2 phase ; see below ) for establishment of proper kinetochore
telomeres in budding yeast tend to localize at the nuclear periphery from the end of mitosis to g1 phase , and this localization depends on the ku- and sir - mediated anchoring mechanisms ( hediger et al . 2002 ; taddei and gasser 2004 ) .
prior to anaphase , however , telomeres localize randomly within the nucleus ( laroche et al .
it was demonstrated that the delocalization of telomeres from the nuclear periphery is triggered by their dna replication , which suppresses the ku - mediated anchoring mechanism in late s phase ( ebrahimi and donaldson 2008 ) .
the detachment of telomeres from the nuclear periphery probably enhances telomere mobility in the nucleus , which has an advantage in subsequent chromosome segregation .
thus , replication at centromeres and telomeres is closely linked to chromosome segregation in mitosis .
this link is probably crucial in budding yeast as it is thought that s phase and mitosis are overlapped , and g2 phase is absent in this organism ( kitamura et al .
dna replication is a spatially regulated process at multiple levels ; i.e. , from replisome architecture to subnuclear chromosome organization . the spatial regulation of dna replication is closely linked to its temporal regulation .
both spatial and temporal regulations seem to be important for efficient duplication of chromosomes , for proper responses to replication stresses and for coupling dna replication with other cellular events such as chromosome segregation .
several new methods have been developed and widely applied over the last decade , making considerable contribution to the research field .
for example , genome - wide approaches have introduced unbiased and thorough landscapes of genome replication in yeast and other organisms ( raghuraman et al .
2004 ) . single - cell and single - molecule assays have enabled analyses of dna replication in high spatial and temporal resolution and have opened a window into how dna replication differs from cell to cell and from chromosome to chromosome ( michalet et al . 1997 ; herrick et al .
further development of these methods and other biochemical , genetic , and cell biological approaches will advance further the research of chromosome duplication . | duplication of chromosomal dna is a temporally and spatially regulated process .
the timing of dna replication initiation at various origins is highly coordinated ; some origins fire early and others late during s phase .
moreover , inside the nuclei , the bulk of dna replication is physically organized in replication factories , consisting of dna polymerases and other replication proteins . in this review article , we discuss how dna replication is organized and regulated spatially within the nucleus and how this spatial organization is linked to temporal regulation .
we focus on dna replication in budding yeast and fission yeast and , where applicable , compare yeast dna replication with that in bacteria and metazoans . | Introduction
Subnuclear localization of replication origins and timing of their firing
Replisome architecture and association of sister replisomes
Possible benefits of the association of sister replisomes
Replication foci and replication factory
Replication factories: regulation, organization, and possible benefits
DNA replication at centromeres and telomeres
Conclusions and perspectives | each origin generates a pair of sister replication forks that subsequently move along parental dna in a bidirectional manner to undergo dna replication . thus , replication initiated at each origin leads to duplication of a discrete dna region , which is called replicon . in budding yeast saccharomyces
cerevisiae , dna replication origins are defined by a 200-bp dna sequence called an autonomously replicating sequence , which was originally identified based on its ability to support the replication of plasmid dna ( newlon and theis 1993 ) . in fission
yeast schizosaccharomyces pombe , replication origins lack a consensus dna sequence but consist of at - rich sequences ( robinson and bell 2005 ) . metazoan cells also lack any dna consensus sequence for replication origins ( robinson and bell 2005 ) , but intriguingly , the initiation points of replication at the nucleotide level show very similar distribution patterns within the origin regions in budding yeast , fission yeast , and humans ( bielinsky and gerbi 2001 ) . despite the difference in the dna sequences of replication origins between yeast and metazoa , the protein components assembling at replication origins and replication forks
the prereplicative complex ( prerc ) is a large protein complex , comprised of the origin recognition complex ( orc ) , cdc6 , cdt1 , and mcm27 ( blow and dutta 2005 ) . the prerc is formed at replication origins from telophase and throughout g1 phase to license the origins for dna replication initiation . at the onset of s phase ,
more proteins such as dna polymerases and a sliding clamp called proliferating cell nuclear antigen ( pcna ) are loaded at origins , establishing a protein complex called the replisome , which subsequently moves with a replication fork to undergo dna replication ( johnson and o'donnell 2005 ) . dna replication initiation at various origins ( origin firing ) occurs by a coordinated temporal program ; some origins fire early and others late during s phase . inside the nuclei ,
duplication of chromosomal dna is physically organized into replication factories , consisting of dna polymerases and other replication proteins . in this review article , we examine the spatial organization and regulation of dna replication within the nucleus and discuss how this spatial organization is linked to temporal regulation . we focus on dna replication in budding yeast and fission yeast and , in chosen topics , compare yeast dna replication with that in bacteria and metazoans . in this context
, we briefly touch upon spatial regulation of dna damage and replication checkpoints , which are , however , reviewed in more detail in herrick and bensimon ( 2008 ) and branzei and foiani ( 2009 ) . when replication origins are isolated and placed on minichromosomes , they normally replicate in early s phase in budding yeast ( ferguson and fangman 1992 ) . however , in their normal chromosomal context , some origins show delayed firing within s phase . this delay is due to proximal cis - acting chromosomal elements , telomeres , and other dna sequences for subtelomeric and nontelomeric late - firing origins , respectively ( ferguson and fangman 1992 ; friedman et al . does this nuclear periphery localization have a causative role in the late firing of replication origins during s phase ? indeed , in various conditions , the nuclear periphery localization of origins is correlated with their delayed replication . moreover , after a subtelomeric late - firing origin was excised from its chromosome locus prior to g1 phase ( in g1 , telomeres localize preferentially at the nuclear periphery ) ; the origin advanced the timing of its firing to early s phase ( raghuraman et al . moreover , genetic screening identified mutants that disrupt telomere localization at the nuclear periphery but still maintain late firing of subtelomeric origins ( hiraga et al . , it does not seem that the subnuclear localization of replication origins per se determines their timing of replication initiation in yeast ; however , underlying chromatin states and structures probably regulate both their localization and initiation timing . for example , euchromatin and heterochromatin undergo dna replication in early and late s phase , respectively ( gilbert 2002 ) . upon replication initiation , dna polymerases and other replication proteins such as pcna and replication factor c assemble at a licensed replication origin , forming a replisome , which subsequently moves together with a replications fork to undergo dna replication ( johnson and o'donnell 2005 ) . in bacteria , one type of dna polymerase ( e.g. in contrast , in eukaryotes , the identity of dna polymerases that synthesize each strand had been unclear until recently . the mutation rates were evaluated using polymerase mutants with reduced replication fidelity in budding yeast , and it was discovered that dna polymerase and primarily synthesize lagging and leading strands , respectively ( pursell et al . however , it was found that on bacterial circular chromosomes where dna replication starts from a single defined origin , sister forks move along dna and normally complete dna replication with similar timing at a defined region on the chromosome ( bussiere and bastia 1999 ) . to explain this coordinated termination of dna replication
, it was proposed that two replisomes at sister forks ( sister replisomes ) remain attached during dna replication ( dingman 1974 ; falaschi 2000 ) . on the other hand , in e. coli , sister replisomes separate shortly after dna replication initiation and undergo dna replication independently ( bates and kleckner 2005 ; reyes - lamothe et al . in contrast to bacteria and viruses , it remained unknown until recently whether sister replisomes are associated together in eukaryotes . in budding yeast
, live - cell imaging was used to analyze the replication timing of chromosome loci ( fig . the fluorescent dots increased their intensity upon their dna replication when the number of teto and laco arrays was doubled , which defined their replication timing by microscopy ( kitamura et al . remarkably , these two loci came close to each other , increased their intensity , and subsequently diverged from each other during s phase ( kitamura et al . this study suggested that human sister replisomes are also associated with each other during dna replication ( ligasov et al . 1sister replisomes are associated with each other during replication in budding yeast . b two loci come close to each other upon dna replication . two loci came close to each other , increased their intensity ( 3 to 1 min ) and subsequently diverged from each other during s phase . ( 2006 ) with permission ( copyright elsevier 2006 ) sister replisomes are associated with each other during replication in budding yeast . b two loci come close to each other upon dna replication . two loci came close to each other , increased their intensity ( 3 to 1 min ) and subsequently diverged from each other during s phase . one possibility is that the close association enables temporal coordination of dna replication between sister replisomes . the velocity of sister replication forks also show significant correlation in budding yeast ( fig . 2002 ) , which represents the time ( minutes ) after release from factor arrest at which 50% of cells complete dna replication , along the chromosomes ( 1-kb intervals ) . b as described in a , we chose 67 replicons out of 260 identified in yabuki et al . the graph indicates that the velocity of replication fork movements shows significant correlation between sister forks ( pearson s correlation , r = 0.4725 , p < 0.0001 , n = 67 ) the velocity of replication fork movements is correlated between sister forks in budding yeast . second , the regions were selected for measurement of the velocity of the leftward and rightward forks ( red lines ) so that they end with the same replication timing ; for example , if the right valley goes deeper than the left , the selected region for the right terminated when the left one ended . third , we chose replicons for the analysis only when their defined regions for measurement span more than 8 kb along a chromosome both at left and right sides , as smaller ones may give larger errors . b as described in a , we chose 67 replicons out of 260 identified in yabuki et al . for example , when dna damage on a chromosomal region halts or terminates the motion of a fork ( branzei and foiani 2005 ) , the behavior of its sister fork would be also affected , widening the adverse effects of the dna damage . intriguingly , however , it was shown that in yeast cells , a replication fork continues to move while its sister fork is halted or terminated due to a dna double - strand break ( doksani et al . once a replication origin is unwound and replication forks are generated , the origin loses its ability to initiate replication , which requires prerc formation at the origin in eukaryotes ( see introduction ) and the origin methylation on both dna strands in bacteria ( boye et al . if avoidance of this problem is a major benefit of associated sister replisomes , this association might not be necessary once both of them start dna replication from an origin . indeed , at least in bacterium e. coli , sister replisomes separate shortly after initiation and undergo dna replication independently ( bates and kleckner 2005 ; reyes - lamothe et al . nonetheless , in other bacteria such as b. subtilis and c. crescentus , or in eukaryotes such as budding yeast and humans , sister replisomes seem to be associated for a longer time , perhaps throughout replication of the whole replicon ( see above ) . this spatial coordination might be particularly important in eukaryotic cells , in which more complex spatial regulation may be required as their multiple replicons are processed for dna replication in a single replication factory ( see below ) . when mammalian cells are pulse - labeled with nucleoside analogs ( such as bromodeoxyuridine ( brdu ) ) or tagged nucleotides during s phase , dna replication appears to start at several discrete sites called replication foci ( nakamura et al . similar replication foci were also observed in budding yeast nuclei . because yeast cells lack a thymidine kinase ( tk ) , they can not utilize brdu or isotope - labeled thymidine , which is widely used to visualize sites of dna replication in intact mammalian cells . 3 ) and may also reflect potentially different chromatin organization between yeast and mammalian cells . 3comparing the size of replication factories and the nucleus between budding yeast and mammalian cells . the subnuclear localization of pcna fused with gfp during s phase in a mouse cell ( top left ; scale bar 1 m ; adapted from leonhardt et al . ( 2000 ) with permission ) and in budding yeast ( top right , asterisks ; scale bar 1 m ) . the nuclei of yeast and mouse cells are outlined in yellow for comparison of their sizes . 2000 ; z series , bottom left ) comparing the size of replication factories and the nucleus between budding yeast and mammalian cells . the subnuclear localization of pcna fused with gfp during s phase in a mouse cell ( top left ; scale bar 1 m ; adapted from leonhardt et al . ( 2000 ) with permission ) and in budding yeast ( top right , asterisks ; scale bar 1 m ) . the nuclei of yeast and mouse cells are outlined in yellow for comparison of their sizes . 2000 ; z series , bottom left ) when replisome components such as dna polymerase a and pcna are visualized by immunolabeling in mammalian cells , they show discrete punctate signals in the nucleus during s phase ( frouin et al . , the sites of ongoing dna replication ; thus , replisome components are concentrated into discrete foci , in which multiple replicons are processed for replication ( hozk et al . the organization and dynamics of replication factories were also examined in live mammalian cells that expressed pcna , fused with a fluorescent protein , ( leonhardt et al . live - cell imaging revealed that replication factories show dynamic assembly and disassembly throughout s phase . replication factories are also formed in the nucleus of budding yeast , as revealed by immunostaining and live - cell imaging ( ohya et al . for example , when pcna or dna polymerases and were visualized with fluorescent proteins , yeast cells showed 1015 globular signals in their nuclei during s phase ( kitamura et al . , replication factory , was up to 200 nm in diameter , which is smaller than the 0.52-mm diameter replication factories of mammalian cells ( leonhardt et al . these replication factories at least partially colocalize with replication foci , visualized with pulse - labeled brdu , in fixed cells ( hiraga et al . , when a teto array ( bound by tetr fusion with a fluorescent protein ) was visualized as a small fluorescent dot on a chromosome locus , the dot increased its intensity specifically upon colocalization with a replication factory , thus , confirming de novo dna replication at factories in live cells ( kitamura et al . fission yeast nuclei also show globular signals of pcna and dna polymerase during s phase ( meister et al . is a replication factory a preformed complex , inside of which replication is initiated ? a number of evidences suggest that the factory is formed only after dna replication initiation . for example , the factory formation is dependent on the activity of cyclin - dependent kinase ( cdk ) 2 that triggers dna replication initiation in vertebrate cells ( cardoso et al . on the other hand , punctate signals of replication protein a ( rpa ) appear prior to dna replication in xenopus egg extract system ( adachi and laemmli 1992 , 1994 ) . however , it turns out that rpa , which binds single - strand dna with dependence on prerc ( and therefore , directly relevant to dna replication ) , forms factories only after replication initiation in s phase ( jackson et al . replication factories are also formed after replication initiation in yeast cells , where the factory formation is delayed if the activation of s - phase cdk is retarded ( kitamura et al . these results suggest that in cells ranging from yeast to vertebrates , the assembly of active replisomes undergoing dna replication leads to the formation of replication factories . as discussed above
, replication factories show dynamic assembly and disassembly during s phase . as a result ,
in mammalian cells , a large number of factories are distributed throughout the nucleus , except for the nucleolus , during early s phase . during mid s phase
then , in late s phase , large factories , composed of several independent small ones ( see fig . 3 ) , are formed inside the nucleus ( leonhardt et al . the change in the distribution of replication factories was also examined in fission yeast ( meister et al . after the onset of s phase , factories appear throughout the nucleus except for the nucleolus . later in s phase ,
interestingly , this temporal pattern is regulated by cds1 ( chk2 ) kinase , a regulator of s - phase checkpoint , even in the absence of replication stress ( meister et al . when dna replication is halted due to replication stress , the replication checkpoint pathway is also required to maintain the organization of replication factories ( dimitrova and gilbert 2000 ) . in mammalian cells ,
a replication focus is considered to represent a cluster of multiple replicons ( 10100 ) that synchronously fire in s phase , although the number of replicons per focus and its synchrony seem to be highly heterogeneous ( berezney et al . what group of replicons forms a replication focus that is processed for replication in a single replication factory ? intriguingly , as s phase proceeds , a replication focus appears in close proximity to a focus replicating earlier , suggesting that replication may proceed to neighboring regions by a domino effect involving local changes of chromatin states ( sporbert et al . in budding yeast , neighboring replicons along a chromosome region can be grouped into clusters , each of which comprises several origins that initiate replication with similar timing and behave similarly after deletion of an s - phase cyclin ( yabuki et al . on the other hand , replicons on different chromosomes , such as those at centromere or telomere regions ( see below ) , might be processed in the same factory due to their proximity in the nucleus . are there any benefits of forming replication factories and undergoing replication at discrete sites ? one possible benefit might be that by concentrating replisome components and dna - building materials such as deoxy - nucleotides , cells may increase the efficiency of dna replication . for example , it is suggested that under a replication stress , the replication initiation from dormant origins is promoted within the factories that have been already formed while replication initiation is suppressed outside of these factories ( ge et al . moreover , when the speed of replication forks changes , this affects the programming of origin firing in the next cell cycle ( courbet et al . in this section , we briefly discuss dna replication at centromeres and telomeres as examples of spatial regulation of replication in particular chromosome contexts . in budding yeast ,
spindle pole bodies ( spbs ; microtubule - organizing centers in yeast ) are embedded in the nuclear envelope , which remains intact throughout the cell cycle ( closed mitosis ; heath 1980 ) , and kinetochores are tethered to spbs by microtubules during most of the cell cycle . however , it was revealed that , upon centromere dna replication , kinetochores are transiently disassembled , causing centromere detachment from microtubules for 12 min ( kitamura et al . because centromeres are replicated in early s phase in budding yeast ( mccarroll and fangman 1988 ; raghuraman et al . the timing of these events is presumably crucial to make a time window sufficient ( even in the absence of g2 phase ; see below ) for establishment of proper kinetochore
telomeres in budding yeast tend to localize at the nuclear periphery from the end of mitosis to g1 phase , and this localization depends on the ku- and sir - mediated anchoring mechanisms ( hediger et al . prior to anaphase , however , telomeres localize randomly within the nucleus ( laroche et al . it was demonstrated that the delocalization of telomeres from the nuclear periphery is triggered by their dna replication , which suppresses the ku - mediated anchoring mechanism in late s phase ( ebrahimi and donaldson 2008 ) . the detachment of telomeres from the nuclear periphery probably enhances telomere mobility in the nucleus , which has an advantage in subsequent chromosome segregation . this link is probably crucial in budding yeast as it is thought that s phase and mitosis are overlapped , and g2 phase is absent in this organism ( kitamura et al . dna replication is a spatially regulated process at multiple levels ; i.e. the spatial regulation of dna replication is closely linked to its temporal regulation . both spatial and temporal regulations seem to be important for efficient duplication of chromosomes , for proper responses to replication stresses and for coupling dna replication with other cellular events such as chromosome segregation . for example , genome - wide approaches have introduced unbiased and thorough landscapes of genome replication in yeast and other organisms ( raghuraman et al . single - cell and single - molecule assays have enabled analyses of dna replication in high spatial and temporal resolution and have opened a window into how dna replication differs from cell to cell and from chromosome to chromosome ( michalet et al . | [
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eight healthy nondiabetic volunteers ( four men and four women aged 23.2 2.5 years with bmi 23.9 4.5 kg / m ) were enrolled for this study .
the study was approved by the institutional review board of the radboud university nijmegen medical centre , and all volunteers gave written informed consent before participation .
all participants were examined on two occasions : once under euglycemic conditions and once under hypoglycemic conditions , scheduled in random order and separated by at least 3 weeks .
female subjects were tested at 4- or 8-week intervals to ensure that experiments took place during corresponding periods of the menstrual cycle .
subjects came to the magnetic resonance research facility at 8:00 a.m. after an overnight fast and after having abstained from alcohol and caffeine - containing substances for 24 h. the brachial artery of the nondominant arm was cannulated under local anesthesia for frequent blood sampling .
an intravenous catheter was inserted in the antecubital vein of the contralateral arm for administration of c - glucose and insulin . after a 30-min equilibration period
, subjects were placed on the scanner bed in supine position with their heads positioned in the magnetic resonance coil .
arterial blood was sampled to obtain baseline variables , and reference spectra were obtained without administration of exogenous c - labeled material during the next 30 min .
subsequently , a hyperinsulinemic ( 60 mu / min / m , equaling approximately 1.5 mu / min / kg ) euglycemic ( 5.0 mmol / l ) or hypoglycemic ( 3.0 mmol / l ) glucose clamp was initiated with [ 1-c]glucose 20% w / w , as described previously ( 12 ) .
briefly , a bolus of 30 ml of 100% enriched [ 1-c]glucose 20% w / w was infused over 10 min at the initiation of the clamp to rapidly increase plasma c enrichment . for the remainder of the experiments , variable infusions of 40 or 50% enriched
[ 1-c]glucose 20% w / w were infused during the euglycemic and hypoglycemic experiments , respectively , in order to maintain plasma glucose levels at predetermined target values .
blood was sampled every 5 min for immediate determination of plasma glucose by the glucose oxidation method ( beckmann glucose analyzer ii ; beckman coulter , fullerton , ca ) and later determination of plasma c isotopic enrichment of glucose and lactate by high - resolution proton nuclear magnetic resonance ( h nmr ) at 500 mhz ( 15,16 ) .
every 30 min , additional blood was sampled for measurement of insulin and counterregulatory hormones as previously described ( 12 ) .
all studies were performed on a 3 t magnetic resonance system ( magnetom trio ; siemens , erlangen , germany ) with a h volume coil and a circularly polarized c surface coil inserted into the h coil ( 17 ) . a distortionless enhanced polarization transfer ( dept )
sequence for h to c polarization transfer combined with proton image - selected in vivo spectroscopy ( h isis ) localization ( 18 ) was used for acquisition of c magnetic resonance spectra .
adiabatic c radiofrequency pulses were used in all sequences to ensure homogeneous excitation using the c surface coil , as well as wideband alternating phase low - power technique for zero residue splitting ( waltz-16 ) proton decoupling ( 19 ) to simplify the spectra and enhance signal - to - noise ratio ( snr ) .
a voxel of 125 ml was placed in occipital brain tissue . in all experiments , one spectrum consisted of 72 repetitions of 2 s , allowing for a time resolution of 2.5 min .
eight reference spectra were obtained before the start of [ 1-c]glucose infusion , during which c mr spectra were acquired continuously .
visual stimulation was avoided by dimming the lights in the magnet room during the experiments .
the eight c mr reference spectra were averaged and subtracted from the dynamic spectra acquired during the c glucose clamp to correct for natural abundance c magnetic resonance signals . to enhance the snr
the resulting spectra were fitted with the advance magnetic resonance ( amares ) algorithm ( 20 ) in the java - based mr user interface ( jmrui ) software package ( 21 ) .
the natural abundance signal of myo - inositol ( mi ) was used to quantify glutamate c4 and glutamate c3 in the spectra based on the premise that mi has a stable concentration of 6 mol / g and is not labeled with c in the time frame of the experiment ( 22 ) .
the natural abundance signal of mi was quantified in jmrui from the spectra obtained by adding all dynamic spectra before correction with reference spectra .
dept c magnetic resonance spectra measured from a phantom were used to eliminate effects of the pulse sequence profile on the experimental spectra .
rates of metabolic fluxes were determined using a standard one - compartment metabolic model ( 14,23,24 ) as depicted in fig .
these rates ( v ) were assessed from the time courses of incorporation of c isotopes into different metabolites .
time courses of glutamate c4 and glutamate c3 concentrations and plasma values and isotopic enrichments of glucose were used as input factors for the model . in this specific situation
, it was also decided to take plasma lactate values and isotopic enrichment into account as input variables for the modeling process .
the model was implemented in matlab r2008b ( mathworks , natick , ma ) and consisted of differential equations describing the inflow and outflow of label from each metabolite pool .
these equations were solved numerically , and a nonlinear least - squares approach was used to fit the model to the experimental data by varying free flux parameters representing vtca , the loss of label through exchange with unlabeled glutamine ( vefflux ) , and the exchange of intracellular lactate with plasma lactate ( vdil ) .
vx , representing the exchange between -ketoglutarate and glutamate , was assumed to be 5 mol / g / min .
this value was fixed to ensure stable fitting . in literature , there is no consensus on the value of vx ; it is either determined to be much higher compared with vtca ( 25 ) or to be in the same order of vtca ( 23,26 ) .
however , for this study comparing two datasets , the choice of vx is not critical ; it will influence absolute values but equally for both datasets and therefore will not compromise the comparison of the two glycemic conditions .
data of all individual volunteers were fitted separately and inspected for the goodness of fit by the cost value , a parameter that represents the difference between the measured concentrations and the corresponding estimates by the model . from these fits ,
one - compartment model for uptake of [ 1-c]glucose and its conversion into labeled metabolites .
brain glucose uptake from the blood was modeled by reversible michaelis - menten kinetics ( 40 ) .
the cycling of glutamate and glutamine between neurons and astroglia is approached by the parameter vgln and assumed equal to vtca .
vtca , vdil , and vefflux are the free parameters of the model , representing tca cycle flux , exchange of plasma , and brain lactate and efflux of labeled glutamine , respectively .
vldh represents lactate dehydrogenase and was assumed to be 3 mol / g / min ; vx represents the exchange between -ketoglutarate and glutamate over the mitochondrial membrane and was assumed to be 5 mol / g / min .
furthermore , the following assumptions were made for total pool concentrations : [ glu ] : 10 mmol / l ; [ gln ] : 2.5 mmol / l ; [ oaa ] ; 0.3 mmol / l ; [ kg ] : 0.25 mmol / l ; [ asp ] : 1.5 mmol / l ; and [ pyr ] : 0.15 mmol / l .
serial data ( e.g. , plasma hormone and lactate levels ) were compared by repeated - measures anova , and differences in means were tested by paired , two - tailed student t tests .
graphpad prism 4 ( graphpad , la jolla , ca ) was used for statistical analysis , and a p value < 0.05 was considered statistically significant .
subjects came to the magnetic resonance research facility at 8:00 a.m. after an overnight fast and after having abstained from alcohol and caffeine - containing substances for 24 h. the brachial artery of the nondominant arm was cannulated under local anesthesia for frequent blood sampling .
an intravenous catheter was inserted in the antecubital vein of the contralateral arm for administration of c - glucose and insulin . after a 30-min equilibration period
, subjects were placed on the scanner bed in supine position with their heads positioned in the magnetic resonance coil .
arterial blood was sampled to obtain baseline variables , and reference spectra were obtained without administration of exogenous c - labeled material during the next 30 min .
subsequently , a hyperinsulinemic ( 60 mu / min / m , equaling approximately 1.5 mu / min / kg ) euglycemic ( 5.0 mmol / l ) or hypoglycemic ( 3.0 mmol / l ) glucose clamp was initiated with [ 1-c]glucose 20% w / w , as described previously ( 12 ) .
briefly , a bolus of 30 ml of 100% enriched [ 1-c]glucose 20% w / w was infused over 10 min at the initiation of the clamp to rapidly increase plasma c enrichment . for the remainder of the experiments , variable infusions of 40 or 50% enriched
[ 1-c]glucose 20% w / w were infused during the euglycemic and hypoglycemic experiments , respectively , in order to maintain plasma glucose levels at predetermined target values .
blood was sampled every 5 min for immediate determination of plasma glucose by the glucose oxidation method ( beckmann glucose analyzer ii ; beckman coulter , fullerton , ca ) and later determination of plasma c isotopic enrichment of glucose and lactate by high - resolution proton nuclear magnetic resonance ( h nmr ) at 500 mhz ( 15,16 ) .
every 30 min , additional blood was sampled for measurement of insulin and counterregulatory hormones as previously described ( 12 ) .
all studies were performed on a 3 t magnetic resonance system ( magnetom trio ; siemens , erlangen , germany ) with a h volume coil and a circularly polarized c surface coil inserted into the h coil ( 17 ) . a distortionless enhanced polarization transfer ( dept )
sequence for h to c polarization transfer combined with proton image - selected in vivo spectroscopy ( h isis ) localization ( 18 ) was used for acquisition of c magnetic resonance spectra .
adiabatic c radiofrequency pulses were used in all sequences to ensure homogeneous excitation using the c surface coil , as well as wideband alternating phase low - power technique for zero residue splitting ( waltz-16 ) proton decoupling ( 19 ) to simplify the spectra and enhance signal - to - noise ratio ( snr ) .
a voxel of 125 ml was placed in occipital brain tissue . in all experiments , one spectrum consisted of 72 repetitions of 2 s , allowing for a time resolution of 2.5 min .
eight reference spectra were obtained before the start of [ 1-c]glucose infusion , during which c mr spectra were acquired continuously .
visual stimulation was avoided by dimming the lights in the magnet room during the experiments .
the eight c mr reference spectra were averaged and subtracted from the dynamic spectra acquired during the c glucose clamp to correct for natural abundance c magnetic resonance signals . to enhance the snr ,
the resulting spectra were fitted with the advance magnetic resonance ( amares ) algorithm ( 20 ) in the java - based mr user interface ( jmrui ) software package ( 21 ) . the natural abundance signal of myo - inositol ( mi ) was used to quantify glutamate c4 and glutamate c3 in the spectra based on the premise that mi has a stable concentration of 6 mol / g and is not labeled with c in the time frame of the experiment ( 22 ) .
the natural abundance signal of mi was quantified in jmrui from the spectra obtained by adding all dynamic spectra before correction with reference spectra .
dept c magnetic resonance spectra measured from a phantom were used to eliminate effects of the pulse sequence profile on the experimental spectra .
rates of metabolic fluxes were determined using a standard one - compartment metabolic model ( 14,23,24 ) as depicted in fig .
these rates ( v ) were assessed from the time courses of incorporation of c isotopes into different metabolites .
time courses of glutamate c4 and glutamate c3 concentrations and plasma values and isotopic enrichments of glucose were used as input factors for the model . in this specific situation , it was also decided to take plasma lactate values and isotopic enrichment into account as input variables for the modeling process .
the model was implemented in matlab r2008b ( mathworks , natick , ma ) and consisted of differential equations describing the inflow and outflow of label from each metabolite pool .
these equations were solved numerically , and a nonlinear least - squares approach was used to fit the model to the experimental data by varying free flux parameters representing vtca , the loss of label through exchange with unlabeled glutamine ( vefflux ) , and the exchange of intracellular lactate with plasma lactate ( vdil ) .
vx , representing the exchange between -ketoglutarate and glutamate , was assumed to be 5 mol / g / min .
this value was fixed to ensure stable fitting . in literature , there is no consensus on the value of vx ; it is either determined to be much higher compared with vtca ( 25 ) or to be in the same order of vtca ( 23,26 ) .
however , for this study comparing two datasets , the choice of vx is not critical ; it will influence absolute values but equally for both datasets and therefore will not compromise the comparison of the two glycemic conditions .
data of all individual volunteers were fitted separately and inspected for the goodness of fit by the cost value , a parameter that represents the difference between the measured concentrations and the corresponding estimates by the model . from these fits ,
one - compartment model for uptake of [ 1-c]glucose and its conversion into labeled metabolites .
brain glucose uptake from the blood was modeled by reversible michaelis - menten kinetics ( 40 ) .
the cycling of glutamate and glutamine between neurons and astroglia is approached by the parameter vgln and assumed equal to vtca .
vtca , vdil , and vefflux are the free parameters of the model , representing tca cycle flux , exchange of plasma , and brain lactate and efflux of labeled glutamine , respectively .
vldh represents lactate dehydrogenase and was assumed to be 3 mol / g / min ; vx represents the exchange between -ketoglutarate and glutamate over the mitochondrial membrane and was assumed to be 5 mol / g / min .
furthermore , the following assumptions were made for total pool concentrations : [ glu ] : 10 mmol / l ; [ gln ] : 2.5 mmol / l ; [ oaa ] ; 0.3 mmol / l ; [ kg ] : 0.25 mmol / l ; [ asp ] : 1.5 mmol / l ; and [ pyr ] : 0.15 mmol / l .
serial data ( e.g. , plasma hormone and lactate levels ) were compared by repeated - measures anova , and differences in means were tested by paired , two - tailed student t tests .
graphpad prism 4 ( graphpad , la jolla , ca ) was used for statistical analysis , and a p value < 0.05 was considered statistically significant .
as a consequence of the [ 1-c]glucose bolus , plasma glucose levels transiently increased to maximally 7.1 0.4
thereafter , plasma glucose levels were allowed to fall and were maintained stable at 5.1 0.3
mmol / l with a coefficient of variation ( cv ) of 4.1 1.0% during the euglycemic clamp and at 3.0 0.4 mmol / l ( cv 5.9 2.2% ) during the hypoglycemic clamp ( fig .
in response to hypoglycemia , plasma levels of glucagon , adrenaline , noradrenaline , cortisol , and growth hormone all significantly increased compared with baseline and compared with similar time points during the euglycemic clamp ( table 1 ) . as expected , glucose infusion rates were approximately threefold lower during hypoglycemia than during euglycemia ( 2.2 0.4 vs. 6.6 0.9 mol / kg / min ; p < 0.001 )
. plasma glucose concentration ( a ) , plasma glucose c enrichment ( b ) , plasma lactate concentration ( c ) , and plasma lactate c enrichment ( d ) as a function of time during the euglycemic and hypoglycemic clamps .
counterregulatory hormone and insulin values during the euglycemic and hypoglycemic clamps data are means sd .
isotopic enrichment of plasma glucose also peaked immediately after the bolus infusion . from t = 20 min onward ,
plasma c glucose labeling was stable during both the euglycemic and the hypoglycemic clamps , although the level was slightly lower during hypoglycemia ( 35.1 1.8% [ cv 3.6 2.1 ] vs. 30.2 5.5% [ cv 6.8 2.4 ] ) ( fig .
after an initial rise , plasma lactate levels remained stable during euglycemia at 1.1 0.3 mmol / l , but tended to increase further in response to hypoglycemia ( from 1.1 0.4 to 1.5 0.6 mmol / l , p = 0.07 vs. euglycemia ) .
conversely , plasma lactate isotopic enrichment remained constant ( after an initial increase ) during hypoglycemia , whereas it increased further during euglycemia ( from 6.1 3.2 at t = 20 min to 8.9 2.7% at t = 100 min ) , p < 0.0001 , versus hypoglycemia ( fig . 2c and d ) .
all c mr spectra were of similar spectral quality with sufficient snr to analyze the signals of the metabolites of interest under both euglycemic and hypoglycemic conditions ( fig .
the c label was progressively incorporated into glutamate c4 , c3 , and c2 ; glutamine c4 , c3 , and c2 ; aspartate c3 and c2 ; and lactate c3 .
figure 4a shows the time courses of glutamate c4 labeling , which appears during the first turn of the tca cycle , and of glutamate c3 labeling , which appears during the second turn of the tca cycle .
the isotopic enrichment of the glutamate pool was lower under hypoglycemic conditions than under euglycemic conditions .
however , after correction for the lower plasma glucose c enrichment level during hypoglycemia , the time courses for both these metabolites were virtually superimposable for the two glycemic conditions ( fig .
representative c magnetic resonance spectra of the human brain measured at the end of a euglycemic experiment and a hypoglycemic experiment .
ppm , parts per million . a : time courses of glutamate c4 and c3 labeling in brain tissue during euglycemic and hypoglycemic clamps .
b : time courses of glutamate c4 and c3 labeling in brain tissue corrected by plasma glucose c enrichment .
c : averaged time courses of glutamate c4 and c3 labeling in brain tissue with natural abundance signal added for modeling purposes together with averaged best fits of individual datasets . the time courses of c label incorporation into glutamate c4 and c3 were fitted with a one - compartment metabolic model ( fig .
the average of individually calculated tca cycle rates ( vtca ) under hypoglycemic clamp conditions was similar to the average values obtained under euglycemic conditions ( table 2 ) . when the tca cycle rates were calculated from averaged datasets ( fig .
1 ) , similar values were obtained ( 0.48 and 0.43 mol / g / min for euglycemia and hypoglycemia , respectively ) . there were also no differences between the two glycemic conditions with regard to other flux parameters .
the absence of an effect of hypoglycemia on metabolic fluxes was similar among men and women in the study ( data not shown ) .
in this study , we used c mrs to investigate the effect of moderate hypoglycemia on glucose handling by the human brain in vivo .
despite the fact that hypoglycemia considerably stimulated glucose counterregulation , which is under control of the central nervous system , metabolism of glucose in the occipital tissue of the brain was remarkably similar under hypoglycemic and euglycemic conditions . indeed ,
the time courses of c labeling of both glutamate c4 and glutamate c3 during hypoglycemia and during euglycemia were superimposable , when corrected for plasma isotopic enrichment , and the rate of tca cycle flux was ~0.5 mol / g / min on both occasions .
these calculated fluxes are in line with previously reported values for vtca under hyperglycemic conditions ( 2729 ) .
this is the first study to investigate and quantify cerebral glucose metabolism under hypoglycemic conditions in humans using c mrs .
the lack of a difference in cerebral glucose metabolism between hypo- and euglycemia contrasts with a h mrs study , which suggested slowing of the tca cycle rate during hypoglycemia based on a reduction of the glutamate - to - creatine ratio ( 7 ) . however , snapshot h magnetic resonance spectra provide steady - state levels of metabolites and can not be used to quantify cerebral glucose metabolic fluxes , as is possible with dynamic c mrs . moreover , our findings are in line with a c mrs study in rats ( 30 ) in which overall cerebral metabolism in control animals was not affected by hypoglycemia .
mathematical modeling of the data was performed to quantify and compare metabolic processes under the two glycemic conditions .
our model showed that the rates for free flux parameters ( vtca , vdil , and vefflux ) were about similar during hypoglycemia and euglycemia .
the vtca values were within the published range ( 2729 ) , but vdil and vefflux were higher than previously reported ( 31,32 ) . because several assumptions for vgln , vldh , and vx had to be made to derive the rates for free flux parameters , these data need to be interpreted with caution .
there is ongoing debate as to which estimated values are appropriate . in the literature , for
vx values below 1 and as high as 57 mol / g / min have been used ( 23,25 ) . in the current study ,
the data were fitted assuming vx = 5 mol / g / min to optimize the fitting . refitting the data with vx = 1 mol / g / min resulted in slightly lower values for vtca of 0.43 mol / g / min during euglycemia and 0.38 mol / g / min during hypoglycemia , but the difference between the two groups remained nonsignificant .
we can not completely exclude the possibility that vgln , vldh , and vx are modulated by the level of glycemia itself , but there are no data available to support such a notion .
thus , identical assumptions for the fixed model parameters were used for both glycemic conditions .
a plausible explanation would be a compensatory increase in cerebral uptake of lactate . in agreement with previous observations ( 33 ) , plasma lactate
lactate can be used by the brain as an alternative energy source and may reduce the cerebral need for glucose ( 3436 ) .
after conversion to pyruvate , lactate carbons may enter the tca cycle as the carbons of glucose do ( 37 ) .
( 6 ) has indicated that brain transporter activity for monocarboxylic substrates such as lactate can be increased twofold during hypoglycemia , which would support the hypothesis of increased lactate consumption during hypoglycemia . moreover , even under resting conditions , increases in lactate availability stimulate consumption of lactate by the brain at the cost of reductions in glucose utilization ( 34 ) .
our model does not include net lactate uptake because this flux is small and can be neglected under physiological conditions .
however , it is possible that the increased plasma lactate level during hypoglycemia could result in increased net lactate uptake in the brain .
therefore , to assess the potential contribution of net lactate uptake , we performed additional modeling incorporating net lactate uptake ( 0.2 mol / g / min , estimated from lactate michaelis - menten kinetics through the blood - brain barrier ) into the model during hypoglycemia and found that it did not affect the values of vtca significantly ( < 5% change ) .
alternatively , we can not exclude the entrance of unlabeled carbons into the tca cycle via glycogen breakdown as was suggested by an in vivo study by oz et al .
breakdown of glycogen located in astrocytes can then provide neurons with lactate to maintain energy metabolism ( 39 ) .
this study provides no evidence for a link between a decline in cognitive function and moderate hypoglycemia .
as c mrs of the brain was usually performed in a limited area of the occipital cortex , we can not exclude the possibility that regional variation in cerebral glucose metabolism occurs .
it is also possible that increased cortical activation in response to hypoglycemia stimulated glucose uptake so that any fall in glucose metabolism was sufficiently compensated for .
mmol / l ) in various regions of the brain in diabetic men ( 8,9 ) but not in the occipital cortex , where reduced rather than increased activation was observed .
it is possible that the relatively mild hypoglycemic condition imposed during this study was only strong enough to stimulate hormonal counterregulation but that deeper hypoglycemia is required to induce impairments in cerebral glucose metabolism .
however , a clamp at much lower glucose levels may be hard to achieve while maintaining stable glucose infusion at a sufficient rate , which is required for administration of the c isotope .
in addition , more severe hypoglycemia would expose participants to significantly more discomfort and risk . in conclusion ,
our results indicate that acute moderate hypoglycemia does not affect cerebral glucose metabolism in healthy human volunteers .
thus , the healthy human brain appears sufficiently resilient to withstand moderate drops in plasma glucose , potentially as a consequence of increased lactate availability .
our study provides new insights into mechanisms that protect the brain from substrate deprivation , which may have important clinical implications if this finding can be reproduced in patients with type 1 diabetes , especially those suffering from repeated hypoglycemic episodes . | objectiveto investigate the effect of acute insulin - induced hypoglycemia on cerebral glucose metabolism in healthy humans , measured by 13c magnetic resonance spectroscopy ( mrs).research design and methodshyperinsulinemic glucose clamps were performed at plasma glucose levels of 5 mmol / l ( euglycemia ) or 3 mmol / l ( hypoglycemia ) in random order in eight healthy subjects ( four women ) on two occasions , separated by at least 3 weeks .
enriched [ 1 - 13c]glucose 20% w / w was used for the clamps to maintain stable plasma glucose labeling .
the levels of the 13c - labeled glucose metabolites glutamate c4 and c3 were measured over time in the occipital cortex during the clamp by continuous 13c mrs in a 3 t magnetic resonance scanner .
time courses of glutamate c4 and c3 labeling were fitted using a one - compartment model to calculate metabolic rates in the brain.resultsplasma glucose 13c isotopic enrichment was stable at 35.1 1.8% during euglycemia and at 30.2 5.5% during hypoglycemia .
hypoglycemia stimulated release of counterregulatory hormones ( all p < 0.05 ) and tended to increase plasma lactate levels ( p = 0.07 ) .
after correction for the ambient 13c enrichment values , label incorporation into glucose metabolites was virtually identical under both glycemic conditions
. calculated tricarboxylic acid cycle rates ( vtca ) were 0.48 0.03 mol / g / min during euglycemia and 0.43 0.08 mol / g / min during hypoglycemia ( p = 0.42).conclusionsthese results indicate that acute moderate hypoglycemia does not affect fluxes through the main pathways of glucose metabolism in the brain of healthy nondiabetic subjects . | RESEARCH DESIGN AND METHODS
Hyperinsulinemic glucose clamps.
MRS.
None
Metabolic modeling.
Statistical analysis.
RESULTS
DISCUSSION | eight healthy nondiabetic volunteers ( four men and four women aged 23.2 2.5 years with bmi 23.9 4.5 kg / m ) were enrolled for this study . all participants were examined on two occasions : once under euglycemic conditions and once under hypoglycemic conditions , scheduled in random order and separated by at least 3 weeks . subjects came to the magnetic resonance research facility at 8:00 a.m. after an overnight fast and after having abstained from alcohol and caffeine - containing substances for 24 h. the brachial artery of the nondominant arm was cannulated under local anesthesia for frequent blood sampling . after a 30-min equilibration period
, subjects were placed on the scanner bed in supine position with their heads positioned in the magnetic resonance coil . arterial blood was sampled to obtain baseline variables , and reference spectra were obtained without administration of exogenous c - labeled material during the next 30 min . subsequently , a hyperinsulinemic ( 60 mu / min / m , equaling approximately 1.5 mu / min / kg ) euglycemic ( 5.0 mmol / l ) or hypoglycemic ( 3.0 mmol / l ) glucose clamp was initiated with [ 1-c]glucose 20% w / w , as described previously ( 12 ) . briefly , a bolus of 30 ml of 100% enriched [ 1-c]glucose 20% w / w was infused over 10 min at the initiation of the clamp to rapidly increase plasma c enrichment . for the remainder of the experiments , variable infusions of 40 or 50% enriched
[ 1-c]glucose 20% w / w were infused during the euglycemic and hypoglycemic experiments , respectively , in order to maintain plasma glucose levels at predetermined target values . blood was sampled every 5 min for immediate determination of plasma glucose by the glucose oxidation method ( beckmann glucose analyzer ii ; beckman coulter , fullerton , ca ) and later determination of plasma c isotopic enrichment of glucose and lactate by high - resolution proton nuclear magnetic resonance ( h nmr ) at 500 mhz ( 15,16 ) . all studies were performed on a 3 t magnetic resonance system ( magnetom trio ; siemens , erlangen , germany ) with a h volume coil and a circularly polarized c surface coil inserted into the h coil ( 17 ) . a distortionless enhanced polarization transfer ( dept )
sequence for h to c polarization transfer combined with proton image - selected in vivo spectroscopy ( h isis ) localization ( 18 ) was used for acquisition of c magnetic resonance spectra . visual stimulation was avoided by dimming the lights in the magnet room during the experiments . the eight c mr reference spectra were averaged and subtracted from the dynamic spectra acquired during the c glucose clamp to correct for natural abundance c magnetic resonance signals . to enhance the snr
the resulting spectra were fitted with the advance magnetic resonance ( amares ) algorithm ( 20 ) in the java - based mr user interface ( jmrui ) software package ( 21 ) . the natural abundance signal of myo - inositol ( mi ) was used to quantify glutamate c4 and glutamate c3 in the spectra based on the premise that mi has a stable concentration of 6 mol / g and is not labeled with c in the time frame of the experiment ( 22 ) . rates of metabolic fluxes were determined using a standard one - compartment metabolic model ( 14,23,24 ) as depicted in fig . these rates ( v ) were assessed from the time courses of incorporation of c isotopes into different metabolites . time courses of glutamate c4 and glutamate c3 concentrations and plasma values and isotopic enrichments of glucose were used as input factors for the model . in this specific situation
, it was also decided to take plasma lactate values and isotopic enrichment into account as input variables for the modeling process . these equations were solved numerically , and a nonlinear least - squares approach was used to fit the model to the experimental data by varying free flux parameters representing vtca , the loss of label through exchange with unlabeled glutamine ( vefflux ) , and the exchange of intracellular lactate with plasma lactate ( vdil ) . vx , representing the exchange between -ketoglutarate and glutamate , was assumed to be 5 mol / g / min . in literature , there is no consensus on the value of vx ; it is either determined to be much higher compared with vtca ( 25 ) or to be in the same order of vtca ( 23,26 ) . however , for this study comparing two datasets , the choice of vx is not critical ; it will influence absolute values but equally for both datasets and therefore will not compromise the comparison of the two glycemic conditions . data of all individual volunteers were fitted separately and inspected for the goodness of fit by the cost value , a parameter that represents the difference between the measured concentrations and the corresponding estimates by the model . from these fits ,
one - compartment model for uptake of [ 1-c]glucose and its conversion into labeled metabolites . vldh represents lactate dehydrogenase and was assumed to be 3 mol / g / min ; vx represents the exchange between -ketoglutarate and glutamate over the mitochondrial membrane and was assumed to be 5 mol / g / min . furthermore , the following assumptions were made for total pool concentrations : [ glu ] : 10 mmol / l ; [ gln ] : 2.5 mmol / l ; [ oaa ] ; 0.3 mmol / l ; [ kg ] : 0.25 mmol / l ; [ asp ] : 1.5 mmol / l ; and [ pyr ] : 0.15 mmol / l . , plasma hormone and lactate levels ) were compared by repeated - measures anova , and differences in means were tested by paired , two - tailed student t tests . graphpad prism 4 ( graphpad , la jolla , ca ) was used for statistical analysis , and a p value < 0.05 was considered statistically significant . subjects came to the magnetic resonance research facility at 8:00 a.m. after an overnight fast and after having abstained from alcohol and caffeine - containing substances for 24 h. the brachial artery of the nondominant arm was cannulated under local anesthesia for frequent blood sampling . after a 30-min equilibration period
, subjects were placed on the scanner bed in supine position with their heads positioned in the magnetic resonance coil . subsequently , a hyperinsulinemic ( 60 mu / min / m , equaling approximately 1.5 mu / min / kg ) euglycemic ( 5.0 mmol / l ) or hypoglycemic ( 3.0 mmol / l ) glucose clamp was initiated with [ 1-c]glucose 20% w / w , as described previously ( 12 ) . briefly , a bolus of 30 ml of 100% enriched [ 1-c]glucose 20% w / w was infused over 10 min at the initiation of the clamp to rapidly increase plasma c enrichment . for the remainder of the experiments , variable infusions of 40 or 50% enriched
[ 1-c]glucose 20% w / w were infused during the euglycemic and hypoglycemic experiments , respectively , in order to maintain plasma glucose levels at predetermined target values . blood was sampled every 5 min for immediate determination of plasma glucose by the glucose oxidation method ( beckmann glucose analyzer ii ; beckman coulter , fullerton , ca ) and later determination of plasma c isotopic enrichment of glucose and lactate by high - resolution proton nuclear magnetic resonance ( h nmr ) at 500 mhz ( 15,16 ) . all studies were performed on a 3 t magnetic resonance system ( magnetom trio ; siemens , erlangen , germany ) with a h volume coil and a circularly polarized c surface coil inserted into the h coil ( 17 ) . a distortionless enhanced polarization transfer ( dept )
sequence for h to c polarization transfer combined with proton image - selected in vivo spectroscopy ( h isis ) localization ( 18 ) was used for acquisition of c magnetic resonance spectra . the eight c mr reference spectra were averaged and subtracted from the dynamic spectra acquired during the c glucose clamp to correct for natural abundance c magnetic resonance signals . to enhance the snr ,
the resulting spectra were fitted with the advance magnetic resonance ( amares ) algorithm ( 20 ) in the java - based mr user interface ( jmrui ) software package ( 21 ) . the natural abundance signal of myo - inositol ( mi ) was used to quantify glutamate c4 and glutamate c3 in the spectra based on the premise that mi has a stable concentration of 6 mol / g and is not labeled with c in the time frame of the experiment ( 22 ) . dept c magnetic resonance spectra measured from a phantom were used to eliminate effects of the pulse sequence profile on the experimental spectra . rates of metabolic fluxes were determined using a standard one - compartment metabolic model ( 14,23,24 ) as depicted in fig . these rates ( v ) were assessed from the time courses of incorporation of c isotopes into different metabolites . time courses of glutamate c4 and glutamate c3 concentrations and plasma values and isotopic enrichments of glucose were used as input factors for the model . in this specific situation , it was also decided to take plasma lactate values and isotopic enrichment into account as input variables for the modeling process . these equations were solved numerically , and a nonlinear least - squares approach was used to fit the model to the experimental data by varying free flux parameters representing vtca , the loss of label through exchange with unlabeled glutamine ( vefflux ) , and the exchange of intracellular lactate with plasma lactate ( vdil ) . vx , representing the exchange between -ketoglutarate and glutamate , was assumed to be 5 mol / g / min . in literature , there is no consensus on the value of vx ; it is either determined to be much higher compared with vtca ( 25 ) or to be in the same order of vtca ( 23,26 ) . however , for this study comparing two datasets , the choice of vx is not critical ; it will influence absolute values but equally for both datasets and therefore will not compromise the comparison of the two glycemic conditions . from these fits ,
one - compartment model for uptake of [ 1-c]glucose and its conversion into labeled metabolites . vldh represents lactate dehydrogenase and was assumed to be 3 mol / g / min ; vx represents the exchange between -ketoglutarate and glutamate over the mitochondrial membrane and was assumed to be 5 mol / g / min . furthermore , the following assumptions were made for total pool concentrations : [ glu ] : 10 mmol / l ; [ gln ] : 2.5 mmol / l ; [ oaa ] ; 0.3 mmol / l ; [ kg ] : 0.25 mmol / l ; [ asp ] : 1.5 mmol / l ; and [ pyr ] : 0.15 mmol / l . graphpad prism 4 ( graphpad , la jolla , ca ) was used for statistical analysis , and a p value < 0.05 was considered statistically significant . as a consequence of the [ 1-c]glucose bolus , plasma glucose levels transiently increased to maximally 7.1 0.4
thereafter , plasma glucose levels were allowed to fall and were maintained stable at 5.1 0.3
mmol / l with a coefficient of variation ( cv ) of 4.1 1.0% during the euglycemic clamp and at 3.0 0.4 mmol / l ( cv 5.9 2.2% ) during the hypoglycemic clamp ( fig . in response to hypoglycemia , plasma levels of glucagon , adrenaline , noradrenaline , cortisol , and growth hormone all significantly increased compared with baseline and compared with similar time points during the euglycemic clamp ( table 1 ) . as expected , glucose infusion rates were approximately threefold lower during hypoglycemia than during euglycemia ( 2.2 0.4 vs. 6.6 0.9 mol / kg / min ; p < 0.001 )
. plasma glucose concentration ( a ) , plasma glucose c enrichment ( b ) , plasma lactate concentration ( c ) , and plasma lactate c enrichment ( d ) as a function of time during the euglycemic and hypoglycemic clamps . isotopic enrichment of plasma glucose also peaked immediately after the bolus infusion . from t = 20 min onward ,
plasma c glucose labeling was stable during both the euglycemic and the hypoglycemic clamps , although the level was slightly lower during hypoglycemia ( 35.1 1.8% [ cv 3.6 2.1 ] vs. 30.2 5.5% [ cv 6.8 2.4 ] ) ( fig . after an initial rise , plasma lactate levels remained stable during euglycemia at 1.1 0.3 mmol / l , but tended to increase further in response to hypoglycemia ( from 1.1 0.4 to 1.5 0.6 mmol / l , p = 0.07 vs. euglycemia ) . conversely , plasma lactate isotopic enrichment remained constant ( after an initial increase ) during hypoglycemia , whereas it increased further during euglycemia ( from 6.1 3.2 at t = 20 min to 8.9 2.7% at t = 100 min ) , p < 0.0001 , versus hypoglycemia ( fig . all c mr spectra were of similar spectral quality with sufficient snr to analyze the signals of the metabolites of interest under both euglycemic and hypoglycemic conditions ( fig . figure 4a shows the time courses of glutamate c4 labeling , which appears during the first turn of the tca cycle , and of glutamate c3 labeling , which appears during the second turn of the tca cycle . the isotopic enrichment of the glutamate pool was lower under hypoglycemic conditions than under euglycemic conditions . however , after correction for the lower plasma glucose c enrichment level during hypoglycemia , the time courses for both these metabolites were virtually superimposable for the two glycemic conditions ( fig . representative c magnetic resonance spectra of the human brain measured at the end of a euglycemic experiment and a hypoglycemic experiment . a : time courses of glutamate c4 and c3 labeling in brain tissue during euglycemic and hypoglycemic clamps . b : time courses of glutamate c4 and c3 labeling in brain tissue corrected by plasma glucose c enrichment . c : averaged time courses of glutamate c4 and c3 labeling in brain tissue with natural abundance signal added for modeling purposes together with averaged best fits of individual datasets . the time courses of c label incorporation into glutamate c4 and c3 were fitted with a one - compartment metabolic model ( fig . the average of individually calculated tca cycle rates ( vtca ) under hypoglycemic clamp conditions was similar to the average values obtained under euglycemic conditions ( table 2 ) . 1 ) , similar values were obtained ( 0.48 and 0.43 mol / g / min for euglycemia and hypoglycemia , respectively ) . the absence of an effect of hypoglycemia on metabolic fluxes was similar among men and women in the study ( data not shown ) . in this study , we used c mrs to investigate the effect of moderate hypoglycemia on glucose handling by the human brain in vivo . despite the fact that hypoglycemia considerably stimulated glucose counterregulation , which is under control of the central nervous system , metabolism of glucose in the occipital tissue of the brain was remarkably similar under hypoglycemic and euglycemic conditions . indeed ,
the time courses of c labeling of both glutamate c4 and glutamate c3 during hypoglycemia and during euglycemia were superimposable , when corrected for plasma isotopic enrichment , and the rate of tca cycle flux was ~0.5 mol / g / min on both occasions . this is the first study to investigate and quantify cerebral glucose metabolism under hypoglycemic conditions in humans using c mrs . the lack of a difference in cerebral glucose metabolism between hypo- and euglycemia contrasts with a h mrs study , which suggested slowing of the tca cycle rate during hypoglycemia based on a reduction of the glutamate - to - creatine ratio ( 7 ) . however , snapshot h magnetic resonance spectra provide steady - state levels of metabolites and can not be used to quantify cerebral glucose metabolic fluxes , as is possible with dynamic c mrs . moreover , our findings are in line with a c mrs study in rats ( 30 ) in which overall cerebral metabolism in control animals was not affected by hypoglycemia . mathematical modeling of the data was performed to quantify and compare metabolic processes under the two glycemic conditions . our model showed that the rates for free flux parameters ( vtca , vdil , and vefflux ) were about similar during hypoglycemia and euglycemia . in the literature , for
vx values below 1 and as high as 57 mol / g / min have been used ( 23,25 ) . in the current study ,
the data were fitted assuming vx = 5 mol / g / min to optimize the fitting . refitting the data with vx = 1 mol / g / min resulted in slightly lower values for vtca of 0.43 mol / g / min during euglycemia and 0.38 mol / g / min during hypoglycemia , but the difference between the two groups remained nonsignificant . thus , identical assumptions for the fixed model parameters were used for both glycemic conditions . in agreement with previous observations ( 33 ) , plasma lactate
lactate can be used by the brain as an alternative energy source and may reduce the cerebral need for glucose ( 3436 ) . however , it is possible that the increased plasma lactate level during hypoglycemia could result in increased net lactate uptake in the brain . therefore , to assess the potential contribution of net lactate uptake , we performed additional modeling incorporating net lactate uptake ( 0.2 mol / g / min , estimated from lactate michaelis - menten kinetics through the blood - brain barrier ) into the model during hypoglycemia and found that it did not affect the values of vtca significantly ( < 5% change ) . as c mrs of the brain was usually performed in a limited area of the occipital cortex , we can not exclude the possibility that regional variation in cerebral glucose metabolism occurs . it is also possible that increased cortical activation in response to hypoglycemia stimulated glucose uptake so that any fall in glucose metabolism was sufficiently compensated for . mmol / l ) in various regions of the brain in diabetic men ( 8,9 ) but not in the occipital cortex , where reduced rather than increased activation was observed . however , a clamp at much lower glucose levels may be hard to achieve while maintaining stable glucose infusion at a sufficient rate , which is required for administration of the c isotope . in conclusion ,
our results indicate that acute moderate hypoglycemia does not affect cerebral glucose metabolism in healthy human volunteers . | [
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is an emergency endoscopy approach beneficial in the treatment of gallstone - induced acute pancreatitis?is one - stage cholecystectomy followed by common bile duct ( cbd ) clearance safer and more effective than endoscopic procedures?when should laparoscopic cholecystectomy be undertaken in patients with gallstone pancreatitis ? is an emergency endoscopy approach beneficial in the treatment of gallstone - induced acute pancreatitis ?
is one - stage cholecystectomy followed by common bile duct ( cbd ) clearance safer and more effective than endoscopic procedures ? when should laparoscopic cholecystectomy be undertaken in patients with gallstone pancreatitis ?
research on the pathophysiology of acute pancreatitis has advanced dramatically during the past 20 years , and , as the number of randomized controlled studies ( rcts ) of severe acute pancreatitis has steadily increased , mainly in the united states and europe , evidence - based management has come to be demanded for acute pancreatitis . in japan , three institutions the japanese society for abdominal emergency medicine , the japan pancreas society , and the research group for intractable diseases and refractory pancreatic diseases , which is sponsored by the japanese ministry of health , labour , and welfare collaborated to publish evidence - based clinical practice guidelines for acute pancreatitis : proposals.1 this article incorporates the latest evidence in relation to gallstone pancreatitis and presents these guidelines ( the jpn guidelines ) for the treatment of gallstone - induced acute pancreatitis , citing the relevant recommendations .
an emergency endoscopy approach is beneficial in patients with acute pancreatitis in whom bile duct obstruction is suspected or there are complications from cholangitis ( recommendation a ) emergency endoscopic approaches to the management of bile duct stones in gallstone - associated acute pancreatitis include endoscopic sphincterotomy ( es ) , endoscopic papillary balloon dilation ( epbd ) , endoscopic nasobiliary drainage ( enbd ) , and stenting .
those procedures are performed to remove gallstones and to drain the bile duct , which mostly results in relieving the obstruction of the pancreatic duct .
the endoscopic approach seems to be most beneficial in those patients with acute pancreatitis suspected of having concomitant gallstones complicated with the occurrence or exacerbation of jaundice , and in patients complicated by cholangitis .
the decision whether or not to perform one - stage choledocholithotomy after es or endoscopic biliary drainage must be based on the pathophysiological condition of the patient and the skill of the physician .
it is important to refer any patient who requires an endoscopic approach to an advanced medical unit if the patient meets the criteria for transfer ( see page ) .
emergency or early endoscopic procedures for acute pancreatitis have been assessed in four randomized controlled trials ( rcts ; level 1b),25 and a metaanalysis of these rcts has been performed ( level 1a).6 the metaanalysis involved a total of 834 patients , comprising 460 patients treated by endoscopic retrograde cholangiopancreatography ( ercp ) followed by es ( ercp / es group ) and 374 patients treated conservatively ( conservative treatment group ) .
the analysis led to the conclusion that the ercp / es group had lower incidences of morbidity and mortality ( table 1 ) , but because pancreatitis patients with treated by ercp or es alone have never been analyzed , it remains unclear whether ercp or es is more useful .
table 1reported morbidity and mortality rates in early ercp / es and conservative treatment groupsauthormorbidity ( % ) mortality ( % ) ercp / es groupconservative treatment groupercp / es groupconservative treatment groupneoptolemos16.933.91.78.1fan17.528.65.29.2nowak16.936.32.312.8folsch46.050.911.16.3pooled data25.038.25.29.1(cited from sharma and howden6 , with some modifications ) reported morbidity and mortality rates in early ercp / es and conservative treatment groups ( cited from sharma and howden6 , with some modifications ) the results of two studies ( level 1b)2,3 that published data stratified by severity indicate that ercp / es is not very useful in patients with mild pancreatitis ( table 2 ) , and a report expressing a negative opinion in regard to early endoscopic treatment ( level 1b)5 excluded patients with jaundice from the analysis , and the percentage of subjects with gallstone pancreatitis was only 19% .
it is concluded from these reports that urgent ercp / es is useful only in severe gallstone pancreatitis .
table 2reported morbidity and mortality rates in early ercp / es and conservative treatment groups , stratified according to severityauthorercp / es groupconservative treatment group number of patientsmorbidity ( % ) mortality ( % ) number of patientsmorbidity ( % ) mortality ( % ) mild pancreatitis groupneoptolemos34403440fan56 ( 34)8 ( 6)058 ( 35)6 ( 6)0pooled data9012 ( 13)09210 ( 11)0severe pancreatitis groupneoptolemos256128175fan41 ( 30)9 ( 4)5 ( 1)40 ( 28)23 ( 15)9 ( 5)pooled data6615 ( 23)6 ( 9)6840 ( 59)14 ( 21 ) patients with cbd stones followed - up with ercp reported morbidity and mortality rates in early ercp / es and conservative treatment groups , stratified according to severity patients with cbd stones followed - up with ercp however , there are differences in severity assessment , particularly of severe cases , between japan and western countries , and no convincing evidence supporting the application of emergency ercp / es in patients with severe cases has been established in japan .
it is currently thought that the indications for emergency ercp / es should be restricted to patients suspected of having prolonged biliary duct obstruction , as manifested by the occurrence or exacerbation of jaundice , and to patients complicated by cholangitis .
various other techniques besides ercp / es are used for biliary drainage and are generally accepted in japan .
a report , entitled evaluation of urgent treatment for impacted bile duct stones ( published in 1992 in japanese ) , claims that endoscopic nasobiliary drainage ( enbd ) is a useful and safe urgent treatment method for impacted bile duct stones , although no high - quality evidence supported by rcts or metaanalysis has yet emerged to support that report . in terms of the safety of ercp / es in the acute stage of pancreatitis
, no difference has been found between the incidence of complications after early intervention ( within 48 h of onset ) and the incidence after delayed intervention ( level 3b),7 and it has been reported that ercp / es within 24 to 72h after onset is safe ( level 2b).8 similar results were reported in a retrospective study of a large number of patients ( level 4).9 the four rcts referred to above25 have suggested bleeding as a complication of ercp / es , but the bleeding occurred in only a few patients with mild pancreatitis and was never a direct cause of death .
based on the reports from overseas , we can not conclude that ercp / es is highly risky in the acute stage of pancreatitis . according to the results of a third nationwide survey in japan ( in 2000 ) of accidental complications from gastrointestinal endoscopy ( level 4 ) ,
the incidence of accidental complications of ercp performed for examination purposes and of es done to treat the papilla of vater was 0.1% and 0.7% , respectively , and the mortality rate was 0.006% and 0.048% , respectively .
the issue of whether or not ercp / es is indicated should be carefully evaluated and adequate care should be taken to prevent accidental complications .
effective performance obviously requires an advanced medical institution with experienced specialists and a special unit whose staff is capable of carrying out emergency ercp / es examinations and dealing with bleeding and other complications .
an emergency endoscopy approach is beneficial in patients with acute pancreatitis in whom bile duct obstruction is suspected or there are complications from cholangitis ( recommendation a ) emergency endoscopic approaches to the management of bile duct stones in gallstone - associated acute pancreatitis include endoscopic sphincterotomy ( es ) , endoscopic papillary balloon dilation ( epbd ) , endoscopic nasobiliary drainage ( enbd ) , and stenting .
those procedures are performed to remove gallstones and to drain the bile duct , which mostly results in relieving the obstruction of the pancreatic duct .
the endoscopic approach seems to be most beneficial in those patients with acute pancreatitis suspected of having concomitant gallstones complicated with the occurrence or exacerbation of jaundice , and in patients complicated by cholangitis .
the decision whether or not to perform one - stage choledocholithotomy after es or endoscopic biliary drainage must be based on the pathophysiological condition of the patient and the skill of the physician .
it is important to refer any patient who requires an endoscopic approach to an advanced medical unit if the patient meets the criteria for transfer ( see page ) .
emergency or early endoscopic procedures for acute pancreatitis have been assessed in four randomized controlled trials ( rcts ; level 1b),25 and a metaanalysis of these rcts has been performed ( level 1a).6 the metaanalysis involved a total of 834 patients , comprising 460 patients treated by endoscopic retrograde cholangiopancreatography ( ercp ) followed by es ( ercp / es group ) and 374 patients treated conservatively ( conservative treatment group ) .
the analysis led to the conclusion that the ercp / es group had lower incidences of morbidity and mortality ( table 1 ) , but because pancreatitis patients with treated by ercp or es alone have never been analyzed , it remains unclear whether ercp or es is more useful .
table 1reported morbidity and mortality rates in early ercp / es and conservative treatment groupsauthormorbidity ( % ) mortality ( % ) ercp / es groupconservative treatment groupercp / es groupconservative treatment groupneoptolemos16.933.91.78.1fan17.528.65.29.2nowak16.936.32.312.8folsch46.050.911.16.3pooled data25.038.25.29.1(cited from sharma and howden6 , with some modifications ) reported morbidity and mortality rates in early ercp / es and conservative treatment groups ( cited from sharma and howden6 , with some modifications ) the results of two studies ( level 1b)2,3 that published data stratified by severity indicate that ercp / es is not very useful in patients with mild pancreatitis ( table 2 ) , and a report expressing a negative opinion in regard to early endoscopic treatment ( level 1b)5 excluded patients with jaundice from the analysis , and the percentage of subjects with gallstone pancreatitis was only 19% .
it is concluded from these reports that urgent ercp / es is useful only in severe gallstone pancreatitis .
table 2reported morbidity and mortality rates in early ercp / es and conservative treatment groups , stratified according to severityauthorercp / es groupconservative treatment group number of patientsmorbidity ( % ) mortality ( % ) number of patientsmorbidity ( % ) mortality ( % ) mild pancreatitis groupneoptolemos34403440fan56 ( 34)8 ( 6)058 ( 35)6 ( 6)0pooled data9012 ( 13)09210 ( 11)0severe pancreatitis groupneoptolemos256128175fan41 ( 30)9 ( 4)5 ( 1)40 ( 28)23 ( 15)9 ( 5)pooled data6615 ( 23)6 ( 9)6840 ( 59)14 ( 21 ) patients with cbd stones followed - up with ercp reported morbidity and mortality rates in early ercp / es and conservative treatment groups , stratified according to severity patients with cbd stones followed - up with ercp however , there are differences in severity assessment , particularly of severe cases , between japan and western countries , and no convincing evidence supporting the application of emergency ercp / es in patients with severe cases has been established in japan .
it is currently thought that the indications for emergency ercp / es should be restricted to patients suspected of having prolonged biliary duct obstruction , as manifested by the occurrence or exacerbation of jaundice , and to patients complicated by cholangitis .
various other techniques besides ercp / es are used for biliary drainage and are generally accepted in japan .
a report , entitled evaluation of urgent treatment for impacted bile duct stones ( published in 1992 in japanese ) , claims that endoscopic nasobiliary drainage ( enbd ) is a useful and safe urgent treatment method for impacted bile duct stones , although no high - quality evidence supported by rcts or metaanalysis has yet emerged to support that report . in terms of the safety of ercp / es in the acute stage of pancreatitis
, no difference has been found between the incidence of complications after early intervention ( within 48 h of onset ) and the incidence after delayed intervention ( level 3b),7 and it has been reported that ercp / es within 24 to 72h after onset is safe ( level 2b).8 similar results were reported in a retrospective study of a large number of patients ( level 4).9 the four rcts referred to above25 have suggested bleeding as a complication of ercp / es , but the bleeding occurred in only a few patients with mild pancreatitis and was never a direct cause of death .
based on the reports from overseas , we can not conclude that ercp / es is highly risky in the acute stage of pancreatitis . according to the results of a third nationwide survey in japan ( in 2000 ) of accidental complications from gastrointestinal endoscopy ( level 4 ) ,
the incidence of accidental complications of ercp performed for examination purposes and of es done to treat the papilla of vater was 0.1% and 0.7% , respectively , and the mortality rate was 0.006% and 0.048% , respectively .
the issue of whether or not ercp / es is indicated should be carefully evaluated and adequate care should be taken to prevent accidental complications .
effective performance obviously requires an advanced medical institution with experienced specialists and a special unit whose staff is capable of carrying out emergency ercp / es examinations and dealing with bleeding and other complications .
open choledochotomy or sphincterotomy ( operative removal of cbd stones is recommended)(recommendation d ) in japan , choledocholithotripsy with emergency endoscopic procedures ( es , epbd ) is performed by endoscopists , as well as by many surgeons , and it has become the treatment of first choice for cbd clearance .
one - stage cholecystectomy and cbd clearance has not been demonstrated to be safer and more effective than endoscopic procedures , and it is not recommended in either the jpn guidelines or the international association of pancreatology ( iap ) guidelines.10 laparoscopic cholecystectomy should be considered after recovery from an attack of gallstone pancreatitis and should be performed during the same hospital stay ( choledochotomy and cbd clearance are performed as required ) ( recommendation b ) cholelithiasis is one of the major causes of acute pancreatitis , and gallstone - induced acute pancreatitis is an absolute indication for cholecystectomy ( and choledochotomy , when necessary ) .
one rct claims that the earlier surgery is performed after onset , the more beneficial it is , while another rct has proposed delayed surgery once the inflammatory process has subsided .
the latter rct ( level 1b)11 compared earlier surgery ( within 72 h of hospital admission ) and delayed surgery , and the results suggested that the earlier surgery was more beneficial . because the results showed no significant difference in the incidence of complications ( 8.3% vs 10.3% ) or the mortality rate ( 2.8% vs 6.9% ) , this rct concluded that surgery on the bile duct system with the aim of removing the cause of the pancreatitis or preventing its progression could be performed safely even in the acute state . according to another rct
( level 1b),12 which is often quoted as grounds to justify delayed surgery , patients subjected to early surgery ( within 48 h of hospitalization ) had a significantly higher complication rate ( 30.1% vs 5.1% ) and mortality rate ( 15.1% vs 2.4% ) than those subjected to delayed surgery .
this conflicting evidence may be explained by potential differences in the severity of the disease in the patients included in the clinical trials .
discussion of the timing of surgery alone , without adequate consideration of the impact of severity , has caused confusion in clinical settings .
however , the introduction of emergency ercp / es2,3 has completely changed the options available for the management of gallstone pancreatitis and has dramatically reduced the need for performing surgery in the acute stage .
there are two options in regard to delayed surgery : ( i ) surgery during the same hospital stay as that for the initial treatment , and ( ii ) surgery during rehospitalization after a sufficient recovery period .
because the rate of recurrence of pancreatitis during the recovery period after discharge is 32% to 61% ( level 3b),1315 patients with mild uncomplicated gallstone pancreatitis should undergo biliary tract exploration and cholecystectomy after the symptoms have been alleviated .
whenever possible , even patients with severe disease should undergo surgery during the same hospital stay as that for the initial treatment .
laparoscopic cholecystectomy ( lc ) has been increasingly used to treat gallstone pancreatitis over the past 10 years . according to prospective cohort studies reported to date (
levels 1b2c),1621 lc was associated with a completion rate of 94.5% ( range , 79% to 100% ) , a complication ( morbidity ) rate of 5.5% ( range , 0% to 10% ) , and a mortality rate of 0.4% ( range , 0% to 2.5% ; table 3 ) , implying that lc resulted in performance equal to or better than that of laparotomy . based on these results ,
the use of lc in mild gallstone pancreatitis is feasible and its clinical usefulness has been demonstrated .
table 3prospective cohort studies of laparoscopic cholecystectomy employed for gallstone - associated acute pancreatitisauthornumber of patientstiming of surgery ( days after onset)completion rate ( % ) conversion rate ( % ) operation time ( min)morbidity ( % ) mortality ( % ) cbd explorationrhodes et al.1610 ( 434)100050 ( 30120)0015/1tate et al.247 ( 324)87.512.576 ( na)8023/0ballestra - lopez et al.403.4/15100086 ( 45210)102.50/40ricci et al.51na1000na1.9040/47uhl et al.4810 ( 429)792180 ( 30225)7.900/33chang et al.59na1000na3.400/58na , not assessed median ( range ) mean mild / severe disease preoperative erc / intraoperative cholangiogram prospective cohort studies of laparoscopic cholecystectomy employed for gallstone - associated acute pancreatitis preoperative erc / intraoperative cholangiogram the conventional standard surgical intervention for gallstone pancreatitis used to be open cholecystectomy and operative cholangiography , followed by choledochotomy when choledocholithiasis was observed .
the introduction of lc , however , has made several options available for biliary tract exploration and the management of choledocholithiasis .
four typical procedures are :
ercp / es prior to surgery , and lc after diagnosing and removing cbd stones . in some patients ,
removal of cbd stones by ercp / es after lc.intraoperative cholangiography ( ioc ) during lc , and conversion to the open method when cbd stones are present.ioc during lc , and when cbd stones are present , the addition of es during or followed by lc.lc , and laparoscopic choledochotomy / lithotripsy when cbd stones are discovered by ioc .
ercp / es prior to surgery , and lc after diagnosing and removing cbd stones . in some patients ,
intraoperative cholangiography ( ioc ) during lc , and conversion to the open method when cbd stones are present .
ioc during lc , and when cbd stones are present , the addition of es during or followed by lc .
however , opinions differ concerning the application of ercp in all patients with gallstone - associated pancreatitis .
some surgeons argue that ercp should be performed only when choledocholithiasis is suspected on the basis of blood studies and other diagnostic imaging , or when it is diagnosed during ioc ( level 1b2b).21,22 the risk inherent in ercp should be noted , particularly in patients with acute pancreatitis ( level 2b).23 it may be possible to substitute ioc for preoperative ercp as a standard method to confirm those patients who are less likely to have choledocholithiasis , if the surgeons in charge of this procedure have sufficient experience and technique to perform ioc successfully .
the choice of which of the above four procedures to opt for when choledocholithiasis is discovered by ioc depends on the skill and technique of the specialist physicians currently at each institution .
collection of more data on the safety , invasiveness , and completion rates associated with each of these procedures is an important task for the future .
open choledochotomy or sphincterotomy ( operative removal of cbd stones is recommended)(recommendation d ) in japan , choledocholithotripsy with emergency endoscopic procedures ( es , epbd ) is performed by endoscopists , as well as by many surgeons , and it has become the treatment of first choice for cbd clearance .
one - stage cholecystectomy and cbd clearance has not been demonstrated to be safer and more effective than endoscopic procedures , and it is not recommended in either the jpn guidelines or the international association of pancreatology ( iap ) guidelines.10
laparoscopic cholecystectomy should be considered after recovery from an attack of gallstone pancreatitis and should be performed during the same hospital stay ( choledochotomy and cbd clearance are performed as required ) ( recommendation b ) cholelithiasis is one of the major causes of acute pancreatitis , and gallstone - induced acute pancreatitis is an absolute indication for cholecystectomy ( and choledochotomy , when necessary ) .
one rct claims that the earlier surgery is performed after onset , the more beneficial it is , while another rct has proposed delayed surgery once the inflammatory process has subsided .
the latter rct ( level 1b)11 compared earlier surgery ( within 72 h of hospital admission ) and delayed surgery , and the results suggested that the earlier surgery was more beneficial . because the results showed no significant difference in the incidence of complications ( 8.3% vs 10.3% ) or the mortality rate ( 2.8% vs 6.9% ) , this rct concluded that surgery on the bile duct system with the aim of removing the cause of the pancreatitis or preventing its progression could be performed safely even in the acute state . according to another rct
( level 1b),12 which is often quoted as grounds to justify delayed surgery , patients subjected to early surgery ( within 48 h of hospitalization ) had a significantly higher complication rate ( 30.1% vs 5.1% ) and mortality rate ( 15.1% vs 2.4% ) than those subjected to delayed surgery .
this conflicting evidence may be explained by potential differences in the severity of the disease in the patients included in the clinical trials .
discussion of the timing of surgery alone , without adequate consideration of the impact of severity , has caused confusion in clinical settings .
however , the introduction of emergency ercp / es2,3 has completely changed the options available for the management of gallstone pancreatitis and has dramatically reduced the need for performing surgery in the acute stage .
there are two options in regard to delayed surgery : ( i ) surgery during the same hospital stay as that for the initial treatment , and ( ii ) surgery during rehospitalization after a sufficient recovery period .
because the rate of recurrence of pancreatitis during the recovery period after discharge is 32% to 61% ( level 3b),1315 patients with mild uncomplicated gallstone pancreatitis should undergo biliary tract exploration and cholecystectomy after the symptoms have been alleviated .
whenever possible , even patients with severe disease should undergo surgery during the same hospital stay as that for the initial treatment .
laparoscopic cholecystectomy ( lc ) has been increasingly used to treat gallstone pancreatitis over the past 10 years . according to prospective cohort studies reported to date ( levels 1b2c),1621 lc
was associated with a completion rate of 94.5% ( range , 79% to 100% ) , a complication ( morbidity ) rate of 5.5% ( range , 0% to 10% ) , and a mortality rate of 0.4% ( range , 0% to 2.5% ; table 3 ) , implying that lc resulted in performance equal to or better than that of laparotomy .
based on these results , the use of lc in mild gallstone pancreatitis is feasible and its clinical usefulness has been demonstrated .
table 3prospective cohort studies of laparoscopic cholecystectomy employed for gallstone - associated acute pancreatitisauthornumber of patientstiming of surgery ( days after onset)completion rate ( % ) conversion rate ( % ) operation time ( min)morbidity ( % ) mortality ( % ) cbd explorationrhodes et al.1610 ( 434)100050 ( 30120)0015/1tate et al.247 ( 324)87.512.576 ( na)8023/0ballestra - lopez et al.403.4/15100086 ( 45210)102.50/40ricci et al.51na1000na1.9040/47uhl et al.4810 ( 429)792180 ( 30225)7.900/33chang et al.59na1000na3.400/58na , not assessed median ( range ) mean mild / severe disease preoperative erc / intraoperative cholangiogram prospective cohort studies of laparoscopic cholecystectomy employed for gallstone - associated acute pancreatitis preoperative erc / intraoperative cholangiogram the conventional standard surgical intervention for gallstone pancreatitis used to be open cholecystectomy and operative cholangiography , followed by choledochotomy when choledocholithiasis was observed .
the introduction of lc , however , has made several options available for biliary tract exploration and the management of choledocholithiasis .
four typical procedures are :
ercp / es prior to surgery , and lc after diagnosing and removing cbd stones . in some patients ,
removal of cbd stones by ercp / es after lc.intraoperative cholangiography ( ioc ) during lc , and conversion to the open method when cbd stones are present.ioc during lc , and when cbd stones are present , the addition of es during or followed by lc.lc , and laparoscopic choledochotomy / lithotripsy when cbd stones are discovered by ioc .
ercp / es prior to surgery , and lc after diagnosing and removing cbd stones . in some patients ,
intraoperative cholangiography ( ioc ) during lc , and conversion to the open method when cbd stones are present .
ioc during lc , and when cbd stones are present , the addition of es during or followed by lc .
however , opinions differ concerning the application of ercp in all patients with gallstone - associated pancreatitis .
some surgeons argue that ercp should be performed only when choledocholithiasis is suspected on the basis of blood studies and other diagnostic imaging , or when it is diagnosed during ioc ( level 1b2b).21,22 the risk inherent in ercp should be noted , particularly in patients with acute pancreatitis ( level 2b).23 it may be possible to substitute ioc for preoperative ercp as a standard method to confirm those patients who are less likely to have choledocholithiasis , if the surgeons in charge of this procedure have sufficient experience and technique to perform ioc successfully .
the choice of which of the above four procedures to opt for when choledocholithiasis is discovered by ioc depends on the skill and technique of the specialist physicians currently at each institution .
collection of more data on the safety , invasiveness , and completion rates associated with each of these procedures is an important task for the future . | gallstones , along with alcohol , are one of the primary etiological factors of acute pancreatitis , and knowledge of the etiology as well as the diagnosis and management of gallstones , is crucial for managing acute pancreatitis . because of this , evidence regarding the management of gallstone - induced pancreatitis in japan was collected , and recommendation levels were established by comparing current clinical practices with optimal clinical practices . the jpn guidelines for managing gallstone - induced acute pancreatitis recommend two procedures : ( 1 ) an urgent endoscopic procedure should be performed in patients in whom biliary duct obstruction is suspected and in patients complicated by cholangitis ( recommendation a ) ; and ( 2 ) after the attack of gallstone pancreatitis has subsided , a laparoscopic cholecystectomy should be performed during the same hospital stay ( recommendation b ) . | Clinical questions
Introduction
Management of bile duct stones in gallstone-associated acute pancreatitis
Clinical questions (CQ) 1. Is an emergency endoscopy approach beneficial in the treatment of gallstone-induced acute pancreatitis?
Surgical approach
CQ2. Is one-stage cholecystectomy followed by common bile duct (CBD) clearance safer and more effective than endoscopic procedures?
CQ3. When should laparoscopic cholecystectomy be undertaken in patients with gallstone pancreatitis? | is an emergency endoscopy approach beneficial in the treatment of gallstone - induced acute pancreatitis?is one - stage cholecystectomy followed by common bile duct ( cbd ) clearance safer and more effective than endoscopic procedures?when should laparoscopic cholecystectomy be undertaken in patients with gallstone pancreatitis ? is an emergency endoscopy approach beneficial in the treatment of gallstone - induced acute pancreatitis ? when should laparoscopic cholecystectomy be undertaken in patients with gallstone pancreatitis ? research on the pathophysiology of acute pancreatitis has advanced dramatically during the past 20 years , and , as the number of randomized controlled studies ( rcts ) of severe acute pancreatitis has steadily increased , mainly in the united states and europe , evidence - based management has come to be demanded for acute pancreatitis . in japan , three institutions the japanese society for abdominal emergency medicine , the japan pancreas society , and the research group for intractable diseases and refractory pancreatic diseases , which is sponsored by the japanese ministry of health , labour , and welfare collaborated to publish evidence - based clinical practice guidelines for acute pancreatitis : proposals.1 this article incorporates the latest evidence in relation to gallstone pancreatitis and presents these guidelines ( the jpn guidelines ) for the treatment of gallstone - induced acute pancreatitis , citing the relevant recommendations . an emergency endoscopy approach is beneficial in patients with acute pancreatitis in whom bile duct obstruction is suspected or there are complications from cholangitis ( recommendation a ) emergency endoscopic approaches to the management of bile duct stones in gallstone - associated acute pancreatitis include endoscopic sphincterotomy ( es ) , endoscopic papillary balloon dilation ( epbd ) , endoscopic nasobiliary drainage ( enbd ) , and stenting . the endoscopic approach seems to be most beneficial in those patients with acute pancreatitis suspected of having concomitant gallstones complicated with the occurrence or exacerbation of jaundice , and in patients complicated by cholangitis . table 1reported morbidity and mortality rates in early ercp / es and conservative treatment groupsauthormorbidity ( % ) mortality ( % ) ercp / es groupconservative treatment groupercp / es groupconservative treatment groupneoptolemos16.933.91.78.1fan17.528.65.29.2nowak16.936.32.312.8folsch46.050.911.16.3pooled data25.038.25.29.1(cited from sharma and howden6 , with some modifications ) reported morbidity and mortality rates in early ercp / es and conservative treatment groups ( cited from sharma and howden6 , with some modifications ) the results of two studies ( level 1b)2,3 that published data stratified by severity indicate that ercp / es is not very useful in patients with mild pancreatitis ( table 2 ) , and a report expressing a negative opinion in regard to early endoscopic treatment ( level 1b)5 excluded patients with jaundice from the analysis , and the percentage of subjects with gallstone pancreatitis was only 19% . table 2reported morbidity and mortality rates in early ercp / es and conservative treatment groups , stratified according to severityauthorercp / es groupconservative treatment group number of patientsmorbidity ( % ) mortality ( % ) number of patientsmorbidity ( % ) mortality ( % ) mild pancreatitis groupneoptolemos34403440fan56 ( 34)8 ( 6)058 ( 35)6 ( 6)0pooled data9012 ( 13)09210 ( 11)0severe pancreatitis groupneoptolemos256128175fan41 ( 30)9 ( 4)5 ( 1)40 ( 28)23 ( 15)9 ( 5)pooled data6615 ( 23)6 ( 9)6840 ( 59)14 ( 21 ) patients with cbd stones followed - up with ercp reported morbidity and mortality rates in early ercp / es and conservative treatment groups , stratified according to severity patients with cbd stones followed - up with ercp however , there are differences in severity assessment , particularly of severe cases , between japan and western countries , and no convincing evidence supporting the application of emergency ercp / es in patients with severe cases has been established in japan . it is currently thought that the indications for emergency ercp / es should be restricted to patients suspected of having prolonged biliary duct obstruction , as manifested by the occurrence or exacerbation of jaundice , and to patients complicated by cholangitis . in terms of the safety of ercp / es in the acute stage of pancreatitis
, no difference has been found between the incidence of complications after early intervention ( within 48 h of onset ) and the incidence after delayed intervention ( level 3b),7 and it has been reported that ercp / es within 24 to 72h after onset is safe ( level 2b).8 similar results were reported in a retrospective study of a large number of patients ( level 4).9 the four rcts referred to above25 have suggested bleeding as a complication of ercp / es , but the bleeding occurred in only a few patients with mild pancreatitis and was never a direct cause of death . according to the results of a third nationwide survey in japan ( in 2000 ) of accidental complications from gastrointestinal endoscopy ( level 4 ) ,
the incidence of accidental complications of ercp performed for examination purposes and of es done to treat the papilla of vater was 0.1% and 0.7% , respectively , and the mortality rate was 0.006% and 0.048% , respectively . an emergency endoscopy approach is beneficial in patients with acute pancreatitis in whom bile duct obstruction is suspected or there are complications from cholangitis ( recommendation a ) emergency endoscopic approaches to the management of bile duct stones in gallstone - associated acute pancreatitis include endoscopic sphincterotomy ( es ) , endoscopic papillary balloon dilation ( epbd ) , endoscopic nasobiliary drainage ( enbd ) , and stenting . the endoscopic approach seems to be most beneficial in those patients with acute pancreatitis suspected of having concomitant gallstones complicated with the occurrence or exacerbation of jaundice , and in patients complicated by cholangitis . table 1reported morbidity and mortality rates in early ercp / es and conservative treatment groupsauthormorbidity ( % ) mortality ( % ) ercp / es groupconservative treatment groupercp / es groupconservative treatment groupneoptolemos16.933.91.78.1fan17.528.65.29.2nowak16.936.32.312.8folsch46.050.911.16.3pooled data25.038.25.29.1(cited from sharma and howden6 , with some modifications ) reported morbidity and mortality rates in early ercp / es and conservative treatment groups ( cited from sharma and howden6 , with some modifications ) the results of two studies ( level 1b)2,3 that published data stratified by severity indicate that ercp / es is not very useful in patients with mild pancreatitis ( table 2 ) , and a report expressing a negative opinion in regard to early endoscopic treatment ( level 1b)5 excluded patients with jaundice from the analysis , and the percentage of subjects with gallstone pancreatitis was only 19% . table 2reported morbidity and mortality rates in early ercp / es and conservative treatment groups , stratified according to severityauthorercp / es groupconservative treatment group number of patientsmorbidity ( % ) mortality ( % ) number of patientsmorbidity ( % ) mortality ( % ) mild pancreatitis groupneoptolemos34403440fan56 ( 34)8 ( 6)058 ( 35)6 ( 6)0pooled data9012 ( 13)09210 ( 11)0severe pancreatitis groupneoptolemos256128175fan41 ( 30)9 ( 4)5 ( 1)40 ( 28)23 ( 15)9 ( 5)pooled data6615 ( 23)6 ( 9)6840 ( 59)14 ( 21 ) patients with cbd stones followed - up with ercp reported morbidity and mortality rates in early ercp / es and conservative treatment groups , stratified according to severity patients with cbd stones followed - up with ercp however , there are differences in severity assessment , particularly of severe cases , between japan and western countries , and no convincing evidence supporting the application of emergency ercp / es in patients with severe cases has been established in japan . it is currently thought that the indications for emergency ercp / es should be restricted to patients suspected of having prolonged biliary duct obstruction , as manifested by the occurrence or exacerbation of jaundice , and to patients complicated by cholangitis . in terms of the safety of ercp / es in the acute stage of pancreatitis
, no difference has been found between the incidence of complications after early intervention ( within 48 h of onset ) and the incidence after delayed intervention ( level 3b),7 and it has been reported that ercp / es within 24 to 72h after onset is safe ( level 2b).8 similar results were reported in a retrospective study of a large number of patients ( level 4).9 the four rcts referred to above25 have suggested bleeding as a complication of ercp / es , but the bleeding occurred in only a few patients with mild pancreatitis and was never a direct cause of death . according to the results of a third nationwide survey in japan ( in 2000 ) of accidental complications from gastrointestinal endoscopy ( level 4 ) ,
the incidence of accidental complications of ercp performed for examination purposes and of es done to treat the papilla of vater was 0.1% and 0.7% , respectively , and the mortality rate was 0.006% and 0.048% , respectively . open choledochotomy or sphincterotomy ( operative removal of cbd stones is recommended)(recommendation d ) in japan , choledocholithotripsy with emergency endoscopic procedures ( es , epbd ) is performed by endoscopists , as well as by many surgeons , and it has become the treatment of first choice for cbd clearance . one - stage cholecystectomy and cbd clearance has not been demonstrated to be safer and more effective than endoscopic procedures , and it is not recommended in either the jpn guidelines or the international association of pancreatology ( iap ) guidelines.10 laparoscopic cholecystectomy should be considered after recovery from an attack of gallstone pancreatitis and should be performed during the same hospital stay ( choledochotomy and cbd clearance are performed as required ) ( recommendation b ) cholelithiasis is one of the major causes of acute pancreatitis , and gallstone - induced acute pancreatitis is an absolute indication for cholecystectomy ( and choledochotomy , when necessary ) . because the results showed no significant difference in the incidence of complications ( 8.3% vs 10.3% ) or the mortality rate ( 2.8% vs 6.9% ) , this rct concluded that surgery on the bile duct system with the aim of removing the cause of the pancreatitis or preventing its progression could be performed safely even in the acute state . however , the introduction of emergency ercp / es2,3 has completely changed the options available for the management of gallstone pancreatitis and has dramatically reduced the need for performing surgery in the acute stage . there are two options in regard to delayed surgery : ( i ) surgery during the same hospital stay as that for the initial treatment , and ( ii ) surgery during rehospitalization after a sufficient recovery period . because the rate of recurrence of pancreatitis during the recovery period after discharge is 32% to 61% ( level 3b),1315 patients with mild uncomplicated gallstone pancreatitis should undergo biliary tract exploration and cholecystectomy after the symptoms have been alleviated . whenever possible , even patients with severe disease should undergo surgery during the same hospital stay as that for the initial treatment . laparoscopic cholecystectomy ( lc ) has been increasingly used to treat gallstone pancreatitis over the past 10 years . according to prospective cohort studies reported to date (
levels 1b2c),1621 lc was associated with a completion rate of 94.5% ( range , 79% to 100% ) , a complication ( morbidity ) rate of 5.5% ( range , 0% to 10% ) , and a mortality rate of 0.4% ( range , 0% to 2.5% ; table 3 ) , implying that lc resulted in performance equal to or better than that of laparotomy . table 3prospective cohort studies of laparoscopic cholecystectomy employed for gallstone - associated acute pancreatitisauthornumber of patientstiming of surgery ( days after onset)completion rate ( % ) conversion rate ( % ) operation time ( min)morbidity ( % ) mortality ( % ) cbd explorationrhodes et al.1610 ( 434)100050 ( 30120)0015/1tate et al.247 ( 324)87.512.576 ( na)8023/0ballestra - lopez et al.403.4/15100086 ( 45210)102.50/40ricci et al.51na1000na1.9040/47uhl et al.4810 ( 429)792180 ( 30225)7.900/33chang et al.59na1000na3.400/58na , not assessed median ( range ) mean mild / severe disease preoperative erc / intraoperative cholangiogram prospective cohort studies of laparoscopic cholecystectomy employed for gallstone - associated acute pancreatitis preoperative erc / intraoperative cholangiogram the conventional standard surgical intervention for gallstone pancreatitis used to be open cholecystectomy and operative cholangiography , followed by choledochotomy when choledocholithiasis was observed . the introduction of lc , however , has made several options available for biliary tract exploration and the management of choledocholithiasis . some surgeons argue that ercp should be performed only when choledocholithiasis is suspected on the basis of blood studies and other diagnostic imaging , or when it is diagnosed during ioc ( level 1b2b).21,22 the risk inherent in ercp should be noted , particularly in patients with acute pancreatitis ( level 2b).23 it may be possible to substitute ioc for preoperative ercp as a standard method to confirm those patients who are less likely to have choledocholithiasis , if the surgeons in charge of this procedure have sufficient experience and technique to perform ioc successfully . open choledochotomy or sphincterotomy ( operative removal of cbd stones is recommended)(recommendation d ) in japan , choledocholithotripsy with emergency endoscopic procedures ( es , epbd ) is performed by endoscopists , as well as by many surgeons , and it has become the treatment of first choice for cbd clearance . one - stage cholecystectomy and cbd clearance has not been demonstrated to be safer and more effective than endoscopic procedures , and it is not recommended in either the jpn guidelines or the international association of pancreatology ( iap ) guidelines.10
laparoscopic cholecystectomy should be considered after recovery from an attack of gallstone pancreatitis and should be performed during the same hospital stay ( choledochotomy and cbd clearance are performed as required ) ( recommendation b ) cholelithiasis is one of the major causes of acute pancreatitis , and gallstone - induced acute pancreatitis is an absolute indication for cholecystectomy ( and choledochotomy , when necessary ) . because the results showed no significant difference in the incidence of complications ( 8.3% vs 10.3% ) or the mortality rate ( 2.8% vs 6.9% ) , this rct concluded that surgery on the bile duct system with the aim of removing the cause of the pancreatitis or preventing its progression could be performed safely even in the acute state . however , the introduction of emergency ercp / es2,3 has completely changed the options available for the management of gallstone pancreatitis and has dramatically reduced the need for performing surgery in the acute stage . there are two options in regard to delayed surgery : ( i ) surgery during the same hospital stay as that for the initial treatment , and ( ii ) surgery during rehospitalization after a sufficient recovery period . because the rate of recurrence of pancreatitis during the recovery period after discharge is 32% to 61% ( level 3b),1315 patients with mild uncomplicated gallstone pancreatitis should undergo biliary tract exploration and cholecystectomy after the symptoms have been alleviated . whenever possible , even patients with severe disease should undergo surgery during the same hospital stay as that for the initial treatment . laparoscopic cholecystectomy ( lc ) has been increasingly used to treat gallstone pancreatitis over the past 10 years . according to prospective cohort studies reported to date ( levels 1b2c),1621 lc
was associated with a completion rate of 94.5% ( range , 79% to 100% ) , a complication ( morbidity ) rate of 5.5% ( range , 0% to 10% ) , and a mortality rate of 0.4% ( range , 0% to 2.5% ; table 3 ) , implying that lc resulted in performance equal to or better than that of laparotomy . table 3prospective cohort studies of laparoscopic cholecystectomy employed for gallstone - associated acute pancreatitisauthornumber of patientstiming of surgery ( days after onset)completion rate ( % ) conversion rate ( % ) operation time ( min)morbidity ( % ) mortality ( % ) cbd explorationrhodes et al.1610 ( 434)100050 ( 30120)0015/1tate et al.247 ( 324)87.512.576 ( na)8023/0ballestra - lopez et al.403.4/15100086 ( 45210)102.50/40ricci et al.51na1000na1.9040/47uhl et al.4810 ( 429)792180 ( 30225)7.900/33chang et al.59na1000na3.400/58na , not assessed median ( range ) mean mild / severe disease preoperative erc / intraoperative cholangiogram prospective cohort studies of laparoscopic cholecystectomy employed for gallstone - associated acute pancreatitis preoperative erc / intraoperative cholangiogram the conventional standard surgical intervention for gallstone pancreatitis used to be open cholecystectomy and operative cholangiography , followed by choledochotomy when choledocholithiasis was observed . the introduction of lc , however , has made several options available for biliary tract exploration and the management of choledocholithiasis . some surgeons argue that ercp should be performed only when choledocholithiasis is suspected on the basis of blood studies and other diagnostic imaging , or when it is diagnosed during ioc ( level 1b2b).21,22 the risk inherent in ercp should be noted , particularly in patients with acute pancreatitis ( level 2b).23 it may be possible to substitute ioc for preoperative ercp as a standard method to confirm those patients who are less likely to have choledocholithiasis , if the surgeons in charge of this procedure have sufficient experience and technique to perform ioc successfully . | [
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] |
inguinal hernia repair is the most frequently performed procedure in general surgery . over the last few years
, the placement of a prosthetic mesh in front of the hernia orifice has become an increasingly popular strategy to prevent recurrence .
several techniques exist that require permanent fixation of the prosthesis to the abdominal wall , usually using tissue - penetrating devices like staples or sutures . however , such techniques can cause post - operative bleeding as well as pain due to nerve compression [ 2 , 3 ] .
in particular , most reports of chronic pain encountered after tension - free groin hernia repair are related to the use of these tissue - penetrating devices [ 35 ] .
the recognised problem of complications associated with permanent mesh fixation methods in groin hernioplasty prompted the search for other fixation techniques .
tisseel1 fibrin sealant ( baxter healthcare , deerfield , illinois , usa ) has been proposed as an alternative , atraumatic method for mesh fixation based on its effective , proven adhesive properties , as well as its potential additional wound - healing properties [ 68 ] .
tisseel is a biodegradable , biological preparation combining highly concentrated , human plasma - derived fibrinogen ( 75115 mg / ml ) and thrombin ( 500 iu / ml ) .
the mixing of these components in the presence of calcium chloride leads to the development of a three - dimensional matrix of polymerised fibrin fibres in a process mimicking the last step of biological coagulation .
fibrin sealant can , therefore , be used as an adjuvant to haemostasis in a variety of surgical applications [ 9 , 10 ] . in 1997 , chevrel and rath first proposed fibrin sealant as an alternate means of mesh fixation in hernia repair , with the aim of reducing the rate of hernia recurrence .
canonico et al . later reported the benefits of fibrin sealant in reducing bleeding complications following hernia repair in patients with impaired coagulation .
have since employed a pig model using a total extraperitoneal ( tep ) technique to evaluate the tensile strength of mesh fixation 12 days after the use of tisseel , demonstrating equal strength to staples .
the results of these studies have encouraged surgeons to use fibrin sealant in daily practice as an atraumatic alternative to mechanical mesh fixation . in france , an estimated 87,500 tension - free hernioplasty procedures were performed in 2000 . in order to assess the feasibility of a new technique of mesh fixation
, we set up an open , multicentre , observational study inviting surgeons across france to report their cases of groin hernia repair involving the fixation of prosthetic meshes with tisseel .
it was intended that this registry of data would represent real - world surgical practice in france at the time of introduction of a surgical sealant , in the absence of data from large randomised controlled trials .
here , we report data from this study focusing on the rate of recurrence , post - operative pain and complications related to haemostasis , i.e. haematoma and seroma following hernia surgery with tisseel as a means of mesh fixation .
this prospective , multicentre , longitudinal study was initiated in 2003 by baxter healthcare under the guidance of a scientific co - ordinator ( bd ) .
an independent clinical research organisation was employed to monitor the study and analyse the data collected .
french general surgeons performing at least 100 hernioplasty procedures per year , who were already using fibrin sealant for mesh fixation , were targeted for recruitment .
surgeons were asked to record data on adult patients who were scheduled to undergo tension - free hernioplasty , either by the open or laparoscopic approach , with tisseel for mesh fixation .
no other criteria were stipulated by the study protocol other than that tisseel was to be used within its approved indication .
the primary outcome of interest in this study of fibrin sealant in hernia repair relates to bleeding complications , i.e. haematoma and seroma .
these were seen to be reliable indicators of short - term effectiveness , given that tisseel s core indication is as an adjunct to local haemostasis .
data from the 2003 cochrane collaboration systematic review of laparoscopic versus open techniques for hernia repair by mccormack et al . indicate that the combined incidence of haematoma and seroma ( excluding bruising ) following either method of hernioplasty is approximately 14% . based on this value
, we estimated that we needed to enrol 1,333 cases to demonstrate a 25% reduction in bleeding complications , given a power level of 80% and a 5% two - sided significance level .
other patient - centred outcome measures studied included recurrence , pain , infections and miscellaneous complications .
rigid definitions of each outcome were not provided , as this study was intended to reflect current practice , so classifications were left to the discretion of individual investigators .
fifty surgeons were targeted for recruitment and each was asked to provide data on 30 cases in order to achieve the target of 1,333 patients , allowing for around 10% drop - out . approximately 2 weeks prior to surgery
, surgeons completed a questionnaire with each patient to collect the following data : demography , hernia risk factors ( prostatism , smoking , chronic constipation , chronic obstructive pulmonary disease ) , type of hernia , haemostasis parameters ( prothrombin time , bleeding time , activated partial thromboplastin time ) , pain assessment ( visual analogue scale [ vas ] , where 0 = no pain and 10 = unbearable pain ) and the use of analgesic treatments . on the day of surgery
, surgeons recorded the type of anaesthesia , surgical technique , mesh , means of fixation , and duration of operation .
they were also asked to complete a vas - graded convenience score to describe tisseel s ease of use ( where 0 = very easy and 10 = very difficult ) .
surgeons were briefed to assess and report any post - operative complications occurring during the first 48 h following surgery . at the one - month follow - up visit
, surgeons examined the patients for evidence of recurrence , including careful questioning to evaluate the level of any post - operative pain relative to pre - operative symptoms .
ultrasound imaging was undertaken at the discretion of the surgeon if it was thought to be necessary to confirm recurrence .
surgeons also completed two additional questionnaires where appropriate : one when sutures had been used in association with fibrin sealant to fix the mesh and the other to describe the previous surgical technique that was used in case of initial recurrent hernia .
this study was run in accordance with the principles outlined in the helsinki declaration of 1964 and its subsequent amendments and in keeping with the good clinical practice guide ( 1988 version ) and article l4113 - 6 of the public health law of france . according to article 78 - 17 of french law on the freedom of access to computerised data of 06/01/78 ,
the study protocol was approved by the french advisory board for the treatment of information related to research in the health sector and the french national computer council . according to article l4113 - 6 of the public health safety law ,
the sponsor submitted the protocol and related documents to the national medical council ( cnom ) , who forwarded a favourable opinion on 27 march 2003 .
this prospective , multicentre , longitudinal study was initiated in 2003 by baxter healthcare under the guidance of a scientific co - ordinator ( bd ) .
an independent clinical research organisation was employed to monitor the study and analyse the data collected .
french general surgeons performing at least 100 hernioplasty procedures per year , who were already using fibrin sealant for mesh fixation , were targeted for recruitment .
surgeons were asked to record data on adult patients who were scheduled to undergo tension - free hernioplasty , either by the open or laparoscopic approach , with tisseel for mesh fixation .
no other criteria were stipulated by the study protocol other than that tisseel was to be used within its approved indication .
the primary outcome of interest in this study of fibrin sealant in hernia repair relates to bleeding complications , i.e. haematoma and seroma .
these were seen to be reliable indicators of short - term effectiveness , given that tisseel s core indication is as an adjunct to local haemostasis .
data from the 2003 cochrane collaboration systematic review of laparoscopic versus open techniques for hernia repair by mccormack et al . indicate that the combined incidence of haematoma and seroma ( excluding bruising ) following either method of hernioplasty is approximately 14% . based on this value
, we estimated that we needed to enrol 1,333 cases to demonstrate a 25% reduction in bleeding complications , given a power level of 80% and a 5% two - sided significance level .
other patient - centred outcome measures studied included recurrence , pain , infections and miscellaneous complications .
rigid definitions of each outcome were not provided , as this study was intended to reflect current practice , so classifications were left to the discretion of individual investigators .
fifty surgeons were targeted for recruitment and each was asked to provide data on 30 cases in order to achieve the target of 1,333 patients , allowing for around 10% drop - out .
approximately 2 weeks prior to surgery , surgeons completed a questionnaire with each patient to collect the following data : demography , hernia risk factors ( prostatism , smoking , chronic constipation , chronic obstructive pulmonary disease ) , type of hernia , haemostasis parameters ( prothrombin time , bleeding time , activated partial thromboplastin time ) , pain assessment ( visual analogue scale [ vas ] , where 0 = no pain and 10 = unbearable pain ) and the use of analgesic treatments . on the day of surgery , surgeons recorded the type of anaesthesia , surgical technique , mesh , means of fixation , and duration of operation .
they were also asked to complete a vas - graded convenience score to describe tisseel s ease of use ( where 0 = very easy and 10 = very difficult ) .
surgeons were briefed to assess and report any post - operative complications occurring during the first 48 h following surgery . at the one - month follow - up visit
, surgeons examined the patients for evidence of recurrence , including careful questioning to evaluate the level of any post - operative pain relative to pre - operative symptoms .
ultrasound imaging was undertaken at the discretion of the surgeon if it was thought to be necessary to confirm recurrence .
surgeons also completed two additional questionnaires where appropriate : one when sutures had been used in association with fibrin sealant to fix the mesh and the other to describe the previous surgical technique that was used in case of initial recurrent hernia .
this study was run in accordance with the principles outlined in the helsinki declaration of 1964 and its subsequent amendments and in keeping with the good clinical practice guide ( 1988 version ) and article l4113 - 6 of the public health law of france . according to article 78 - 17 of french law on the freedom of access to computerised data of 06/01/78 ,
the study protocol was approved by the french advisory board for the treatment of information related to research in the health sector and the french national computer council . according to article l4113 - 6 of the public health safety law ,
the sponsor submitted the protocol and related documents to the national medical council ( cnom ) , who forwarded a favourable opinion on 27 march 2003 .
forty - five surgeons from 42 public and private institutions of varying sizes completed study questionnaires on 1,201 patients within the allocated recruitment period of 1 year . in addition , 51
associated sutures and 29 initial recurrent hernia questionnaires were completed . of the 1,201 patients ,
the mean age was 57 years , with a mean height of 1.72 m and mean body weight of 73.8 kg .
a total of 38% of patients had at least one hernia risk factor ( among them , 39% of patients smoked and 24% had prostatism ) .
in the vast majority of cases , hernias were inguinal ( 99% ) and primary ( 93% ) ; 79% of hernias were unilateral .
the mean pre - operative vas pain rating was 3.2 , indicating moderate pain ( table 1).table 1assessment of pain on a visual analogue scale ( vas ) pre - operatively , at 12 days and at 1 month follow - upmedian time of assessmentnumber of answersnumber ( % ) of patients reporting painvas scoremean ( sd)vas scoremin maxnumber ( % ) of patients with vas score > 3pre - operative ( 15 days before surgery)1,1581,049 ( 90.6)3.2 ( 2.1)010459 ( 39.6)short - term follow - up ( 2 days)1,1801,021 ( 86.5)2.3 ( 1.7)010232 ( 19.7)mid - term follow - up ( 34 days)1,185341 ( 28.8)1.8 ( 1.2)0925 ( 2.1)vas scale : 0 = no pain , 10 = unbearable painpain > 3 on the vas is considered to be moderate or severe assessment of pain on a visual analogue scale ( vas ) pre - operatively , at 12 days and at 1 month follow - up vas scale : 0 = no pain , 10 = unbearable pain pain > 3 on the vas is considered to be moderate or severe general anaesthesia was performed in 84% of the cases .
the techniques used ( table 2 ) were fairly evenly divided between the open approach ( lichtenstein , 34.1% ; plug and patch , 4.4% ; various other open techniques such as stoppa , 5.4% ) and laparoscopic repairs ( trans - abdominal pre - peritoneal patch repair [ tapp ] , 28.4% ; totally extraperitoneal repair [ tep ] , 27.8% ] .
the overall mean duration of surgery was 39 min , with laparoscopic procedures taking slightly longer than open techniques , a result consistent with other trials .table
2surgical techniques used , duration of operation and percentage of patients experiencing complications that could be related to fibrin sealant at 34-day follow - uppatientsopen techniqueslaparoscopic techniquestotallichtensteinplug and patchothertapptepnumber(%)408(34.1)53(4.4)65(5.4)341(28.4)334(27.8)1,201(100)duration of operation ( min ) , mean [ sd]33.3 [ 15.9]30.8 [ 11.7]40.0 [ 17.9]46.0 [ 19.1]39.2 [ 21.3]38.8 [ 19.3]patients with haematomas or seromas ( % ) 5.43.84.93.02.74.4tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair surgical techniques used , duration of operation and percentage of patients experiencing complications that could be related to fibrin sealant at 34-day follow - up tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair in total , 23 different brands of mesh were used ; the most frequently used mesh was the composite parietex ( 24% ) .
surgeons used sutures or staples in addition to tisseel in 28% of patients , mainly ( in 53% of cases ) during lichtenstein repair to fix the prosthesis to the pubic angle or to reduce the hernia orifice in direct hernia .
fixations were located on the pubis or on cooper s ligament , generally with one suture / staple as per standard practice .
the other reason cited for the use of sutures in addition to tisseel was in order to reduce the size of the inguinal orifice , and not for mesh fixation to the abdominal wall .
the amount of fibrin glue used was 1 ml in 0.4% of cases , 2 ml in 74.6% of cases and 5 ml in 25% of cases ( mostly for bilateral hernia ) .
application devices were adapted to each kind of surgery ( application needle or spray for the open techniques , soft or rigid dual - lumen catheters for laparoscopic techniques ) .
regardless of the application device used , surgeons found that tisseel was easy to apply , with a mean vas - graded convenience score of 1.6 .
assessed 2448 h after surgery , patients mean vas - graded pain score was 2.3 , indicating mild pain ( table 1 ) .
analgesics were prescribed to 86.3% of patients reporting pain , mainly paracetamol ( 63.2% of prescriptions ) and dextropropoxyphene ( 25.3% of prescriptions ) .
follow - up data were collected at a median of 34 days after surgery ( table 2 ) .
local complications that could be influenced by the use of fibrin sealant were recorded in 4.7% of patients overall : 3.0% of patients had haematoma , 1.4% seroma and 0.3% ( four patients ) recurrence .
no cases of neuralgia at the operating site were recorded ; one infection was noted .
only 2.1% of the patients scored pain > 3 on the vas ( moderate or severe ) .
the mean ( sd ) duration of sick leave was 25.2 ( 13.5 ) days and was comparable between active and sedentary workers .
forty - five surgeons from 42 public and private institutions of varying sizes completed study questionnaires on 1,201 patients within the allocated recruitment period of 1 year . in addition , 51
of the 1,201 patients , 90% were male and 47% were sedentary workers or retired .
the mean age was 57 years , with a mean height of 1.72 m and mean body weight of 73.8 kg .
a total of 38% of patients had at least one hernia risk factor ( among them , 39% of patients smoked and 24% had prostatism ) .
in the vast majority of cases , hernias were inguinal ( 99% ) and primary ( 93% ) ; 79% of hernias were unilateral .
the mean pre - operative vas pain rating was 3.2 , indicating moderate pain ( table 1).table 1assessment of pain on a visual analogue scale ( vas ) pre - operatively , at 12 days and at 1 month follow - upmedian time of assessmentnumber of answersnumber ( % ) of patients reporting painvas scoremean ( sd)vas scoremin maxnumber ( % ) of patients with vas score > 3pre - operative ( 15 days before surgery)1,1581,049 ( 90.6)3.2 ( 2.1)010459 ( 39.6)short - term follow - up ( 2 days)1,1801,021 ( 86.5)2.3 ( 1.7)010232 ( 19.7)mid - term follow - up ( 34 days)1,185341 ( 28.8)1.8 ( 1.2)0925 ( 2.1)vas scale : 0 = no pain , 10 = unbearable painpain > 3 on the vas is considered to be moderate or severe assessment of pain on a visual analogue scale ( vas ) pre - operatively , at 12 days and at 1 month follow - up vas scale : 0 = no pain , 10 = unbearable pain pain > 3 on the vas is considered to be moderate or severe
general anaesthesia was performed in 84% of the cases . the techniques used ( table 2 ) were fairly evenly divided between the open approach ( lichtenstein , 34.1% ; plug and patch , 4.4% ; various other open techniques such as stoppa , 5.4% ) and laparoscopic repairs ( trans - abdominal pre - peritoneal patch repair [ tapp ] , 28.4% ; totally extraperitoneal repair [ tep ] , 27.8% ] .
the overall mean duration of surgery was 39 min , with laparoscopic procedures taking slightly longer than open techniques , a result consistent with other trials .table
2surgical techniques used , duration of operation and percentage of patients experiencing complications that could be related to fibrin sealant at 34-day follow - uppatientsopen techniqueslaparoscopic techniquestotallichtensteinplug and patchothertapptepnumber(%)408(34.1)53(4.4)65(5.4)341(28.4)334(27.8)1,201(100)duration of operation ( min ) , mean [ sd]33.3 [ 15.9]30.8 [ 11.7]40.0 [ 17.9]46.0 [ 19.1]39.2 [ 21.3]38.8 [ 19.3]patients with haematomas or seromas ( % ) 5.43.84.93.02.74.4tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair surgical techniques used , duration of operation and percentage of patients experiencing complications that could be related to fibrin sealant at 34-day follow - up tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair in total , 23 different brands of mesh were used ; the most frequently used mesh was the composite parietex ( 24% ) .
surgeons used sutures or staples in addition to tisseel in 28% of patients , mainly ( in 53% of cases ) during lichtenstein repair to fix the prosthesis to the pubic angle or to reduce the hernia orifice in direct hernia .
fixations were located on the pubis or on cooper s ligament , generally with one suture / staple as per standard practice .
the other reason cited for the use of sutures in addition to tisseel was in order to reduce the size of the inguinal orifice , and not for mesh fixation to the abdominal wall .
the amount of fibrin glue used was 1 ml in 0.4% of cases , 2 ml in 74.6% of cases and 5 ml in 25% of cases ( mostly for bilateral hernia ) .
application devices were adapted to each kind of surgery ( application needle or spray for the open techniques , soft or rigid dual - lumen catheters for laparoscopic techniques ) .
regardless of the application device used , surgeons found that tisseel was easy to apply , with a mean vas - graded convenience score of 1.6 .
assessed 2448 h after surgery , patients mean vas - graded pain score was 2.3 , indicating mild pain ( table 1 ) .
analgesics were prescribed to 86.3% of patients reporting pain , mainly paracetamol ( 63.2% of prescriptions ) and dextropropoxyphene ( 25.3% of prescriptions ) .
follow - up data were collected at a median of 34 days after surgery ( table 2 ) .
local complications that could be influenced by the use of fibrin sealant were recorded in 4.7% of patients overall : 3.0% of patients had haematoma , 1.4% seroma and 0.3% ( four patients ) recurrence .
no cases of neuralgia at the operating site were recorded ; one infection was noted .
only 2.1% of the patients scored pain > 3 on the vas ( moderate or severe ) .
the mean ( sd ) duration of sick leave was 25.2 ( 13.5 ) days and was comparable between active and sedentary workers .
initial studies with tisseel fibrin sealant as a method of mesh fixation in tension - free hernia repair were promising , prompting further investigation of its utility in large , controlled trials . such a trial
has recently been completed in open inguinal hernia repair ( timeli trial , campanelli et al .
tisseel was introduced to france in 1982 and began to be used in hernia repair from 2002 , a time when french surgeons were performing over 87,000 hernia repair operations per year , and quickly became popular as an alternative means of mesh fixation . in the absence of randomised controlled trial data , in 2003 ,
it was decided to set up a registry to collect data on the use of tisseel as a means of mesh fixation in hernioplasty , in order to assess its feasibility .
this led to the design of this large , multicentre , observational study , which concluded in 2005 . in our study of 1,201 patients , we noted the following low rates of complications at 34 days follow - up : 3.0% haematoma , 1.4% seroma , 0.3% short - term recurrence , with a mean vas pain score of 1.8 .
there was one infection noted , and no cases of neuralgia at the site of operation . since the completion of our study , many studies that assess the use of fibrin sealant in the most popular forms of inguinal hernia repair have been published ( table 3 ) .
compared fibrin sealant with staples as a means of mesh fixation in 197 patients undergoing tapp repair of inguinal or femoral hernia . at 12-month follow - up ,
the mean vas pain score ( the primary endpoint ) was significantly lower in the sealant group ( 19 vs. 26 mm ; p < 0.05 ) .
one haematoma / seroma was noted in the staples group and one recurrence in the sealant group . in another italian
randomised controlled trial of tapp hernioplasty , 600 patients received fixation with one of three different tack systems or fibrin sealant .
after 1 month , no recurrences were observed in any group , but morbidity was generally lower with tisseel , with more rapid return to work noted.table 3recently published prospective studies on the use of fibrin sealant in hernia repair , by surgical techniquetechniqueauthor [ references]yearnumber of patients ( hernias ) treatedfollow - up ( months)recurrencehaematomachronic paintapplovisetto et al .
2005551200.02%0tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair , ipom intra - peritoneal onlay meshrandomised controlled trial recently published prospective studies on the use of fibrin sealant in hernia repair , by surgical technique tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair , ipom intra - peritoneal onlay mesh randomised controlled trial a french study by topart et al . focused on tep repair : fibrin sealant was used for mesh fixation in 66 patients , compared with staple fixation in 102 patients .
adequate mesh fixation was achieved with a lower incidence of chronic post - operative pain with fibrin sealant versus staples ( 4.5 vs. 11.8% , respectively ) .
lau compared outcomes with fibrin sealant and staple fixation following simultaneous bilateral tep in 93 patients , demonstrating a significant reduction of analgesic consumption in the fibrin sealant group .
there was a small increase in the incidence of post - operative seroma , although this was not considered to be clinically significant .
finally , olmi et al . performed intra - peritoneal onlay mesh ( ipom ) fixation with fibrin sealant in 60 selected patients .
after an average of 23.7-months follow - up , one patient experienced trocar - site haematoma , but no other complications were observed . regarding the lichtenstein technique , canonico et al . assessed the use of fibrin sealant in 80 patients in an italian study with 12 months follow - up .
no complications were observed , and the use of fibrin sealant was considered to be effective for the prevention of local haemorrhagic complications after herniorrhaphy in patients with coagulation disorders . a spanish study by hidalgo et al .
assessed mesh fixation using fibrin sealant compared with polypropylene sutures in 55 patients treated for bilateral hernia using the lichtenstein technique .
similar overall outcomes were reported in both inguinal regions , but there was less post - operative pain and less inflammatory reaction associated with fibrin - fixed hernia repairs .
once again , there were no recurrences after 1 year of follow - up .
the results of our observational study complement the findings of other studies comparing fibrin sealant with standard mesh fixation methods , and suggest that fibrin sealant may yield fewer haematomas , seromas , recurrences and less post - operative pain than mechanical means of mesh fixation . the surgeons in our study found the sealant to be easy to use and operation times appear similar to studies reporting on mechanical forms of mesh in both open and laparoscopic hernia repair fixation . in terms of cost , the price of 2 ml of fibrin sealant ( the most commonly used dosage ) is similar to that of staples .
clearly , large controlled trials are needed to confirm or disprove these promising indications and we eagerly await the results of the timeli trial . in terms of study limitations , we achieved only 90% of the target patient population before the end of the 1-year recruitment period precluded further enrolment . however , a cohort of 1,201 patients still represents a very large sample . given the design of this study , some other limitations deserve mention .
in particular , the short - term follow - up is a major shortcoming , and a comprehensive attempt to obtain 12-month follow - up data would have yielded worthwhile information .
perhaps one might have expected to see slightly higher recurrence rates at 12-months follow - up .
it is also possible that some small seromas or haematomas may have arisen and resolved between the 2- and 34-day follow - up assessments .
the absence of a pre - defined classification system for each complication may also be criticised , as might be the absence of standardised operating procedures , including precise methods for mesh fixation .
however , it is important to remember that this study was initiated to collect data reflecting the widespread use of tisseel for hernia repair in day - to - day surgical practice in 20032004 , in the absence of randomised control data .
this is the first study of its type in france and remains the largest of its kind in this country .
data from 42 institutions were collected , providing a robust and highly generalisable representation of tisseel use in hernia surgery from across the nation .
with a total of 1,201 patients enrolled , this study is the only large - scale french study on the use of fibrin sealant in hernia repair and provides epidemiological support for the use of tisseel fibrin sealant in the fixation of prosthetic mesh during groin hernia repair .
fibrin sealant is an appropriate , atraumatic , easy - to - use alternative to the traditional , tissue - penetrating mesh fixation devices used during common hernia repair techniques ( lichtenstein , totally extraperitoneal repair [ tep ] and trans - abdominal pre - peritoneal patch repair [ tapp ] ) . in our study , the use of tisseel was associated with a very low recurrence rate ( 0.3% ) , as well as a low rate of local complications ( 4.7% overall ) , and few patients experienced post - operative pain at 1-month follow - up , which has an important impact on the overall patient satisfaction with surgery .
fibrin sealant appears to be a promising alternative to stapling / suturing for mesh fixation during inguinal hernioplasty . | purposea prospective , multicentre , observational study was undertaken to assess tisseel fibrin sealant for atraumatic mesh fixation in inguinal hernia repair throughout france.methodssurgeons recorded data on patients undergoing tension - free inguinal hernioplasty with mesh fixation with tisseel , regardless of the hernioplasty technique used .
assessments were made at 2 days and 1 month after surgery .
data on local complications , operation times and ease of product use were collected.resultsin total , 1,201 patients were recruited ( 90% men , mean age 57 years ) , among which 526 procedures were performed using open techniques and 675 using laparoscopic repairs .
local complications occurred in 4.7% of patients : 3.0% haematoma , 1.4% seroma , 0.3% recurrence .
the mean visual analogue scale ( vas)-rated pain scores were 3.2 pre - operatively , 2.3 immediately after surgery and 1.8 at 1 month .
surgeons rated the product as very easy to use.conclusionstisseel fibrin sealant appears to be a well - tolerated and easy - to - use alternative to traditional , tissue - penetrating devices for mesh fixation in hernia repair techniques . | Introduction
Materials and methods
Design
Assessments
Statistical analysis
Ethical considerations
Results
Collection of data
Pre-operative assessment
Surgical technique
Short-term follow-up
One-month follow-up
Discussion
Conclusion | inguinal hernia repair is the most frequently performed procedure in general surgery . several techniques exist that require permanent fixation of the prosthesis to the abdominal wall , usually using tissue - penetrating devices like staples or sutures . in particular , most reports of chronic pain encountered after tension - free groin hernia repair are related to the use of these tissue - penetrating devices [ 35 ] . tisseel1 fibrin sealant ( baxter healthcare , deerfield , illinois , usa ) has been proposed as an alternative , atraumatic method for mesh fixation based on its effective , proven adhesive properties , as well as its potential additional wound - healing properties [ 68 ] . in 1997 , chevrel and rath first proposed fibrin sealant as an alternate means of mesh fixation in hernia repair , with the aim of reducing the rate of hernia recurrence . later reported the benefits of fibrin sealant in reducing bleeding complications following hernia repair in patients with impaired coagulation . have since employed a pig model using a total extraperitoneal ( tep ) technique to evaluate the tensile strength of mesh fixation 12 days after the use of tisseel , demonstrating equal strength to staples . the results of these studies have encouraged surgeons to use fibrin sealant in daily practice as an atraumatic alternative to mechanical mesh fixation . in france , an estimated 87,500 tension - free hernioplasty procedures were performed in 2000 . in order to assess the feasibility of a new technique of mesh fixation
, we set up an open , multicentre , observational study inviting surgeons across france to report their cases of groin hernia repair involving the fixation of prosthetic meshes with tisseel . haematoma and seroma following hernia surgery with tisseel as a means of mesh fixation . this prospective , multicentre , longitudinal study was initiated in 2003 by baxter healthcare under the guidance of a scientific co - ordinator ( bd ) . french general surgeons performing at least 100 hernioplasty procedures per year , who were already using fibrin sealant for mesh fixation , were targeted for recruitment . surgeons were asked to record data on adult patients who were scheduled to undergo tension - free hernioplasty , either by the open or laparoscopic approach , with tisseel for mesh fixation . the primary outcome of interest in this study of fibrin sealant in hernia repair relates to bleeding complications , i.e. data from the 2003 cochrane collaboration systematic review of laparoscopic versus open techniques for hernia repair by mccormack et al . approximately 2 weeks prior to surgery
, surgeons completed a questionnaire with each patient to collect the following data : demography , hernia risk factors ( prostatism , smoking , chronic constipation , chronic obstructive pulmonary disease ) , type of hernia , haemostasis parameters ( prothrombin time , bleeding time , activated partial thromboplastin time ) , pain assessment ( visual analogue scale [ vas ] , where 0 = no pain and 10 = unbearable pain ) and the use of analgesic treatments . they were also asked to complete a vas - graded convenience score to describe tisseel s ease of use ( where 0 = very easy and 10 = very difficult ) . ultrasound imaging was undertaken at the discretion of the surgeon if it was thought to be necessary to confirm recurrence . this study was run in accordance with the principles outlined in the helsinki declaration of 1964 and its subsequent amendments and in keeping with the good clinical practice guide ( 1988 version ) and article l4113 - 6 of the public health law of france . according to article l4113 - 6 of the public health safety law ,
the sponsor submitted the protocol and related documents to the national medical council ( cnom ) , who forwarded a favourable opinion on 27 march 2003 . this prospective , multicentre , longitudinal study was initiated in 2003 by baxter healthcare under the guidance of a scientific co - ordinator ( bd ) . french general surgeons performing at least 100 hernioplasty procedures per year , who were already using fibrin sealant for mesh fixation , were targeted for recruitment . surgeons were asked to record data on adult patients who were scheduled to undergo tension - free hernioplasty , either by the open or laparoscopic approach , with tisseel for mesh fixation . the primary outcome of interest in this study of fibrin sealant in hernia repair relates to bleeding complications , i.e. approximately 2 weeks prior to surgery , surgeons completed a questionnaire with each patient to collect the following data : demography , hernia risk factors ( prostatism , smoking , chronic constipation , chronic obstructive pulmonary disease ) , type of hernia , haemostasis parameters ( prothrombin time , bleeding time , activated partial thromboplastin time ) , pain assessment ( visual analogue scale [ vas ] , where 0 = no pain and 10 = unbearable pain ) and the use of analgesic treatments . they were also asked to complete a vas - graded convenience score to describe tisseel s ease of use ( where 0 = very easy and 10 = very difficult ) . ultrasound imaging was undertaken at the discretion of the surgeon if it was thought to be necessary to confirm recurrence . of the 1,201 patients ,
the mean age was 57 years , with a mean height of 1.72 m and mean body weight of 73.8 kg . the mean pre - operative vas pain rating was 3.2 , indicating moderate pain ( table 1).table 1assessment of pain on a visual analogue scale ( vas ) pre - operatively , at 12 days and at 1 month follow - upmedian time of assessmentnumber of answersnumber ( % ) of patients reporting painvas scoremean ( sd)vas scoremin maxnumber ( % ) of patients with vas score > 3pre - operative ( 15 days before surgery)1,1581,049 ( 90.6)3.2 ( 2.1)010459 ( 39.6)short - term follow - up ( 2 days)1,1801,021 ( 86.5)2.3 ( 1.7)010232 ( 19.7)mid - term follow - up ( 34 days)1,185341 ( 28.8)1.8 ( 1.2)0925 ( 2.1)vas scale : 0 = no pain , 10 = unbearable painpain > 3 on the vas is considered to be moderate or severe assessment of pain on a visual analogue scale ( vas ) pre - operatively , at 12 days and at 1 month follow - up vas scale : 0 = no pain , 10 = unbearable pain pain > 3 on the vas is considered to be moderate or severe general anaesthesia was performed in 84% of the cases . the techniques used ( table 2 ) were fairly evenly divided between the open approach ( lichtenstein , 34.1% ; plug and patch , 4.4% ; various other open techniques such as stoppa , 5.4% ) and laparoscopic repairs ( trans - abdominal pre - peritoneal patch repair [ tapp ] , 28.4% ; totally extraperitoneal repair [ tep ] , 27.8% ] . the overall mean duration of surgery was 39 min , with laparoscopic procedures taking slightly longer than open techniques , a result consistent with other trials .table
2surgical techniques used , duration of operation and percentage of patients experiencing complications that could be related to fibrin sealant at 34-day follow - uppatientsopen techniqueslaparoscopic techniquestotallichtensteinplug and patchothertapptepnumber(%)408(34.1)53(4.4)65(5.4)341(28.4)334(27.8)1,201(100)duration of operation ( min ) , mean [ sd]33.3 [ 15.9]30.8 [ 11.7]40.0 [ 17.9]46.0 [ 19.1]39.2 [ 21.3]38.8 [ 19.3]patients with haematomas or seromas ( % ) 5.43.84.93.02.74.4tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair surgical techniques used , duration of operation and percentage of patients experiencing complications that could be related to fibrin sealant at 34-day follow - up tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair in total , 23 different brands of mesh were used ; the most frequently used mesh was the composite parietex ( 24% ) . the other reason cited for the use of sutures in addition to tisseel was in order to reduce the size of the inguinal orifice , and not for mesh fixation to the abdominal wall . regardless of the application device used , surgeons found that tisseel was easy to apply , with a mean vas - graded convenience score of 1.6 . assessed 2448 h after surgery , patients mean vas - graded pain score was 2.3 , indicating mild pain ( table 1 ) . follow - up data were collected at a median of 34 days after surgery ( table 2 ) . local complications that could be influenced by the use of fibrin sealant were recorded in 4.7% of patients overall : 3.0% of patients had haematoma , 1.4% seroma and 0.3% ( four patients ) recurrence . the mean ( sd ) duration of sick leave was 25.2 ( 13.5 ) days and was comparable between active and sedentary workers . the mean age was 57 years , with a mean height of 1.72 m and mean body weight of 73.8 kg . the mean pre - operative vas pain rating was 3.2 , indicating moderate pain ( table 1).table 1assessment of pain on a visual analogue scale ( vas ) pre - operatively , at 12 days and at 1 month follow - upmedian time of assessmentnumber of answersnumber ( % ) of patients reporting painvas scoremean ( sd)vas scoremin maxnumber ( % ) of patients with vas score > 3pre - operative ( 15 days before surgery)1,1581,049 ( 90.6)3.2 ( 2.1)010459 ( 39.6)short - term follow - up ( 2 days)1,1801,021 ( 86.5)2.3 ( 1.7)010232 ( 19.7)mid - term follow - up ( 34 days)1,185341 ( 28.8)1.8 ( 1.2)0925 ( 2.1)vas scale : 0 = no pain , 10 = unbearable painpain > 3 on the vas is considered to be moderate or severe assessment of pain on a visual analogue scale ( vas ) pre - operatively , at 12 days and at 1 month follow - up vas scale : 0 = no pain , 10 = unbearable pain pain > 3 on the vas is considered to be moderate or severe
general anaesthesia was performed in 84% of the cases . the techniques used ( table 2 ) were fairly evenly divided between the open approach ( lichtenstein , 34.1% ; plug and patch , 4.4% ; various other open techniques such as stoppa , 5.4% ) and laparoscopic repairs ( trans - abdominal pre - peritoneal patch repair [ tapp ] , 28.4% ; totally extraperitoneal repair [ tep ] , 27.8% ] . the overall mean duration of surgery was 39 min , with laparoscopic procedures taking slightly longer than open techniques , a result consistent with other trials .table
2surgical techniques used , duration of operation and percentage of patients experiencing complications that could be related to fibrin sealant at 34-day follow - uppatientsopen techniqueslaparoscopic techniquestotallichtensteinplug and patchothertapptepnumber(%)408(34.1)53(4.4)65(5.4)341(28.4)334(27.8)1,201(100)duration of operation ( min ) , mean [ sd]33.3 [ 15.9]30.8 [ 11.7]40.0 [ 17.9]46.0 [ 19.1]39.2 [ 21.3]38.8 [ 19.3]patients with haematomas or seromas ( % ) 5.43.84.93.02.74.4tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair surgical techniques used , duration of operation and percentage of patients experiencing complications that could be related to fibrin sealant at 34-day follow - up tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair in total , 23 different brands of mesh were used ; the most frequently used mesh was the composite parietex ( 24% ) . the other reason cited for the use of sutures in addition to tisseel was in order to reduce the size of the inguinal orifice , and not for mesh fixation to the abdominal wall . regardless of the application device used , surgeons found that tisseel was easy to apply , with a mean vas - graded convenience score of 1.6 . local complications that could be influenced by the use of fibrin sealant were recorded in 4.7% of patients overall : 3.0% of patients had haematoma , 1.4% seroma and 0.3% ( four patients ) recurrence . the mean ( sd ) duration of sick leave was 25.2 ( 13.5 ) days and was comparable between active and sedentary workers . initial studies with tisseel fibrin sealant as a method of mesh fixation in tension - free hernia repair were promising , prompting further investigation of its utility in large , controlled trials . such a trial
has recently been completed in open inguinal hernia repair ( timeli trial , campanelli et al . tisseel was introduced to france in 1982 and began to be used in hernia repair from 2002 , a time when french surgeons were performing over 87,000 hernia repair operations per year , and quickly became popular as an alternative means of mesh fixation . in the absence of randomised controlled trial data , in 2003 ,
it was decided to set up a registry to collect data on the use of tisseel as a means of mesh fixation in hernioplasty , in order to assess its feasibility . this led to the design of this large , multicentre , observational study , which concluded in 2005 . in our study of 1,201 patients , we noted the following low rates of complications at 34 days follow - up : 3.0% haematoma , 1.4% seroma , 0.3% short - term recurrence , with a mean vas pain score of 1.8 . since the completion of our study , many studies that assess the use of fibrin sealant in the most popular forms of inguinal hernia repair have been published ( table 3 ) . compared fibrin sealant with staples as a means of mesh fixation in 197 patients undergoing tapp repair of inguinal or femoral hernia . in another italian
randomised controlled trial of tapp hernioplasty , 600 patients received fixation with one of three different tack systems or fibrin sealant . after 1 month , no recurrences were observed in any group , but morbidity was generally lower with tisseel , with more rapid return to work noted.table 3recently published prospective studies on the use of fibrin sealant in hernia repair , by surgical techniquetechniqueauthor [ references]yearnumber of patients ( hernias ) treatedfollow - up ( months)recurrencehaematomachronic paintapplovisetto et al . 2005551200.02%0tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair , ipom intra - peritoneal onlay meshrandomised controlled trial recently published prospective studies on the use of fibrin sealant in hernia repair , by surgical technique tapp trans - abdominal pre - peritoneal patch repair , tep totally extraperitoneal repair , ipom intra - peritoneal onlay mesh randomised controlled trial a french study by topart et al . focused on tep repair : fibrin sealant was used for mesh fixation in 66 patients , compared with staple fixation in 102 patients . adequate mesh fixation was achieved with a lower incidence of chronic post - operative pain with fibrin sealant versus staples ( 4.5 vs. 11.8% , respectively ) . performed intra - peritoneal onlay mesh ( ipom ) fixation with fibrin sealant in 60 selected patients . no complications were observed , and the use of fibrin sealant was considered to be effective for the prevention of local haemorrhagic complications after herniorrhaphy in patients with coagulation disorders . assessed mesh fixation using fibrin sealant compared with polypropylene sutures in 55 patients treated for bilateral hernia using the lichtenstein technique . the results of our observational study complement the findings of other studies comparing fibrin sealant with standard mesh fixation methods , and suggest that fibrin sealant may yield fewer haematomas , seromas , recurrences and less post - operative pain than mechanical means of mesh fixation . the surgeons in our study found the sealant to be easy to use and operation times appear similar to studies reporting on mechanical forms of mesh in both open and laparoscopic hernia repair fixation . the absence of a pre - defined classification system for each complication may also be criticised , as might be the absence of standardised operating procedures , including precise methods for mesh fixation . however , it is important to remember that this study was initiated to collect data reflecting the widespread use of tisseel for hernia repair in day - to - day surgical practice in 20032004 , in the absence of randomised control data . data from 42 institutions were collected , providing a robust and highly generalisable representation of tisseel use in hernia surgery from across the nation . with a total of 1,201 patients enrolled , this study is the only large - scale french study on the use of fibrin sealant in hernia repair and provides epidemiological support for the use of tisseel fibrin sealant in the fixation of prosthetic mesh during groin hernia repair . fibrin sealant is an appropriate , atraumatic , easy - to - use alternative to the traditional , tissue - penetrating mesh fixation devices used during common hernia repair techniques ( lichtenstein , totally extraperitoneal repair [ tep ] and trans - abdominal pre - peritoneal patch repair [ tapp ] ) . in our study , the use of tisseel was associated with a very low recurrence rate ( 0.3% ) , as well as a low rate of local complications ( 4.7% overall ) , and few patients experienced post - operative pain at 1-month follow - up , which has an important impact on the overall patient satisfaction with surgery . fibrin sealant appears to be a promising alternative to stapling / suturing for mesh fixation during inguinal hernioplasty . | [
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] |
due to the increasing number of multilinguals in modern society , the incidence of language impairments induced by brain lesions ( aphasia ) in this population is growing rapidly [ 1 , 2 ] .
the rehabilitation of multilingual aphasic patients represents an important challenge for clinicians because ( i ) since the representation of first ( l1 ) and second ( l2 ) languages partly overlaps in bilinguals ' brains , brain lesions do not necessarily affect l1 and l2 equally ; and ( ii ) recovery patterns for each language in multilingual aphasic patients vary considerably and so far are unpredictable .
most of the current literature indicates that language recovery in bilingual aphasic patients depends on the degree of language mastery or language - specific factors [ 57 ] .
for example , similarities in typology , phonological , morphological , lexical , and syntactic aspects between languages are shown to affect the pattern of recovery of languages in bilingual aphasic patients [ 1 , 6 ] .
such an approach is also supported by evidence that changes in second language expertise and use are associated with an increase of connectivity within the language network of healthy subjects .
however , growing evidence suggests that the control system may also play a key role in this process [ 5 , 8 , 9 ] . in healthy bilingual speakers ,
cognitive control system is strongly involved in language production because language representations must be manipulated and monitored both within the language being spoken and across languages to select the appropriate vocabulary and syntax and to inhibit the nontarget language .
dynamic view in which the pattern of language recovery in bilingual aphasia depends on the patient 's ability to select and control language activation [ 10 , 12 ] : ( i ) a parallel recovery , in which both impaired languages improve to a similar extent , and , concurrently , occurs when both languages are inhibited to the same degree ; ( ii ) an antagonistic recovery , in which the patient is able to speak in one language on one day while on the next day only in the other , occurs when inhibition affects only one language for a period of time and then shifts to the other language ( with disinhibition of the previously inhibited language ) ; ( iii ) a selective recovery , in which one language remains impaired while the other recovers , occurs if the lesion has permanently raised the activation threshold for one language ; and ( iv ) a pathological mixing , in which the elements of the two languages are involuntarily mixed during language production , occurs when languages can no longer be selectively inhibited [ 9 , 10 , 13 ] . while this theory accounts for the large variability in recovery patterns of multilingual aphasia , there is only sparse evidence for any association between control function and language recovery since control functions are rarely specifically assessed in aphasic patients .
reported the case of a bilingual aphasic patient who showed a greater deficit in her more used l1 than in her less practiced l2 , following lesions mainly involving the left basal ganglia .
the authors suggest that the patient 's deficit in l1 may be considered as a pathological fixation on a foreign language resulting from a deficit in switching between languages . however , the patient had a normal performance in the wisconsin card - sorting test ( wcs ) , a nonverbal task testing the ability to change from one criterion of choice to another .
this result suggested that , in the absence of a remarkable impairment in control functions ( shown in wcs which evaluated shifting , a part of control functions ) , the patient 's fixation behavior was mostly linguistic .
moreover , since the assessment of executive functions was conducted one year after the stroke , anatomofunctional plastic reorganization of the language and control networks could already have taken place and likely confounded the results . an earlier evaluation ( e.g. , at acute or subacute phase )
following the stroke could have better shown whether this so - called pathological fixation on l2 and the l1 impairment has resulted from impairment in cognitive control function .
verreyt et al . reported the case of an early french - dutch bilingual aphasic who , following a lesion to the left thalamus , presented larger impairment in dutch . by showing cognate facilitation and cognate interference effects in different lexical decision tasks and an impaired performance in the flanker task , the authors suggested that the differential pattern of impairment in language could be explained by a language - control deficit .
in addition , abutalebi et al . , in a longitudinal , single - case study of a chronic bilingual aphasic patient combining fmri and dynamic causal modelling ( dcm ) , showed an increased connectivity within the control and language networks for the treated and recovered language . in line with the paradis 's activation threshold theory , which holds that lesions that do not completely damage language areas but cause an imbalance in activating and inhibiting languages are responsible for aphasia in bilinguals , they found that the engagement of the areas mediating language control played a crucial role in language recovery in bilingual aphasic patients .
they showed that connections between language and control areas were stronger in the language that recovered better , probably because it received more resources for its functioning .
the network underlying language control described by abutalebi and green and abutalebi et al . includes the prefrontal cortex ( mainly inferior prefrontal cortex including lifgorb ( left inferior frontal gyrus pars orbitalis , ba47 ) ) , the anterior cingulate cortex ( acc ) ( bas24 , 32 , 33 ) , and the basal ganglia .
this network is interconnected with language areas involved in word production ( lifgtri : left inferior frontal gyrus pars triangularis , ba45 ) and basal temporal language area , btla involved in semantic decoding during picture naming ( posterior part of the left inferior temporal gyrus bas19 and 37 ) . in the bilingual brain ,
the prefrontal cortex is involved in word production in the less proficient language and in inhibiting responses from the more proficient language .
together with the anterior cingulate that detects response conflicts , it constitutes a control loop in which the identification of conflict triggers a top - down signal from the prefrontal cortex to modulate the nontarget representation ( see [ 10 , 15 , 16 ] ) .
the left caudate and the acc are strongly connected to the prefrontal cortex and work together with this structure to inhibit interferences from the nontarget language .
the acc signals potential response conflicts or errors to the prefrontal cortex ( i.e. , in the case that an erroneous language has been chosen ) and the prefrontal cortex then seeks to avoid incorrect selection . finally , the basal ganglia may subserve language planning , that is , the activation of a given language as a main function of the left caudate and the control of articulatory processes in the left putamen ( see [ 18 , 19 ] ) . using linguistic and nonlinguistic switching tasks
, it has been shown that the neuroanatomical bases of language control and domain - general cognitive control share the partially overlapping structures , although their involvement may vary [ 20 , 21 ] .
it is worth noting that understanding neural mechanisms underlying patterns of recovery has many implications for the therapeutic approach .
based on the hypothesis of a key role for cognitive control in bilingual language production and in the recovery of bilingual aphasia , our study aims to test whether among the different control areas proposed by abutalebi et al .
, changes in certain connections between control and language areas influence the recovery of language ( namely , between lifgtri and lifgorb and lc and acc ) . to this aim
, we tested five late bilingual patients who suffered from aphasia following a focal left - hemispheric brain lesion .
the patients were evaluated at two time points ( subacute and chronic phases , three months apart ) .
three main analyses were conducted to examine the pattern of changes in patients ' language and control functions , connectivity within language - control network , and possible correlation between behavioral performances and connectivity with language - control network.as a descriptive marker of behavioral improvement / changes in language and control functions , the patients were behaviorally evaluated for their pattern of recovery of language and executive functions using general aphasia evaluation ( gae ) , picture naming and executive tasks ( linguistic and nonlinguistic switching).in order to investigate the connections within the language - control network , we used fmri analyses and applied dynamic causal modelling in the fmri picture naming task in l1 and l2 to examine whole brain activation patterns and the effective connectivity between the control areas ( acc , left caudate nucleus , and lifgorb ) and the regions involved in language production ( especially lifgtri ) .
we further examined whether global changes in connectedness within language - control network are associated with the recovery of languages.to directly assess the hypothesis advanced in the language - control model , we examined the correlations between the recovery of language functions and the changes in the strength of connections between the above - mentioned areas using group analyses .
in fact , as meier et al . in a dcm study on chronic aphasic patients and a group of controls have found that language network parameters are specifically associated with naming abilities in picture naming task , we consider that there should be a difference in connection strength in l1 and l2 and also according to naming improvement across time . as a descriptive marker of behavioral improvement
/ changes in language and control functions , the patients were behaviorally evaluated for their pattern of recovery of language and executive functions using general aphasia evaluation ( gae ) , picture naming and executive tasks ( linguistic and nonlinguistic switching ) . in order to investigate the connections within the language - control network ,
we used fmri analyses and applied dynamic causal modelling in the fmri picture naming task in l1 and l2 to examine whole brain activation patterns and the effective connectivity between the control areas ( acc , left caudate nucleus , and lifgorb ) and the regions involved in language production ( especially lifgtri ) .
we further examined whether global changes in connectedness within language - control network are associated with the recovery of languages . to directly assess the hypothesis advanced in the language - control model
, we examined the correlations between the recovery of language functions and the changes in the strength of connections between the above - mentioned areas using group analyses .
in fact , as meier et al . in a dcm study on chronic aphasic patients and a group of controls have found that language network parameters are specifically associated with naming abilities in picture naming task , we consider that there should be a difference in connection strength in l1 and l2 and also according to naming improvement across time .
we chose to evaluate bilingual aphasic patients during the subacute phase since this population has rarely been studied in the acute and subacute phases .
this will allow us to better understand the contribution of the control system in the recovery of language in bilingual aphasia , especially during the period when spontaneous recovery process mainly takes place [ 6 , 23 ] .
in addition , in this phase , the spontaneous recovery and neural plasticity processes are ongoing and given that bilingual population is strongly relied on cognitive control system , we assume that the changes in cognitive control system and its interconnection with the language system probably play a role in the recovery of aphasia .
we recruited right - handed late ( age of acquisition ( aoa ) of l2 after 6 y / o ) bilingual patients aged between 18 and 85 years old , who suffered from aphasia following a focal left - sided ischemic or hemorrhagic stroke .
the following languages were included in the study : french ( in each case as subjects ' l1 or l2 ) and english , german , spanish , or italian . during the recruitment procedure , we excluded patients with a history of premorbid language impairment , several brain lesions , or severe aphasia .
however , only six patients completed all the steps of the study and , among them , five subjects fulfilled our criteria of the selection of regions of interest ( rois ) for the dcm analyses and therefore are reported in this paper .
five more patients performed the first session of the study and then declined to participate in the second session ( see section 2.3 for details of the steps of the study ) and were therefore not included in the analyses . among the five patients included in this paper ( aged 61.6 ( 6.9 ) years old and including two females ) ,
three patients were french ( l1 ) and english ( l2 ) and two patients were italian ( l1 ) and french ( l2 ) .
the lesion of each patient is shown in a figure specifically designed for each of them ( figures 3(a)7(a ) ) .
the study procedure was approved by the local ethics committees of geneva university hospital ( ce 12 - 274 ) and fribourg cantonal hospital ( 018/12-cer - fr ) .
yl is a 61-year - old man who is a french ( l1)-english ( l2 ) bilingual .
yl estimates an advanced level of english for reading , speaking , and comprehension ( all between 95 and 100% according to the self - evaluation scale of l2 level ) . before the stroke , his language usage was mainly in french
however , his reading was 50% in french and 50% in english ( readings in english are mainly work - related books and documents ) , and he used mainly english at his workplace ( 80% ) .
yl was admitted to geneva university hospital ( hug ) with right sensorimotor hemiparesis , right facial palsy , and impaired comprehension and language production mainly manifested in l2 following a left frontotemporal ischemic stroke . a secondary hemorrhagic event in the ischemic area
a first language evaluation showed a transcortical sensory aphasia ; he presented mainly auditory comprehension problems and produced repeated semantic errors .
she moved to the french - speaking part of switzerland at the age of 24 , and then she has taken some courses to learn french . before the stroke , she used italian and french quite equally ; she used french at work ( 100% ) , and italian for tv or radio programs ( 100% ) .
she used 50% in italian and 50% in french to speak with her family and friends and to read books and journals .
mr was admitted to hug for resection of a meningioma on the left greater wing of the sphenoid bone .
two days after the resection of the meningioma , she presented a right sensorimotor hemiparesis and a severe language production problem plus a lesser degree of comprehension problems in both languages , caused by an epidural hematoma with pressure over the operation site and ischemic changes in the left frontobasal area ( figure 4(a ) ) .
ca is a 63-year - old woman who is an italian ( l1)-french ( l2 ) bilingual .
she moved to the french - speaking part of switzerland at the age of 10 ; thereafter she started to learn french .
she has also basic knowledge in english and spanish , which she has learned at school .
before the stroke , the main language of conversation was french with her husband and children ( 90% ) and at work ( 75% ) and she spoke italian with her parents ( 100% ) .
she was admitted to hug because of right hemiparesis and severe global aphasia due to a left basal ganglia hemorrhagic stroke with no evidence of midline shift ( figure 5(a ) ) . within a few days , global aphasia developed into severe anomia with hypophonia mainly affecting l2 .
rg is a 49-year - old bilingual french ( l1)-english ( l2 ) male patient .
he used english quite frequently in his daily life ; he used french and english equally at work ( 50% french and 50% english for teaching and customer care ) .
he followed tv and radio programs and also read books and journals 50% in french and 50% in english .
however , he spoke only in french with his friends and family . according to the self - evaluation questionnaire filled in by his wife ,
his language abilities were estimated as follows : speaking 50% , comprehension 70% , reading 85% , and writing 30% .
he was admitted to fribourg cantonal hospital with a sudden right hemiparesis and anomia and no other language symptoms due to a left sylvian ischemic stroke ( figure 6(a ) ) .
gh is a 79-year - old bilingual french ( l1)-english ( l2 ) male patient .
he has learned english around the age of 18 when he first travelled to the us and england .
he has been working in sweden for about 18 years teaching guitar in both english and swedish .
he used both french and english in his teachings ( 50% french and 50% english ) . with his family
he spoke only in french ; however with his friends he spoke 50% in french and 50% in english .
he followed tv and radio program mostly in french ( 75% in french and 25% in english ) and he read books and journals only in french . he was admitted to hug with a paresthesia in his left arm and global aphasia .
the cerebral ct scan after the acute event confirmed an ischemic lesion in the left frontal , insula , and sylvian areas ( figure 7(a ) ) . within a few days , global aphasia developed into severe anomia and increased switching behavior .
more details of patients can be found in tables 1 and 2 and supplementary data 1 available online at http://dx.doi.org/10.1155/2016/8797086 .
the data and results on the control subjects are presented in supplementary data 2 and 3 .
subjects were assessed using a questionnaire on their immersion in both l1 and l2 , aoa , how long they had lived in a region where predominantly the second language was spoken , which language they spoke with their family members , in school , and in present activities ( watching tv / listening to radio , reading books , and mental arithmetic ) , and if the language was acquired in school or out of school only . in the self - evaluation part , subjects ( or their family members ) had to indicate in percentages how well they would estimate their reading , speaking , comprehension , and writing skills .
patients were assessed at subacute ( three to five weeks after stroke onset , t1 ) and chronic ( three months after t1 evaluation , t2 ) phases . in both sessions
we used the same procedures , listed as follows : ( 1 ) behavioral assessment of the severity of aphasia as well as a combination of language - control function evaluations ; ( 2 ) in an fmri recording session , the patients performed a language production task ( picture naming ) in each language ( see section 2.6.1 for picture naming task ) .
general aphasia evaluation ( gae ) : global severity of the aphasia and language capacities was assessed using a separate evaluation of language capacities in each language ( i.e. , l1 and l2 were evaluated separately , one day apart ) .
this evaluation consisted of a brief test of object naming ( ten objects to name ) , automatic speech ( series : days of the week , counting from 1 to 25 ) , word and phrase repetition , yes / no questions , object recognition , following oral and written instructions ( simple , semicomplex , and complex commands ) , description , and verbal fluency .
all these tests were extracted from the bilingual aphasia test ( bat ) except for yes / no questions which were extracted from the mississippi aphasia screening test ( mast ) .
this evaluation material has been already used in our previously published works , for example , . as a result , a production index of maximum 52 scores ( i.e. , the sum of the scores obtained from production tasks including object naming , series , verbal fluency , word and phrase repetition , and description ) and a total score ( maximum 96 scores ) was obtained .
language - control functions were evaluated using the following : a linguistic switching task ( adapted from abutalebi et al .
2008 for aphasic patients ): forty images ( black and white line drawing picture ) of snodgrass and vanderwart ( all noncognate words ) were used for each list .
eight pairs of lists were prepared ( a combination of french as first or second language and the other four languages ) .
the subjects were asked to name , as quickly as possible , the images in l1 when the image appeared on the upper part and name the image in l2 when the image appeared on the lower part of the screen . after a fixation cross of 500 ms ,
the images were presented on the screen for 5,000 ms and were followed by a blank screen of variable duration of 3,0007,000 ms ( to provide a random duration of the interstimulus interval ) .
therefore , the subjects had at most between 8,000 and 12,000 ms to respond .
however , only first - attempt correct responses within five seconds of the presentation of the image were scored as correct .
the first six trials of the task were cued with the language in which the image should be named ( l1 or l2 ) written on the left of the image ( figure 1(a ) ) .
this task lasted between 10 and 12 minutes depending on patients ' response time.a nonlinguistic switching task : four images ( a red or blue circle or square ) were presented on the upper or the lower part of the screen .
subjects were instructed to name , as quickly as possible , the color of the image when the image was presented on the upper part of the screen and to say the shape of the image when it was presented on the lower part of the screen . after a fixation cross of 500 ms ,
the images were presented on the screen for 5,000 ms and were followed by a blank screen of variable duration of 3,0007,000 ms ( to provide a random duration of the interstimulus interval ) .
. however , only first - attempt correct responses within five seconds of the presentation of the image were scored as correct .
the first six trials of the task were cued with the category in which the image should be named ( color or shape ) written on the left of the image ( figure 1(b ) ) .
the task lasted around 1012 minutes depending on patient 's response time . a linguistic switching task ( adapted from abutalebi et al .
2008 for aphasic patients ): forty images ( black and white line drawing picture ) of snodgrass and vanderwart ( all noncognate words ) were used for each list .
eight pairs of lists were prepared ( a combination of french as first or second language and the other four languages ) .
the subjects were asked to name , as quickly as possible , the images in l1 when the image appeared on the upper part and name the image in l2 when the image appeared on the lower part of the screen . after a fixation cross of 500 ms ,
the images were presented on the screen for 5,000 ms and were followed by a blank screen of variable duration of 3,0007,000 ms ( to provide a random duration of the interstimulus interval ) .
therefore , the subjects had at most between 8,000 and 12,000 ms to respond .
however , only first - attempt correct responses within five seconds of the presentation of the image were scored as correct .
each trial was started manually by the experimenter when the word ready ? was presented on the screen .
the first six trials of the task were cued with the language in which the image should be named ( l1 or l2 ) written on the left of the image ( figure 1(a ) ) .
this task lasted between 10 and 12 minutes depending on patients ' response time . a nonlinguistic switching task : four images ( a red or blue circle or square ) were presented on the upper or the lower part of the screen .
subjects were instructed to name , as quickly as possible , the color of the image when the image was presented on the upper part of the screen and to say the shape of the image when it was presented on the lower part of the screen . after a fixation cross of 500 ms ,
the images were presented on the screen for 5,000 ms and were followed by a blank screen of variable duration of 3,0007,000 ms ( to provide a random duration of the interstimulus interval ) .
. however , only first - attempt correct responses within five seconds of the presentation of the image were scored as correct .
each trial was started manually by the experimenter when the word ready ? was presented on the screen .
the first six trials of the task were cued with the category in which the image should be named ( color or shape ) written on the left of the image ( figure 1(b ) ) .
for all the tasks , instructions were given both written on the screen and orally , and the subjects performed a short training session just before starting the task .
the evaluation of the language - control function was performed in the more proficient language ( usually l1 ) . moreover , because of slowness of patients and fatigability , for all the tasks we did not record reaction times , and the analyses were focused on response accuracy . because of the limited number of patients , differences in lesion size and site , and variability of symptoms , we used primarily a multiple single - case approach for our analyses between t1 and t2 . for comparison of the patients ' scores in the two sessions
( i.e. , the average response accuracy percentage in picture naming and production score of the gae ) are assessed as language performances . specifically , we focused on the combined production score which could better represent language production performance
five lists ( one list per language ) of 40 noncognate words ( black and white line drawing pictures ) were selected from snodgrass and vanderwart .
each fmri session started with a picture naming in l1 ; in this part , the subjects were instructed to name the pictures that appeared on the screen in their l1 . after a fixation cross of 500 ms ,
the images were presented on the screen for 5,000 ms and were followed by a blank screen of variable duration of 4,1006,100 ms ( to provide a random duration of the interstimulus interval ) . therefore , the subjects had at most between 9,100 and 11,100 ms to respond
. however , only first - attempt correct responses within five seconds of the presentation of the image were scored as correct .
each task lasted around 7 - 8 minutes ( a total of around 15 minutes for picture naming in both l1 and l2 ) .
after about 30 seconds of rest , the subjects started their second task in which they had to name the pictures in their second language .
the first six trials of the task were cued with the language in which the image should be named ( l1 or l2 ) written on the left of the image .
for the fmri tasks , a short training was performed before the subjects entered the scanner . in this training , which contained 10 trials ,
the subjects were presented with black and white line drawing pictures selected from snodgrass list and were asked to name the pictures in their l1 or l2 to become familiar with the task .
data of the aphasic patients were acquired using three different 3 t scanners on two different sites ; site 1 : fribourg cantonal hospital ( hfr ) and site 2 : university hospital of geneva ( hug ) .
the scanners which were used were ( 1 ) discovery mr750 ; ge healthcare , waukesha , wisconsin , with a 32-channel receive head coil ( site 1 ) , ( 2 ) magneton trio , siemens medical solutions , erlangen , germany , with a 12-channel receive head coil ( site 2 ) , and ( 3 ) magneton prisma , siemens medical solutions , erlangen , germany , with a 20-channel receive head ( site 2 ) .
subjects were in a supine position with their heads stabilized by foam to reduce head movements .
they wore headphones ( mkii system from mr confon , magdeburg , germany ) coupled with an mri - compatible microphone ( fomri - iii system from optoacoustics , israel ) to record oral response during the experiment . in the first scanner , visual stimuli were presented on an lcd screen ( nordicneurolab , bergen , norway ) . in the other two scanners , the stimuli were displayed on a screen by a video projector ( hitachi cp - x1200 with long focal distance hitachi ll-504 , hitachi ltd . , tokyo , japan ) through a mirror system . in all three cases ,
the stimuli resolution was 1024 768 with a refresh rate of 60 hz .
the e - prime 2 software ( psychology software tools , pittsburgh , usa ) was used to show stimuli and record behavioral data .
mri acquisition parameters were optimized for each site . from the first site in fribourg ( scanner 1 ) ,
t1-weighted images were acquired with a fspgr bravo sequence , voxel size : 0.86 0.86 1 mm , field of view ( fov ) = 220 mm , number of coronal slices : 276 , tr / te = 7300/2.8 ms , flip angle = 9 , phase acceleration factor ( paf ) = 1.5 , and intensity correction ( scic ) . functional t2weighted echo planar images ( epi ) with blood oxygenation level - dependent ( bold ) contrast
were acquired with voxel size : 2.3 2.3 3 mm , fov = 220 mm , 37 ascending axial slices , interslice spacing = 0.2 mm , tr / te = 2000/30 ms , flip angle = 85 , and piaf : 2 .
in addition , a b0 field inhomogeneity mapping sequence was acquired to correct for geometrical distortion that occurred along the phase - encoding direction ( using a gradient echo protocol ) with the same scan coverage as the functional scan : number of slices = 37 , fov = 220 mm , tr / te1/te2 = 50/4.9/7.3 ms . from the second site ( scanners 2 and 3 ) ,
t1 weighted images were acquired with an mp rage sequence , voxel size : 0.86 0.86 1.1 mm , fov = 220 mm , number of coronal slices : 208 , tr / te = 2500/2.94 ms for scanner 2 and 2500/2.97 for scanner 3 , flip angle = 9 , and paf : 2 .
functional t2weighted epi with bold contrast were acquired with voxel size : 2 2 3.5 mm , fov = 240 mm , 29 ascending axial slices , interslice spacing = 0.35 mm , tr / te = 2000/30 ms , flip angle = 85 , and piaf : 2 .
a b0 field inhomogeneity mapping sequence was also acquired with the same scan coverage as the functional mri sequences : number of slices = 29 , fov = 240 mm , and tr / te1/te2 = 400/5.19/7.65 ms . on average ,
a total of 248 volumes were acquired during the picture naming in l1 and picture naming in l2 .
each fmri acquisition session started with six seconds of dummy scans to ensure a steady - state magnetization of the tissues .
we used the spm8 software ( welcome trust centre for neuroimaging , institute of neurology , university college london ) , running on matlab 2012b ( mathworks , inc .
preprocessing steps included a spatial realignment , unwrapping ( using the fieldmap 2.1 toolbox ) , slice timing ( with middle temporal slice as reference ) , coregistration on t1 image , normalization on the montreal neurological institute ( mni ) space with 3 3 3 mm voxel size , and smoothing with a gaussian kernel of 8 mm full width at half maximum ( fwhm ) . in order to exclude the brain lesion from the analyses , a mask file of the brain lesion of each subject
was manually drawn on axial slices of the standard montreal neurological institute 's ( mni ) brain template using the mricron software ( https://www.nitrc.org/projects/mricron ) and used during the preprocessing of data on spm .
the preprocessed volumes were submitted to fixed effects analyses at the subject level by applying the general linear model to each voxel .
each stimulus onset was modelled as an event encoded in condition - specific stick - functions and convolved with a canonical hemodynamic response function .
a separate model was built for picture naming in l1 and picture naming in l2 .
time series from all voxels were submitted to a high - pass filter with a 1/250 hz threshold , and an autoregressive function ( ar ( 1 ) ) was applied .
dcm is a widely used method for investigating context - dependent causal interactions between brain regions and it describes the architecture of the network ( i.e. , the rois and the connections ) . in dcm , the brain is treated as a dynamic input - state - output system . a given experiment is considered as a designed perturbation of neuronal dynamics that is propagated throughout a network of interconnected nodes .
three sets of parameters are estimated in dcm : the direct influence of stimuli on regional activity ( driving input ) , the intrinsic connections between regions , and the changes in the intrinsic connectivity between regions induced by adding or removing a modulatory influence ( modulatory effect ) .
we based our analyses on this model , which has been defined by abutalebi et al . .
because of the variability of lesion site in our patients , in order to be able to compare changes in connectivity with the same model across all subjects / conditions , we have defined this model for all subjects and conditions .
we have not selected a fully connected model ( i.e. , with all possible connections within the network ) to avoid having a very complex model and overfitting of the data .
in addition the structure of this model was designed based on a priori hypotheses already tested by previous works .
therefore , the selection of the rois and the intrinsic connections was based exactly on the work by this group .
accordingly , the following five rois were selected for the network : btla , lifgtri ( areas related to language processing ) , head of left caudate , acc ( areas involved in cognitive control function ) , and lifgorb as a part of both language and cognitive control systems . as per abutalebi
, we also included only left - hemispheric regions as our main focus was the effect of control areas on the intrahemispheric reorganization of language areas ( see figure 2 for the model structure ) .
the same model was used for all subjects ( patients and controls ) and for both testing sessions . using td - icbm - mni template atlas , we prepared the mask for the rois .
individual subject time series data from each subject 's individual activation map threshold at p < 0.05 uncorrected were extracted from each 7 mm spherical roi centered at the subject 's local maximum inside the rois . for the patients ,
when the patients or control subjects did not fulfill our criteria ( showing activation with threshold < 0.05 uncorrected in all 5 rois and/or absence of lesion in the rois ) they were removed from the analyses .
this way , we have removed one patient ( as one of the rois was inside the lesion ) and one control participant ( as he did not show activation in one of the rois in the desired threshold ) . however , in order to take into account the modulatory effect of the language task on the network ( which was not included previously in the model by abutalebi et al . ) , we inserted the modulatory effect of the task over btla ( as the sensory input of the network ) and lifgtri as two different models .
we compared the three models ( two models with modulatory effect of picture naming on lifgtri or btla and a model with no modulatory effect ) using a bayesian model selection with a fixed effect strategy which assumes that the optimal structure is assumed to be identical across subjects , and the model with modulatory effect over the btla best explained fmri activation through the different patients and controls ( separately ) during the naming in l1 and l2 according to this comparison .
the analyses of dcm were performed using spm12 and using the data preprocessed in spm8 .
considering the limited number of patients and the effect of the different scanners used in this study , we primarily performed the analyses at a single - case level .
patterns of brain activation in the four different conditions ( picture naming in l1 and l2 at t1 : subacute phase and t2 : chronic phase ) for each patient are shown in the figure representing the data related to the patient ( figures 3(b)7(b ) ) . regarding the dcm analyses , in order to investigate how connection strength changes over time for single intrinsic connections within the network , the differences in the strength of connection between l1 and l2 ( l2 l1 ) at each session are presented in a graph for each patient .
these graphs represent the pattern of difference in connection strength in language - control network while performing picture naming in l1 and l2 across time ( these graphs are shown in figures 3(d)7(d ) ) . at the group level ,
correlation analyses were performed with aphasic patients to investigate possible correlations between the changes in connection strength ( especially for the connections between control and language areas ) and the changes in combined production scores for each language separately .
( a ) we first conducted mcnemar chi - squared tests comparing language performance ( gae and picture naming scores ) in l1 and l2 and control function ( linguistic and nonlinguistic switch task scores ) across time ; ( b ) using dcm on fmri , we compared the strength of connectivity within the language - control network between l1 and l2 across time at single subject level ; ( c ) at the group level , we then performed a correlation analysis between the recovery of language production scores and the changes in the strength of connection between language and control areas .
a description of the main results of the analyses is provided here , and a complete reporting of the scores and results is provided in table 3 and supplementary data 1 .
the combined production score showed improvement in l1 ( : 12.7 , p : 0.005 ) but no changes in l2 .
in addition , no improvement was found in linguistic and nonlinguistic switching tasks accuracy ( figure 3(c ) ) .
( b ) changes in connectivity in the language - control network . for each single intrinsic connection within the network , the differences in connection strengths between l1 and l2 ( l2 l1 ) at each session are shown in figure 3(d ) . at t1 ( subacute phase ) , seven connection strengths were greater for l1 and eight connections had greater coupling values for l2 . at t2 ( chronic phase ) , however , the majority of connections ( 10 out of 15 ) had stronger coupling values for l1 compared to l2 ( i.e. , the following five connections had higher strength values in l2 at t2 ( chronic phase ) : connections from lc to acc , lifgtri , and lifgorb , from lifgtri to lc , and from btla to lifgtri ) .
the rest of the 15 connections had higher strength values in l1 at t2 ( chronic phase ) .
these changes in strength values indicated a globally higher connectedness inside the language - control network for l1 .
( a ) behavioral scores . at t2 ( chronic phase ) , the combined production score improved in both l1 ( : 9.09 , p : 0.002 ) and l2 ( : 5.14 , p : 0.023 )
. however , no significant improvement was found in linguistic ( : 3.2 , p : 0.07 ) and nonlinguistic switching tasks ( : 0.5 , p : 0.47 ) ( figure 4(c ) ) ( see table 3 for details of the patient 's performance ) .
( b ) changes in connectivity in the language - control network . regarding the dcm analyses for each single intrinsic connection within the network ,
importantly , a notable change was seen in the language - control network in the pattern of differences in connection strengths between l1 and l2 from t1 ( subacute phase ) and t2 ( chronic phase ) : at t1 , five connections had greater strength values for l1 and 10 connections had greater strength for l2 . at t2 ( chronic phase ) ,
seven connections had greater strength values for l1 and eight connections had greater strength values for l2 . across time , eight connections showed different patterns of difference between l1 and l2 .
in particular , the connections from acc to lifgtri , from lifgorb to lifgtri , and from acc to lc had higher strength values for l2 compared to l1 at t2 ( chronic phase ) , while the connections from lc to acc , lifgtri , and lifgorb , from acc to lifgorb , and from lifgtri to ab47 showed greater strength values for l1 compared to l2 at t2 ( chronic phase ) . although reorganization happened in the connection strengths for l1 and l2 at t2 ( chronic phase ) , there was a similar degree of connectedness within the language - control network for l1 and l2 .
the combined production score showed improvement in both l1 ( : 25.07 , p < 0.0001 ) and l2 ( : 4.16 , p : 0.041 ) at t2 ( chronic phase ) .
the patient also showed a significant improvement in both linguistic and nonlinguistic switching tasks across time ( : 17.05 , p < 0.0001 and : 21.04 , p < 0.0001 , resp . ) ( figure 5(c ) ) ( see table 3 and supplementary data 1 for details of the patient 's performance ) .
the differences between l1 and l2 ( l2 l1 ) in the strength of single intrinsic connections within the network are shown in figure 5(d ) .
the raw differences in the strength of connections within the language - control network in this patient also indicated differing patterns in the connection strengths between l1 and l2 from t1 ( subacute phase ) and t2 ( chronic phase ) in half of the connections ; notably , the connections from acc to lifgtri and forward and backward connections between lifgtri and lifgorb showed greater connection strengths for l1 compared to l2 at t2 ( chronic phase ) . however , forward and backward connections between lc and lifgorb and the connection from btla to lifgtri and lifgorb had higher connection strength values for l2 compared to l1 at t2 ( chronic phase ) .
overall , at t1 ( subacute phase ) , seven connections had higher strength values in l1 while at t2 ( chronic phase ) , nine connections had higher strength values in l1 .
altogether , there was a similar degree of connectedness within the language - control network for l1 and l2 at t2 ( chronic phase ) .
( a ) behavioral scores . at t2 ( chronic phase ) , the combined production score improved in l2 ( : 8.16 , p : 0.004 ) and no improvement was seen in l1 ( already spared at t1 ( subacute phase ) ) .
his performance in the linguistic switching task improved significantly ( : 4.16 , p : 0.041 ) and his nonlinguistic switching performance was spared at t1 ( subacute phase ) ( figure 6(c ) ) .
( b ) changes in connectivity in the language - control network . at the single intrinsic connection level ,
the differences between l1 and l2 ( l2 l1 ) in strength of single intrinsic connections within the network for each session are shown in figure 6(d ) . at t1 ( subacute phase ) ,
around half of connections had higher strength values for l1 ( eight out of 15 ) , while at t2 ( chronic phase ) , only three connections had greater values for l1 ( i.e. , connection from lifgtri to lc , lifgtri to lifgorb , and btla to lifgorb ) and the rest of the connections showed higher coupling values for l2 .
these changes indicated a globally higher connectedness inside the language - control network for l2 at t2 ( chronic phase ) .
the combined production score improved in both l1 ( : 9.09 , p : 0.002 ) and l2 ( : 12.07 , p : 0.0005 ) at t2 ( chronic phase ) , although the patient still made several language switching errors .
however , no improvement was seen in the linguistic and nonlinguistic switching task performances ( figure 7(c ) ; more details can be found in table 3 and supplementary data 1 ) .
( b ) changes in connectivity in the language - control network . at the single intrinsic connection level ,
the differences between l1 and l2 ( l2 l1 ) in the strength of single intrinsic connections within the network for each session are shown in figure 7(d ) .
importantly , several connections showed inverse patterns between t1 ( subacute phase ) and t2 ( chronic phase ) ; that is , four connections ( from acc to lifgtri , acc to lifgorb , lifgtri to acc , and lifgtri to lifgorb ) had higher strength values for l1 at t2 ( chronic phase ) , and four connections ( from lc to lifgtri , lifgorb to acc , lifgorb to lifgtri , and btla to lifgtri ) had greater strength values for l2 at t2 ( chronic phase ) .
as with the changes seen in patients 2 and 3 , there was a similar connectedness within the language - control network in l1 and l2 at t2 ( chronic phase ) .
for the aphasic patients , the patterns of activation at each session of picture naming in l1 and l2 were presented for each patient separately ; a threshold of uncorrected p < 0.001 was selected to visualize the main effects ( figures 3(b)7(b ) ) . as our main aim of the fmri study was to perform dcm analysis based on a previously published model , we did not statistically compare the activations in the different conditions .
( c ) correlation analysis between language production recovery and changes in the strength of connection . at the group level , in the aphasic patients , the changes in the strength of intrinsic connections between language and control areas ( specifically between acc , lc , and lifgorb from control subnetwork to lifgtri in language subnetwork ) were implemented to correlate with the changes in combined production scores . in the aphasic patients , we found a significant correlation between changes in the combined production scores in l1 and changes in the strength of connection from acc to lifgtri ( while performing picture naming in l1 ) ( spearman 's rho : 0.921 , p : 0.026 ) .
moreover , changes in the combined productions score in l2 were negatively correlated to the changes in the strength of connections from lifgtri to lifgorb ( spearman 's rho : 0.900 , p : 0.037 ) . to better compare the changes in the number of connections with higher strength values between the improved versus unimproved language across time
, we concatenated the data of patients 1 and 4 ( who improved language production in only one language ) . this combined analysis showed a higher number of connections in the improved language at t2 ( chronic phase ) ( : 4.44 , p 0.035 ) .
using a longitudinal design , we examined language production recovery in five late bilingual patients suffering from poststroke aphasia at subacute and chronic phases following a stroke . from three weeks to four months following a stroke , ( a ) we monitored modifications in language and control performance to identify whether language recovery was linked with the recovery of control functions .
moreover , ( b ) using a dcm approach , we examined how the interconnections between language and control areas changed with the recovery of language production , and ( c ) we then investigated the possible correlation between changes in language production performances and changes in the strength of each single connection within language - control network across time .
considering the changes in the combined production scores , three of our five patients recovered in both l1 and l2 ( patients 2 , 3 , and 5 ) , one patient recovered in l1 ( patient 1 ) , and one ( patient 4 ) in l2 only ( the latter patient already had a high accuracy score in l1 at the subacute phase ) .
two patients ( patient 3 with recovery in both languages and patient 4 with recovery in l2 ) showed improvement in language - control functions ( table 4 ) .
descriptive analyses of the dcm suggested a relationship between the pattern of recovery of language production and changes in the strength of connections across time . in patient 1 , who recovered only l1 production score across time ,
the majority of connections within language - control network ( 10 out of 15 connections ) had higher connection strength values at the chronic phase , indicating a higher connectedness within the language - control network while picture naming in l1 .
the similar pattern of changes in the connectedness within language - control network took place in patient 4 who recovered only l2 ( i.e. , at the chronic phase he showed higher connectedness within language - control network while picture naming in l2 ) . in these two patients with recovery of only one language , combined analyses revealed that improvement in production score in one language was associated with an increase in the number of connections with higher strength values at t2 ( chronic phase ) while performing the task in that specific language .
in addition , showing a similar pattern of changes in the language - control network connectedness , patients 2 and 5 at t1 ( subacute phase ) in the majority of connections had higher coupling values for picture naming in l2 , while at t2 , the coupling values of 7 connections were higher in l1 and 8 connections had higher coupling values in l2 task .
also , patient 3 showed higher coupling values for the majority of connections for l1 picture naming at t1 ( subacute phase ) , while at t2 , the coupling values of 7 connections were higher in l1 and 8 connections had higher coupling values in l2 task .
taken together , in patients 2 , 3 , and 5 who recovered both l1 and l2 , a redistribution of the connection strength occurred across time ; the strength of the connections between language and control areas was similarly distributed at t2 ( chronic phase ) over the network during picture naming in l1 and l2 . in the control group with main l2 exposure and usage in daily life ,
a higher connectedness was seen within the network for l1 compared to l2 ( see supplementary data 2 and 3 ) .
although the role of control functions in the recovery of bilingual aphasia has been suggested in several studies [ 8 , 9 ] , in our patients , at the behavioral level , the improvement in language - control functions alone could not explain their patterns of language recovery . the observed pattern of language and control recovery does not directly support paradis ' statement that when language - control function is intact , one can expect a parallel recovery of languages , and in the presence of language - control problems one may expect the weaker language in the premorbid stage to be impaired .
however , green and abutalebi suggest that , along with premorbid proficiency , languages that were mostly used following stroke may become more proficient and easily manageable , especially in the case of reduced resources for controlling the use of two languages .
our findings on the changes in the differences in connectedness between l1 and l2 within the language - control network are in line with the results of abutalebi et al .
, in the case of a bilingual aphasic patient for whom the language which recovered better showed increased connections between language and control networks .
our dcm results support a role for language - control interconnections in language recovery in bilingual aphasic patients [ 9 , 10 , 35 ] and are in line with the dynamic view of language production , which posits that patterns of language recovery are related to alterations in language control .
interestingly , for patients in whom both languages recovered ( patients 2 , 3 , and 5 ) , the two languages were connected to the control system to the same extent . additionally , when one language recovered better , there was a greater engagement of language - control interconnections in this language .
previous studies of the association between global patterns of brain connectivity and the recovery of language functions have suggested that decreased functional connectivity between anterior and posterior areas of the default mode network ( dmn ) is associated with cognitive impairment .
accordingly , therapy - induced increases in functional connectivity between anterior and posterior areas of the dmn have been reported in a group of chronic monolingual aphasic patients . in a further study ,
sebastian and colleagues evaluated the recovery of naming functions from acute to chronic phase and showed that the degree of functional connectivity between language - specific areas in both hemispheres was important for optimal recovery of naming functions .
furthermore , our results suggest that a change in connection strength from acc to lifgtri during picture naming in l1 was associated with l1 recovery ; the coupling between these two areas became stronger when l1 recovered .
lifgtri , along with the lifg pars opercularis ( ba44 ) , is known to be involved in different steps of language production , namely , in syntactic encoding , speech praxis , and verb retrieval .
acc is known to be involved in conflict and error monitoring , including domain - general control functions in healthy populations . in the normally functioning bilingual brain , acc , in connection with the prefrontal cortex ,
is a component of the circuit involved in inhibiting interference from the nontarget language [ 1 , 18 , 43 ] while in a healthy brain , this interference is caused mainly by the more proficient language ( usually l1 ) ; our results could be explained by the fact that in the presence of language and control dysfunction ( e.g. , following a stroke ) , conflicts may arise between l1 and l2 even in the case of different proficiencies .
therefore , a higher engagement of the circuit between acc and lifgtri could possibly facilitate performance in the recovery of l1 by blocking the interference of information from l2
. analyses of the changes in connectivity strengths across time suggest that , in patients with l2 recovery ( four out of five patients ) , the connection from lifgtri to lifgorb becomes weaker for l2 compared to l1 .
this finding is also supported by the result of a correlation analysis showing that the recovery of combined production scores in l2 negatively correlates with changes in the strength of connection from lifgtri to lifgorb . in other words , when l2 recovers , the coupling from lifgtri to lifgorb decreases .
lifgtri is selected as the main language production area and lifgorb is a part of language - control network , which is involved in both language production and language - control processes ( lexical semantic processes along with lifgtri and selecting among lexical competitors ) [ 44 , 45 ] .
the reason for the decrease of coupling from lifgtri to lifgorb in l2 production could be explained by the revised hierarchical model of lexical and conceptual representation in the bilingual brain [ 46 , 47 ] . in this psycholinguistic model
however , l1 is hypothesized to have privileged access to the conceptual system , favored by a strong connection between the areas involved in lexical and semantic processing ( resp . ,
hence , a weaker connectivity between these two areas may help in the process of l2 recovery .
first , we could not implement certain language and control tasks since they were too demanding for aphasic patients , especially in the acute and subacute phases .
a comprehensive evaluation of language and control would have helped better understand the possible correlation between the recovery of control functions and the recovery of language performance following a stroke . even though we narrowed our selection of tasks to a limited series of evaluation materials , half of the initially recruited patients dropped out of the second session of the study .
another limitation of this study was the small number of included patients ; their results may not be easily applicable to all bilingual aphasic patients .
in addition , we could not control the age of the patients in the study ( the age of patients ranged from 49 to 79 years old ) .
it is known that the age factor can affect behavioral performance , functional brain activity , and connectivity within brain areas as a result of alteration in neuronal activity and connectivity in aging brain [ 48 , 49 ] .
however , as the design is mainly within - subject the age does not seem to affect importantly the results .
in addition , regressing out the effect of age from the analyses would not let any significant results due to the small sample size .
moreover , the use of different mri scanners in this study restricted us in carrying out a direct group comparison of brain activation in different conditions .
finally , as obtaining an accurate measure of premorbid proficiency following their stroke was impossible , the evaluation of premorbid second language proficiency was restricted to a detailed questionnaire filled in by a family member or the patient himself .
it worth noting that , in the present study , only three patients followed language therapy sessions and the therapy was computer assisted to improve lexical access and in turn improve naming performances .
one patient ( patient 1 ) received therapy in his l1 ( french ) and he then improved in l1 production .
this lack of improvement in l2 could be explained by the very low l2 usage and immersion by this subject .
it has been previously suggested by edmonds and kiran that the effect of therapy in less mastered language is more likely to transfer to the untreated language as the subject is more relied on borrowing word from the more proficient language .
another explanation of the absence of transfer of the effect of language therapy in l1 to the untreated l2 is the fact that he did not show improvement in control functions across time .
patient 3 received therapy in her l2 ( french ) , which she has been used and was immersed in an equal level as her l1 since 53 years ago .
her improvement in both languages can be explained by high immersion in both languages as well as improvement in the cognitive control functions .
patient 5 attended to a limited number of therapy sessions ( 10 sessions ) in his l1 ( french ) and he improved in both l1 and l2 ( english ) . in this patient , the recovery of both languages can not be explained by the choice of therapy or the pattern of changes in cognitive control functions .
therefore , no consistent pattern of the effect of therapy and possible cross language transfer of the effect of therapy was found .
therefore , our interpretation of these results is not based on the language therapy . moreover , because of the timing of the study sessions ( at three weeks and around four months following the stroke ) , the process of spontaneous recovery should be still ongoing [ 6 , 23 ] . accordingly
, this recovery takes place as the result of a combination of spontaneous recovery and language therapy .
taken together , our findings supply additional evidence that the engagement of the interconnected language - control network is crucial for the recovery of languages . furthermore , we suggest that l1 recovery is improved by increased connectivity between acc and lifgtri , which prevents conflicts from the second language
. however , l2 recovery requires a decrease in connectivity from lifgtri to lifgorb in order to decrease the automatic activation of the l1 lexical system , which , according to the revised hierarchical model , has stronger links with the conceptual system . | aphasia in bilingual patients is a therapeutic challenge since both languages can be impacted by the same lesion . language control has been suggested to play an important role in the recovery of first ( l1 ) and second ( l2 ) language in bilingual aphasia following stroke . to test this hypothesis , we collected behavioral measures of language production ( general aphasia evaluation and picture naming ) in each language and language control ( linguistic and nonlinguistic switching tasks ) , as well as fmri during a naming task at one and four months following stroke in five bilingual patients suffering from poststroke aphasia .
we further applied dynamic causal modelling ( dcm ) analyses to the connections between language and control brain areas .
three patients showed parallel recovery in language production , one patient improved in l1 , and one improved in l2 only .
language - control functions improved in two patients .
consistent with the dynamic view of language recovery , dcm analyses showed a higher connectedness between language and control areas in the language with the better recovery .
moreover , similar degrees of connectedness between language and control areas were found in the patients who recovered in both languages .
our data suggest that engagement of the interconnected language - control network is crucial in the recovery of languages . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion | the rehabilitation of multilingual aphasic patients represents an important challenge for clinicians because ( i ) since the representation of first ( l1 ) and second ( l2 ) languages partly overlaps in bilinguals ' brains , brain lesions do not necessarily affect l1 and l2 equally ; and ( ii ) recovery patterns for each language in multilingual aphasic patients vary considerably and so far are unpredictable . most of the current literature indicates that language recovery in bilingual aphasic patients depends on the degree of language mastery or language - specific factors [ 57 ] . dynamic view in which the pattern of language recovery in bilingual aphasia depends on the patient 's ability to select and control language activation [ 10 , 12 ] : ( i ) a parallel recovery , in which both impaired languages improve to a similar extent , and , concurrently , occurs when both languages are inhibited to the same degree ; ( ii ) an antagonistic recovery , in which the patient is able to speak in one language on one day while on the next day only in the other , occurs when inhibition affects only one language for a period of time and then shifts to the other language ( with disinhibition of the previously inhibited language ) ; ( iii ) a selective recovery , in which one language remains impaired while the other recovers , occurs if the lesion has permanently raised the activation threshold for one language ; and ( iv ) a pathological mixing , in which the elements of the two languages are involuntarily mixed during language production , occurs when languages can no longer be selectively inhibited [ 9 , 10 , 13 ] . moreover , since the assessment of executive functions was conducted one year after the stroke , anatomofunctional plastic reorganization of the language and control networks could already have taken place and likely confounded the results . by showing cognate facilitation and cognate interference effects in different lexical decision tasks and an impaired performance in the flanker task , the authors suggested that the differential pattern of impairment in language could be explained by a language - control deficit . , in a longitudinal , single - case study of a chronic bilingual aphasic patient combining fmri and dynamic causal modelling ( dcm ) , showed an increased connectivity within the control and language networks for the treated and recovered language . in line with the paradis 's activation threshold theory , which holds that lesions that do not completely damage language areas but cause an imbalance in activating and inhibiting languages are responsible for aphasia in bilinguals , they found that the engagement of the areas mediating language control played a crucial role in language recovery in bilingual aphasic patients . they showed that connections between language and control areas were stronger in the language that recovered better , probably because it received more resources for its functioning . this network is interconnected with language areas involved in word production ( lifgtri : left inferior frontal gyrus pars triangularis , ba45 ) and basal temporal language area , btla involved in semantic decoding during picture naming ( posterior part of the left inferior temporal gyrus bas19 and 37 ) . using linguistic and nonlinguistic switching tasks
, it has been shown that the neuroanatomical bases of language control and domain - general cognitive control share the partially overlapping structures , although their involvement may vary [ 20 , 21 ] . based on the hypothesis of a key role for cognitive control in bilingual language production and in the recovery of bilingual aphasia , our study aims to test whether among the different control areas proposed by abutalebi et al . , changes in certain connections between control and language areas influence the recovery of language ( namely , between lifgtri and lifgorb and lc and acc ) . three main analyses were conducted to examine the pattern of changes in patients ' language and control functions , connectivity within language - control network , and possible correlation between behavioral performances and connectivity with language - control network.as a descriptive marker of behavioral improvement / changes in language and control functions , the patients were behaviorally evaluated for their pattern of recovery of language and executive functions using general aphasia evaluation ( gae ) , picture naming and executive tasks ( linguistic and nonlinguistic switching).in order to investigate the connections within the language - control network , we used fmri analyses and applied dynamic causal modelling in the fmri picture naming task in l1 and l2 to examine whole brain activation patterns and the effective connectivity between the control areas ( acc , left caudate nucleus , and lifgorb ) and the regions involved in language production ( especially lifgtri ) . we further examined whether global changes in connectedness within language - control network are associated with the recovery of languages.to directly assess the hypothesis advanced in the language - control model , we examined the correlations between the recovery of language functions and the changes in the strength of connections between the above - mentioned areas using group analyses . as a descriptive marker of behavioral improvement
/ changes in language and control functions , the patients were behaviorally evaluated for their pattern of recovery of language and executive functions using general aphasia evaluation ( gae ) , picture naming and executive tasks ( linguistic and nonlinguistic switching ) . in order to investigate the connections within the language - control network ,
we used fmri analyses and applied dynamic causal modelling in the fmri picture naming task in l1 and l2 to examine whole brain activation patterns and the effective connectivity between the control areas ( acc , left caudate nucleus , and lifgorb ) and the regions involved in language production ( especially lifgtri ) . we further examined whether global changes in connectedness within language - control network are associated with the recovery of languages . to directly assess the hypothesis advanced in the language - control model
, we examined the correlations between the recovery of language functions and the changes in the strength of connections between the above - mentioned areas using group analyses . this will allow us to better understand the contribution of the control system in the recovery of language in bilingual aphasia , especially during the period when spontaneous recovery process mainly takes place [ 6 , 23 ] . in addition , in this phase , the spontaneous recovery and neural plasticity processes are ongoing and given that bilingual population is strongly relied on cognitive control system , we assume that the changes in cognitive control system and its interconnection with the language system probably play a role in the recovery of aphasia . among the five patients included in this paper ( aged 61.6 ( 6.9 ) years old and including two females ) ,
three patients were french ( l1 ) and english ( l2 ) and two patients were italian ( l1 ) and french ( l2 ) . two days after the resection of the meningioma , she presented a right sensorimotor hemiparesis and a severe language production problem plus a lesser degree of comprehension problems in both languages , caused by an epidural hematoma with pressure over the operation site and ischemic changes in the left frontobasal area ( figure 4(a ) ) . subjects were assessed using a questionnaire on their immersion in both l1 and l2 , aoa , how long they had lived in a region where predominantly the second language was spoken , which language they spoke with their family members , in school , and in present activities ( watching tv / listening to radio , reading books , and mental arithmetic ) , and if the language was acquired in school or out of school only . in both sessions
we used the same procedures , listed as follows : ( 1 ) behavioral assessment of the severity of aphasia as well as a combination of language - control function evaluations ; ( 2 ) in an fmri recording session , the patients performed a language production task ( picture naming ) in each language ( see section 2.6.1 for picture naming task ) . general aphasia evaluation ( gae ) : global severity of the aphasia and language capacities was assessed using a separate evaluation of language capacities in each language ( i.e. the first six trials of the task were cued with the language in which the image should be named ( l1 or l2 ) written on the left of the image ( figure 1(a ) ) . the first six trials of the task were cued with the language in which the image should be named ( l1 or l2 ) written on the left of the image ( figure 1(a ) ) . the evaluation of the language - control function was performed in the more proficient language ( usually l1 ) . the first six trials of the task were cued with the language in which the image should be named ( l1 or l2 ) written on the left of the image . on average ,
a total of 248 volumes were acquired during the picture naming in l1 and picture naming in l2 . this way , we have removed one patient ( as one of the rois was inside the lesion ) and one control participant ( as he did not show activation in one of the rois in the desired threshold ) . these graphs represent the pattern of difference in connection strength in language - control network while performing picture naming in l1 and l2 across time ( these graphs are shown in figures 3(d)7(d ) ) . at the group level ,
correlation analyses were performed with aphasic patients to investigate possible correlations between the changes in connection strength ( especially for the connections between control and language areas ) and the changes in combined production scores for each language separately . ( a ) we first conducted mcnemar chi - squared tests comparing language performance ( gae and picture naming scores ) in l1 and l2 and control function ( linguistic and nonlinguistic switch task scores ) across time ; ( b ) using dcm on fmri , we compared the strength of connectivity within the language - control network between l1 and l2 across time at single subject level ; ( c ) at the group level , we then performed a correlation analysis between the recovery of language production scores and the changes in the strength of connection between language and control areas . in addition , no improvement was found in linguistic and nonlinguistic switching tasks accuracy ( figure 3(c ) ) . ( b ) changes in connectivity in the language - control network . these changes in strength values indicated a globally higher connectedness inside the language - control network for l1 . however , no significant improvement was found in linguistic ( : 3.2 , p : 0.07 ) and nonlinguistic switching tasks ( : 0.5 , p : 0.47 ) ( figure 4(c ) ) ( see table 3 for details of the patient 's performance ) . ( b ) changes in connectivity in the language - control network . regarding the dcm analyses for each single intrinsic connection within the network ,
importantly , a notable change was seen in the language - control network in the pattern of differences in connection strengths between l1 and l2 from t1 ( subacute phase ) and t2 ( chronic phase ) : at t1 , five connections had greater strength values for l1 and 10 connections had greater strength for l2 . although reorganization happened in the connection strengths for l1 and l2 at t2 ( chronic phase ) , there was a similar degree of connectedness within the language - control network for l1 and l2 . the patient also showed a significant improvement in both linguistic and nonlinguistic switching tasks across time ( : 17.05 , p < 0.0001 and : 21.04 , p < 0.0001 , resp . ) the raw differences in the strength of connections within the language - control network in this patient also indicated differing patterns in the connection strengths between l1 and l2 from t1 ( subacute phase ) and t2 ( chronic phase ) in half of the connections ; notably , the connections from acc to lifgtri and forward and backward connections between lifgtri and lifgorb showed greater connection strengths for l1 compared to l2 at t2 ( chronic phase ) . altogether , there was a similar degree of connectedness within the language - control network for l1 and l2 at t2 ( chronic phase ) . at t2 ( chronic phase ) , the combined production score improved in l2 ( : 8.16 , p : 0.004 ) and no improvement was seen in l1 ( already spared at t1 ( subacute phase ) ) . ( b ) changes in connectivity in the language - control network . , connection from lifgtri to lc , lifgtri to lifgorb , and btla to lifgorb ) and the rest of the connections showed higher coupling values for l2 . these changes indicated a globally higher connectedness inside the language - control network for l2 at t2 ( chronic phase ) . however , no improvement was seen in the linguistic and nonlinguistic switching task performances ( figure 7(c ) ; more details can be found in table 3 and supplementary data 1 ) . ( b ) changes in connectivity in the language - control network . as with the changes seen in patients 2 and 3 , there was a similar connectedness within the language - control network in l1 and l2 at t2 ( chronic phase ) . at the group level , in the aphasic patients , the changes in the strength of intrinsic connections between language and control areas ( specifically between acc , lc , and lifgorb from control subnetwork to lifgtri in language subnetwork ) were implemented to correlate with the changes in combined production scores . in the aphasic patients , we found a significant correlation between changes in the combined production scores in l1 and changes in the strength of connection from acc to lifgtri ( while performing picture naming in l1 ) ( spearman 's rho : 0.921 , p : 0.026 ) . using a longitudinal design , we examined language production recovery in five late bilingual patients suffering from poststroke aphasia at subacute and chronic phases following a stroke . from three weeks to four months following a stroke , ( a ) we monitored modifications in language and control performance to identify whether language recovery was linked with the recovery of control functions . moreover , ( b ) using a dcm approach , we examined how the interconnections between language and control areas changed with the recovery of language production , and ( c ) we then investigated the possible correlation between changes in language production performances and changes in the strength of each single connection within language - control network across time . considering the changes in the combined production scores , three of our five patients recovered in both l1 and l2 ( patients 2 , 3 , and 5 ) , one patient recovered in l1 ( patient 1 ) , and one ( patient 4 ) in l2 only ( the latter patient already had a high accuracy score in l1 at the subacute phase ) . two patients ( patient 3 with recovery in both languages and patient 4 with recovery in l2 ) showed improvement in language - control functions ( table 4 ) . descriptive analyses of the dcm suggested a relationship between the pattern of recovery of language production and changes in the strength of connections across time . in patient 1 , who recovered only l1 production score across time ,
the majority of connections within language - control network ( 10 out of 15 connections ) had higher connection strength values at the chronic phase , indicating a higher connectedness within the language - control network while picture naming in l1 . the similar pattern of changes in the connectedness within language - control network took place in patient 4 who recovered only l2 ( i.e. , at the chronic phase he showed higher connectedness within language - control network while picture naming in l2 ) . in addition , showing a similar pattern of changes in the language - control network connectedness , patients 2 and 5 at t1 ( subacute phase ) in the majority of connections had higher coupling values for picture naming in l2 , while at t2 , the coupling values of 7 connections were higher in l1 and 8 connections had higher coupling values in l2 task . taken together , in patients 2 , 3 , and 5 who recovered both l1 and l2 , a redistribution of the connection strength occurred across time ; the strength of the connections between language and control areas was similarly distributed at t2 ( chronic phase ) over the network during picture naming in l1 and l2 . although the role of control functions in the recovery of bilingual aphasia has been suggested in several studies [ 8 , 9 ] , in our patients , at the behavioral level , the improvement in language - control functions alone could not explain their patterns of language recovery . the observed pattern of language and control recovery does not directly support paradis ' statement that when language - control function is intact , one can expect a parallel recovery of languages , and in the presence of language - control problems one may expect the weaker language in the premorbid stage to be impaired . our findings on the changes in the differences in connectedness between l1 and l2 within the language - control network are in line with the results of abutalebi et al . , in the case of a bilingual aphasic patient for whom the language which recovered better showed increased connections between language and control networks . our dcm results support a role for language - control interconnections in language recovery in bilingual aphasic patients [ 9 , 10 , 35 ] and are in line with the dynamic view of language production , which posits that patterns of language recovery are related to alterations in language control . interestingly , for patients in whom both languages recovered ( patients 2 , 3 , and 5 ) , the two languages were connected to the control system to the same extent . in a further study ,
sebastian and colleagues evaluated the recovery of naming functions from acute to chronic phase and showed that the degree of functional connectivity between language - specific areas in both hemispheres was important for optimal recovery of naming functions . lifgtri , along with the lifg pars opercularis ( ba44 ) , is known to be involved in different steps of language production , namely , in syntactic encoding , speech praxis , and verb retrieval . in the normally functioning bilingual brain , acc , in connection with the prefrontal cortex ,
is a component of the circuit involved in inhibiting interference from the nontarget language [ 1 , 18 , 43 ] while in a healthy brain , this interference is caused mainly by the more proficient language ( usually l1 ) ; our results could be explained by the fact that in the presence of language and control dysfunction ( e.g. therefore , a higher engagement of the circuit between acc and lifgtri could possibly facilitate performance in the recovery of l1 by blocking the interference of information from l2
. this finding is also supported by the result of a correlation analysis showing that the recovery of combined production scores in l2 negatively correlates with changes in the strength of connection from lifgtri to lifgorb . lifgtri is selected as the main language production area and lifgorb is a part of language - control network , which is involved in both language production and language - control processes ( lexical semantic processes along with lifgtri and selecting among lexical competitors ) [ 44 , 45 ] . a comprehensive evaluation of language and control would have helped better understand the possible correlation between the recovery of control functions and the recovery of language performance following a stroke . another explanation of the absence of transfer of the effect of language therapy in l1 to the untreated l2 is the fact that he did not show improvement in control functions across time . her improvement in both languages can be explained by high immersion in both languages as well as improvement in the cognitive control functions . in this patient , the recovery of both languages can not be explained by the choice of therapy or the pattern of changes in cognitive control functions . moreover , because of the timing of the study sessions ( at three weeks and around four months following the stroke ) , the process of spontaneous recovery should be still ongoing [ 6 , 23 ] . taken together , our findings supply additional evidence that the engagement of the interconnected language - control network is crucial for the recovery of languages . | [
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